US20200109430A1 - Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof - Google Patents
Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof Download PDFInfo
- Publication number
- US20200109430A1 US20200109430A1 US16/706,592 US201916706592A US2020109430A1 US 20200109430 A1 US20200109430 A1 US 20200109430A1 US 201916706592 A US201916706592 A US 201916706592A US 2020109430 A1 US2020109430 A1 US 2020109430A1
- Authority
- US
- United States
- Prior art keywords
- amino acid
- cell wall
- bacteria
- modified amino
- modified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 80
- 210000002421 cell wall Anatomy 0.000 title claims abstract description 69
- 239000000203 mixture Substances 0.000 title abstract description 33
- 238000002372 labelling Methods 0.000 title description 63
- 238000011065 in-situ storage Methods 0.000 title description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 110
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 claims abstract description 89
- 108010013639 Peptidoglycan Proteins 0.000 claims abstract description 89
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 81
- 150000008574 D-amino acids Chemical class 0.000 claims abstract description 78
- 241000894006 Bacteria Species 0.000 claims abstract description 77
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 39
- 238000012216 screening Methods 0.000 claims abstract description 17
- 150000001413 amino acids Chemical class 0.000 claims description 80
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 27
- 238000010348 incorporation Methods 0.000 claims description 24
- 239000007850 fluorescent dye Substances 0.000 claims description 15
- 230000004260 plant-type cell wall biogenesis Effects 0.000 claims description 13
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 claims description 12
- IGHBXJSNZCFXNK-UHFFFAOYSA-N 4-chloro-7-nitrobenzofurazan Chemical compound [O-][N+](=O)C1=CC=C(Cl)C2=NON=C12 IGHBXJSNZCFXNK-UHFFFAOYSA-N 0.000 claims description 10
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 claims description 10
- 239000002243 precursor Substances 0.000 claims description 10
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims description 9
- MNLRQHMNZILYPY-MDMHTWEWSA-N N-acetyl-alpha-D-muramic acid Chemical compound OC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O)[C@@H]1NC(C)=O MNLRQHMNZILYPY-MDMHTWEWSA-N 0.000 claims description 8
- 241000192125 Firmicutes Species 0.000 claims description 6
- LKLWLDOUZJEHDY-UHFFFAOYSA-N 7-hydroxy-2-oxochromene-3-carboxylic acid Chemical compound C1=C(O)C=C2OC(=O)C(C(=O)O)=CC2=C1 LKLWLDOUZJEHDY-UHFFFAOYSA-N 0.000 claims description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 5
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 claims description 4
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 claims description 4
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 claims description 4
- 229950006780 n-acetylglucosamine Drugs 0.000 claims description 4
- ZCVAGTPWBAZXAL-UHFFFAOYSA-N 4-nitro-2,1,3-benzoxadiazole Chemical compound [O-][N+](=O)C1=CC=CC2=NON=C12 ZCVAGTPWBAZXAL-UHFFFAOYSA-N 0.000 claims description 3
- XUJNEKJLAYXESH-UWTATZPHSA-N D-Cysteine Chemical compound SC[C@@H](N)C(O)=O XUJNEKJLAYXESH-UWTATZPHSA-N 0.000 claims description 3
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 claims description 3
- CKLJMWTZIZZHCS-UWTATZPHSA-N D-aspartic acid Chemical compound OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 claims description 3
- 101100502320 Arabidopsis thaliana FAD4 gene Proteins 0.000 claims description 2
- PLVOEKQNBIXHDI-SBSPUUFOSA-N (2R)-2-amino-3-[(7-hydroxy-2-oxochromene-3-carbonyl)amino]propanoic acid hydrochloride Chemical compound Cl.N[C@H](CNC(=O)c1cc2ccc(O)cc2oc1=O)C(O)=O PLVOEKQNBIXHDI-SBSPUUFOSA-N 0.000 claims 1
- SLVOVFVZZFUEAS-UHFFFAOYSA-N 2-[2-[2-[bis(carboxymethyl)amino]ethoxy]ethyl-(carboxymethyl)amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCN(CC(O)=O)CC(O)=O SLVOVFVZZFUEAS-UHFFFAOYSA-N 0.000 claims 1
- MVVPIAAVGAWJNQ-DOFZRALJSA-N Arachidonoyl dopamine Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)NCCC1=CC=C(O)C(O)=C1 MVVPIAAVGAWJNQ-DOFZRALJSA-N 0.000 claims 1
- 102100021587 Embryonic testis differentiation protein homolog A Human genes 0.000 claims 1
- 241000630665 Hada Species 0.000 claims 1
- 101000898120 Homo sapiens Embryonic testis differentiation protein homolog A Proteins 0.000 claims 1
- 229940024606 amino acid Drugs 0.000 description 49
- 235000001014 amino acid Nutrition 0.000 description 49
- 235000014469 Bacillus subtilis Nutrition 0.000 description 34
- NEPLBHLFDJOJGP-BYPYZUCNSA-N (2s)-2-(5-fluoro-2,4-dinitroanilino)propanamide Chemical compound NC(=O)[C@H](C)NC1=CC(F)=C([N+]([O-])=O)C=C1[N+]([O-])=O NEPLBHLFDJOJGP-BYPYZUCNSA-N 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 27
- 241000588724 Escherichia coli Species 0.000 description 24
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 22
- MDBGGTQNNUOQRC-UHFFFAOYSA-N Allidochlor Chemical compound ClCC(=O)N(CC=C)CC=C MDBGGTQNNUOQRC-UHFFFAOYSA-N 0.000 description 21
- 230000012010 growth Effects 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 18
- -1 alkyne azide Chemical class 0.000 description 15
- 230000007935 neutral effect Effects 0.000 description 15
- 238000003786 synthesis reaction Methods 0.000 description 15
- 125000000524 functional group Chemical group 0.000 description 14
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 150000008575 L-amino acids Chemical class 0.000 description 11
- 239000002253 acid Substances 0.000 description 11
- 239000000975 dye Substances 0.000 description 11
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 150000001345 alkine derivatives Chemical class 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 150000001540 azides Chemical class 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 241000894007 species Species 0.000 description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 7
- 238000000799 fluorescence microscopy Methods 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 6
- 244000063299 Bacillus subtilis Species 0.000 description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 6
- 150000007513 acids Chemical class 0.000 description 6
- 125000003277 amino group Chemical group 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 238000010869 super-resolution microscopy Methods 0.000 description 6
- KRCVJTNGGWBYEW-UWTATZPHSA-N (2r)-2-(2-diazohydrazinyl)propanoic acid Chemical compound OC(=O)[C@@H](C)NN=[N+]=[N-] KRCVJTNGGWBYEW-UWTATZPHSA-N 0.000 description 5
- 239000012099 Alexa Fluor family Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 241001538234 Nala Species 0.000 description 5
- 240000002390 Pandanus odoratissimus Species 0.000 description 5
- 235000005311 Pandanus odoratissimus Nutrition 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 238000004132 cross linking Methods 0.000 description 5
- 230000009977 dual effect Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- 241000010804 Caulobacter vibrioides Species 0.000 description 4
- 125000000030 D-alanine group Chemical group [H]N([H])[C@](C([H])([H])[H])(C(=O)[*])[H] 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 244000057717 Streptococcus lactis Species 0.000 description 4
- 241000531819 Streptomyces venezuelae Species 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 238000006352 cycloaddition reaction Methods 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- AWJUIBRHMBBTKR-UHFFFAOYSA-N isoquinoline Chemical compound C1=NC=CC2=CC=CC=C21 AWJUIBRHMBBTKR-UHFFFAOYSA-N 0.000 description 4
- XBGNERSKEKDZDS-UHFFFAOYSA-N n-[2-(dimethylamino)ethyl]acridine-4-carboxamide Chemical compound C1=CC=C2N=C3C(C(=O)NCCN(C)C)=CC=CC3=CC2=C1 XBGNERSKEKDZDS-UHFFFAOYSA-N 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000000717 retained effect Effects 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000004885 tandem mass spectrometry Methods 0.000 description 4
- 238000002287 time-lapse microscopy Methods 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- KVKVTWIEKGPCPZ-UHFFFAOYSA-N (2e)-1-[6-(3-azidopropylamino)-6-oxohexyl]-2-[(e)-3-[3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indol-1-ium-2-yl]prop-2-enylidene]-3,3-dimethylindole-5-sulfonate Chemical compound [N-]=[N+]=NCCCNC(=O)CCCCCN1C2=CC=C(S([O-])(=O)=O)C=C2C(C)(C)\C1=C/C=C/C1=[N+](CCCS(O)(=O)=O)C2=CC=C(S(O)(=O)=O)C=C2C1(C)C KVKVTWIEKGPCPZ-UHFFFAOYSA-N 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 241000218943 Brachybacterium conglomeratum Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- KGZHFKDNSAEOJX-WIFQYKSHSA-N Ramoplanin Chemical class C([C@H]1C(=O)N[C@H](CCCN)C(=O)N[C@H](C(=O)N[C@@H](C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C)C(=O)N[C@H](C(=O)O[C@@H]([C@@H](C(N[C@@H](C(=O)N[C@H](CCCN)C(=O)N[C@@H](C(=O)N[C@H](C(=O)N[C@@H](C(=O)N[C@H](C(=O)N1)[C@H](C)O)C=1C=CC(O)=CC=1)C=1C=CC(O)=CC=1)[C@@H](C)O)C=1C=CC(O)=CC=1)=O)NC(=O)[C@H](CC(N)=O)NC(=O)\C=C/C=C/CC(C)C)C(N)=O)C=1C=C(Cl)C(O)=CC=1)C=1C=CC(O)=CC=1)[C@@H](C)O)C=1C=CC(O[C@@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)=CC=1)C1=CC=CC=C1 KGZHFKDNSAEOJX-WIFQYKSHSA-N 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 241000207195 Verrucomicrobium spinosum Species 0.000 description 3
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 3
- 125000000304 alkynyl group Chemical group 0.000 description 3
- 229910052786 argon Inorganic materials 0.000 description 3
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 3
- 239000012267 brine Substances 0.000 description 3
- 230000032823 cell division Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
- 239000010949 copper Substances 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000019439 ethyl acetate Nutrition 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000003068 molecular probe Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 229950003551 ramoplanin Drugs 0.000 description 3
- 108010076689 ramoplanin Proteins 0.000 description 3
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000001488 sodium phosphate Substances 0.000 description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 3
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- KRJLRVZLNABMAT-RXMQYKEDSA-N (2r)-3-azaniumyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CC(C)(C)OC(=O)N[C@H](C[NH3+])C([O-])=O KRJLRVZLNABMAT-RXMQYKEDSA-N 0.000 description 2
- KRJLRVZLNABMAT-YFKPBYRVSA-N (2s)-3-amino-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](CN)C(O)=O KRJLRVZLNABMAT-YFKPBYRVSA-N 0.000 description 2
- VGIRNWJSIRVFRT-UHFFFAOYSA-N 2',7'-difluorofluorescein Chemical compound OC(=O)C1=CC=CC=C1C1=C2C=C(F)C(=O)C=C2OC2=CC(O)=C(F)C=C21 VGIRNWJSIRVFRT-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000157902 Brachybacterium Species 0.000 description 2
- 241001453380 Burkholderia Species 0.000 description 2
- 241000863012 Caulobacter Species 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 2
- 101710116957 D-alanyl-D-alanine carboxypeptidase Proteins 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 241000194036 Lactococcus Species 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- 239000007832 Na2SO4 Substances 0.000 description 2
- 241000037463 Paraburkholderia phytofirmans Species 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 150000004775 coumarins Chemical class 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 101150038944 dacA gene Proteins 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 101150014046 disA gene Proteins 0.000 description 2
- 238000000132 electrospray ionisation Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 230000007773 growth pattern Effects 0.000 description 2
- 125000005980 hexynyl group Chemical group 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000010829 isocratic elution Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 125000005981 pentynyl group Chemical group 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 230000006098 transglycosylation Effects 0.000 description 2
- 238000005918 transglycosylation reaction Methods 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- LXAXDNARFSIPLO-SCSAIBSYSA-N (2r)-2-(ethynylamino)propanoic acid Chemical compound OC(=O)[C@@H](C)NC#C LXAXDNARFSIPLO-SCSAIBSYSA-N 0.000 description 1
- IVWWFWFVSWOTLP-YVZVNANGSA-N (3'as,4r,7'as)-2,2,2',2'-tetramethylspiro[1,3-dioxolane-4,6'-4,7a-dihydro-3ah-[1,3]dioxolo[4,5-c]pyran]-7'-one Chemical compound C([C@@H]1OC(O[C@@H]1C1=O)(C)C)O[C@]21COC(C)(C)O2 IVWWFWFVSWOTLP-YVZVNANGSA-N 0.000 description 1
- 125000001399 1,2,3-triazolyl group Chemical class N1N=NC(=C1)* 0.000 description 1
- WORJRXHJTUTINR-UHFFFAOYSA-N 1,4-dioxane;hydron;chloride Chemical compound Cl.C1COCCO1 WORJRXHJTUTINR-UHFFFAOYSA-N 0.000 description 1
- LULFUERYRGYWRH-UHFFFAOYSA-N 1-(3-azidopropoxy)-7-(methylamino)phenoxazin-3-one Chemical compound [N-]=[N+]=NCCCOC1=CC(=O)C=C2OC3=CC(NC)=CC=C3N=C21 LULFUERYRGYWRH-UHFFFAOYSA-N 0.000 description 1
- IOOMXAQUNPWDLL-UHFFFAOYSA-N 2-[6-(diethylamino)-3-(diethyliminiumyl)-3h-xanthen-9-yl]-5-sulfobenzene-1-sulfonate Chemical compound C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=C(S(O)(=O)=O)C=C1S([O-])(=O)=O IOOMXAQUNPWDLL-UHFFFAOYSA-N 0.000 description 1
- BNBQQYFXBLBYJK-UHFFFAOYSA-N 2-pyridin-2-yl-1,3-oxazole Chemical class C1=COC(C=2N=CC=CC=2)=N1 BNBQQYFXBLBYJK-UHFFFAOYSA-N 0.000 description 1
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- PECYZEOJVXMISF-REOHCLBHSA-N 3-amino-L-alanine Chemical compound [NH3+]C[C@H](N)C([O-])=O PECYZEOJVXMISF-REOHCLBHSA-N 0.000 description 1
- MCSXGCZMEPXKIW-UHFFFAOYSA-N 3-hydroxy-4-[(4-methyl-2-nitrophenyl)diazenyl]-N-(3-nitrophenyl)naphthalene-2-carboxamide Chemical compound Cc1ccc(N=Nc2c(O)c(cc3ccccc23)C(=O)Nc2cccc(c2)[N+]([O-])=O)c(c1)[N+]([O-])=O MCSXGCZMEPXKIW-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- TXUWMXQFNYDOEZ-UHFFFAOYSA-N 5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylidene-4-imidazolidinone Chemical compound O=C1N(C)C(=S)NC1CC1=CNC2=CC=CC=C12 TXUWMXQFNYDOEZ-UHFFFAOYSA-N 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 244000144725 Amygdalus communis Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- JHFNSBBHKSZXKB-VKHMYHEASA-N Asp-Gly Chemical compound OC(=O)C[C@H](N)C(=O)NCC(O)=O JHFNSBBHKSZXKB-VKHMYHEASA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000219307 Atriplex rosea Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000588807 Bordetella Species 0.000 description 1
- 241000589968 Borrelia Species 0.000 description 1
- 241000589562 Brucella Species 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- VMQMZMRVKUZKQL-UHFFFAOYSA-N Cu+ Chemical compound [Cu+] VMQMZMRVKUZKQL-UHFFFAOYSA-N 0.000 description 1
- XPDXVDYUQZHFPV-UHFFFAOYSA-N Dansyl Chloride Chemical compound C1=CC=C2C(N(C)C)=CC=CC2=C1S(Cl)(=O)=O XPDXVDYUQZHFPV-UHFFFAOYSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 description 1
- 206010056740 Genital discharge Diseases 0.000 description 1
- DXJZITDUDUPINW-WHFBIAKZSA-N Gln-Asn Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(O)=O DXJZITDUDUPINW-WHFBIAKZSA-N 0.000 description 1
- XITLYYAIPBBHPX-ZKWXMUAHSA-N Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(N)=O XITLYYAIPBBHPX-ZKWXMUAHSA-N 0.000 description 1
- SIGGQAHUPUBWNF-BQBZGAKWSA-N Gln-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(N)=O SIGGQAHUPUBWNF-BQBZGAKWSA-N 0.000 description 1
- PABVKUJVLNMOJP-WHFBIAKZSA-N Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(O)=O PABVKUJVLNMOJP-WHFBIAKZSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 241000589989 Helicobacter Species 0.000 description 1
- MDCTVRUPVLZSPG-BQBZGAKWSA-N His-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CNC=N1 MDCTVRUPVLZSPG-BQBZGAKWSA-N 0.000 description 1
- 101000610640 Homo sapiens U4/U6 small nuclear ribonucleoprotein Prp3 Proteins 0.000 description 1
- WMDZARSFSMZOQO-DRZSPHRISA-N Ile-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 WMDZARSFSMZOQO-DRZSPHRISA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 239000004166 Lanolin Substances 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 241000589248 Legionella Species 0.000 description 1
- 208000007764 Legionnaires' Disease Diseases 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 208000016604 Lyme disease Diseases 0.000 description 1
- UGTZHPSKYRIGRJ-YUMQZZPRSA-N Lys-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O UGTZHPSKYRIGRJ-YUMQZZPRSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- IXQIUDNVFVTQLJ-UHFFFAOYSA-N Naphthofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C(C=CC=1C3=CC=C(O)C=1)=C3OC1=C2C=CC2=CC(O)=CC=C21 IXQIUDNVFVTQLJ-UHFFFAOYSA-N 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 102100032132 Neuroendocrine convertase 1 Human genes 0.000 description 1
- 101710151472 Neuroendocrine convertase 1 Proteins 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 108090000279 Peptidyltransferases Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241000425347 Phyla <beetle> Species 0.000 description 1
- BEPSGCXDIVACBU-IUCAKERBSA-N Pro-His Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H]1NCCC1)C1=CN=CN1 BEPSGCXDIVACBU-IUCAKERBSA-N 0.000 description 1
- 108010059712 Pronase Proteins 0.000 description 1
- 241000186429 Propionibacterium Species 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000669298 Pseudaulacaspis pentagona Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 101001110823 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-A Proteins 0.000 description 1
- 101000712176 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-B Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- RZEQTVHJZCIUBT-WDSKDSINSA-N Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N RZEQTVHJZCIUBT-WDSKDSINSA-N 0.000 description 1
- LZLREEUGSYITMX-JQWIXIFHSA-N Ser-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)N)C(O)=O)=CNC2=C1 LZLREEUGSYITMX-JQWIXIFHSA-N 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000187392 Streptomyces griseus Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000192584 Synechocystis Species 0.000 description 1
- 241000192581 Synechocystis sp. Species 0.000 description 1
- DSGIVWSDDRDJIO-ZXXMMSQZSA-N Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DSGIVWSDDRDJIO-ZXXMMSQZSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- JAQGKXUEKGKTKX-HOTGVXAUSA-N Tyr-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 JAQGKXUEKGKTKX-HOTGVXAUSA-N 0.000 description 1
- 102100040374 U4/U6 small nuclear ribonucleoprotein Prp3 Human genes 0.000 description 1
- JKHXYJKMNSSFFL-IUCAKERBSA-N Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN JKHXYJKMNSSFFL-IUCAKERBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 241001261005 Verrucomicrobia Species 0.000 description 1
- AQEMQEKCGYGLRM-UHFFFAOYSA-M [9-(3-azidopropoxy)-7-piperidin-1-ylphenoxazin-3-ylidene]-dimethylazanium;perchlorate Chemical compound [O-]Cl(=O)(=O)=O.C1=C2OC3=CC(=[N+](C)C)C=CC3=NC2=C(OCCCN=[N+]=[N-])C=C1N1CCCCC1 AQEMQEKCGYGLRM-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000010256 biochemical assay Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000007942 carboxylates Chemical group 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- CZPLANDPABRVHX-UHFFFAOYSA-N cascade blue Chemical compound C=1C2=CC=CC=C2C(NCC)=CC=1C(C=1C=CC(=CC=1)N(CC)CC)=C1C=CC(=[N+](CC)CC)C=C1 CZPLANDPABRVHX-UHFFFAOYSA-N 0.000 description 1
- PTIUZRZHZRYCJE-UHFFFAOYSA-N cascade yellow Chemical compound C1=C(S([O-])(=O)=O)C(OC)=CC=C1C1=CN=C(C=2C=C[N+](CC=3C=C(C=CC=3)C(=O)ON3C(CCC3=O)=O)=CC=2)O1 PTIUZRZHZRYCJE-UHFFFAOYSA-N 0.000 description 1
- 235000019994 cava Nutrition 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- KJOZJSGOIJQCGA-UHFFFAOYSA-N dichloromethane;2,2,2-trifluoroacetic acid Chemical compound ClCCl.OC(=O)C(F)(F)F KJOZJSGOIJQCGA-UHFFFAOYSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 101150096961 dltA gene Proteins 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002101 electrospray ionisation tandem mass spectrometry Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 238000002169 hydrotherapy Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 229940039717 lanolin Drugs 0.000 description 1
- 235000019388 lanolin Nutrition 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- ULXTYUPMJXVUHQ-OVTFQNCVSA-N lipid II Chemical compound OC(=O)[C@@H](C)NC(=O)[C@@H](C)NC(=O)[C@H](CCCCN)NC(=O)CC[C@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@@H]1[C@@H](NC(C)=O)[C@@H](OP(O)(=O)OP(O)(=O)OC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 ULXTYUPMJXVUHQ-OVTFQNCVSA-N 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 description 1
- 229940107698 malachite green Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- VWKNUUOGGLNRNZ-UHFFFAOYSA-N methylbimane Chemical compound CC1=C(C)C(=O)N2N1C(C)=C(C)C2=O VWKNUUOGGLNRNZ-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000003158 microbiostatic effect Effects 0.000 description 1
- 231100000324 minimal toxicity Toxicity 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 229940051921 muramidase Drugs 0.000 description 1
- ZFIFHAKCBWOSRN-UHFFFAOYSA-N naphthalene-1-sulfonamide Chemical compound C1=CC=C2C(S(=O)(=O)N)=CC=CC2=C1 ZFIFHAKCBWOSRN-UHFFFAOYSA-N 0.000 description 1
- 150000002790 naphthalenes Chemical class 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000001048 orange dye Substances 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 150000003220 pyrenes Chemical class 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- XLXOKMFKGASILN-UHFFFAOYSA-N rhodamine red-X Chemical compound C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=C(S(=O)(=O)NCCCCCC(O)=O)C=C1S([O-])(=O)=O XLXOKMFKGASILN-UHFFFAOYSA-N 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 238000010857 super resolution fluorescence microscopy Methods 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241001624918 unidentified bacterium Species 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000007794 visualization technique Methods 0.000 description 1
- 235000014101 wine Nutrition 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/74—Benzo[b]pyrans, hydrogenated in the carbocyclic ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
- C07K9/003—Peptides being substituted by heterocyclic radicals, e.g. bleomycin, phleomycin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Definitions
- the invention relates generally to microbiology, and more particularly to compositions and methods for assessing cell wall synthesis in bacteria, for identifying bacteria, and for screening for cell wall-acting/-disrupting agents.
- PG domain-specific peptidoglycan
- DAA D-amino acid
- antibiotic concentration needs to be carefully controlled to avoid damage to the cell.
- a modified amino acid in a first respect, includes a D-amino acid covalently attached to a fluorescent label.
- a muramylpentapeptide precursor unit that includes an N-acetyl muramic acid (NAM) moiety having a stem peptide of three to five amino acids.
- NAM N-acetyl muramic acid
- One or more of the amino acids in the stem peptide includes a modified amino acid that includes a D-amino acid covalently attached to a fluorescent label and optionally an additional modified amino acid.
- the additional modified amino acid includes a clickable D-amino acid.
- a peptidoglycan unit in a third respect, includes a muramylpentapeptide precursor unit as described above in the second respect that is covalently linked to an N-acetyl glucosamine (NAG) moiety.
- NAG N-acetyl glucosamine
- a method of assessing bacterial cell wall synthesis in real time includes the step of providing live bacteria with a first amount of at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally a second amount of at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient for bacterial cell wall synthesis.
- the bacteria covalently incorporate the at least one modified amino acid and optionally the at least one additional modified amino acid into a stem peptide of peptidoglycan of the bacterial cell wall.
- a method of screening for a putative cell wall-acting agent includes the step of co-contacting bacteria with an effective amount of an agent and an amount of at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally an amount at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient to permit ongoing peptidoglycan biosynthesis in a bacterial cell wall.
- the agent comprises a cell wall-acting agent if the agent interferes with ongoing peptidoglycan biosynthesis in the bacterial cell wall.
- a method of screening for a putative cell wall-disrupting agent includes the step contacting modified bacteria with an amount of an agent.
- the agent is a cell wall-disrupting agent if the agent weakens integrity of peptidoglycan in an existing bacterial cell wall.
- the modified bacteria have a modified cell wall containing modified peptidoglycan having at least one stem peptide containing at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally at least one additional modified amino acid comprising a clickable D-amino acid.
- a method of identifying bacteria includes two steps.
- the first step includes contacting live bacteria with an amount of at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally an amount of at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient for ongoing bacterial cell wall synthesis.
- the bacteria covalently incorporate into peptidoglycan of a bacterial cell wall the at least one modified amino acid, and optionally the at least one additional modified amino acid.
- Each of the least one modified amino acid and optionally the at least one additional modified amino acid comprises a spectrally distinct fluorescent label.
- the second step includes visualizing the spectrally distinct fluorescent labels to determine an incorporation pattern of the at least one modified amino acid, and optionally the at least one additional modified amino acid, wherein the incorporation pattern identifies the bacteria.
- kits for incorporating labeled D-amino acids into live bacteria includes at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label and a positive bacterial control.
- the kit can include an optional negative bacterial control.
- the positive bacterial control has at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label incorporated into a stem peptide of peptidoglycan of the bacterial cell wall.
- the optional negative bacterial control if included, does not have the modified amino acid comprising a D-amino acid covalently attached to a fluorescent label incorporated into a stem peptide of peptidoglycan of the bacterial cell wall.
- FIG. 1 shows the three general stages of PG biosynthesis and general structures of the NAM and NAG units of PG.
- FIG. 2 shows exemplary D-Ala-based FDAAs.
- D-NBD and D-HCC (based on (R)-diaminopropionic acid) emit in the green and blue regions, respectively.
- FIG. 3 shows results of control experiments in which the cell walls of Agrobacterium tumefaciens (“ A. tumefaciens ” top row), Bacillus subtilis (“ B. subtilis ” middle row) and Escherichia coli (“ E. coli ” bottom row) were fluorescently labeled with fluorescent D-Ala (D-HCC) or fluorescent L-Ala (L-HCC).
- D-HCC fluorescent D-Ala
- L-HCC fluorescent L-Ala
- FIG. 4 shows results of a pulse chase experiment with a fluorescent D-Ala in B. subtilis (top row) or A. tumefaciens (bottom)).
- FIG. 5 shows results of a short pulse experiment with fluorescent D-Ala in B. subtilis
- FIG. 6 shows results of a fluorescent D-Ala derivative in a dual-labeling format.
- FIG. 7 shows exemplary structures for FDAAs, such as HCC-OH-labeled 3-amino-D-Ala (HADA), NBD-Cl-labeled 3-amino-D-Ala (NADA), F-labeled D-Lys (FDL) and T-labeled D-Lys (TDL), as well as exemplary structures for CDAAs, such as EDA and ADA.
- FDAAs such as HCC-OH-labeled 3-amino-D-Ala (HADA), NBD-Cl-labeled 3-amino-D-Ala (NADA), F-labeled D-Lys (FDL) and T-labeled D-Lys (TDL), as well as exemplary structures for CDAAs, such as EDA and ADA.
- FIG. 8 shows that long labeling pulses with HADA uniformly label PG in live Escherichia coli (left), Bacillus subtilis (center) and Agrobacterium tumefaciens (left).
- the FDAA fluorescence was retained in isolated sacculi, which also stained with a NAG-specific wheat germ agglutinin (WAG) lectin conjugated to Alexa Fluor® 594 (red). Scale bars, 2 ⁇ m.
- FIGS. 9A-D show FDAA incorporation into the stem peptide of the PG unit.
- FIG. 9A shows a schematic representing the muramylpentapeptide precursor as incorporated into a nascent PG unit and a modified D-amino acid (FDAA).
- FIG. 9B shows HPLC detection of modified muropeptides in E. coli incubated with HADA, HALA and NADA, or NALA.
- Samples were monitored using a dual wavelength UV monitor set fix general muropeptide detection and for FDAA-specific wavelengths.
- Peaks HEC-1 (panel (i)) and NEC-1 (panel (ii)) correspond to HADA- or NADA-modified muropeptides in E. coli that were further characterized by electrospray ionization MS/MS (ESI-MS/MS).
- FIG. 9C shows percentage of FDAA incorporation into the total muropeptides varies among bacteria as revealed by HPLC analysis.
- FIG. 9D shows a schematic representing MS/MS analyses of FDAAs exclusively incorporated into the 5th position in B. subtilis (panel (i)) and the 4th position of muropeptides in E. coli and A. tumefaciens (panel (ii)).
- FIGS. 10A-F show FDAAs label diverse bacterial growth patterns. Arrows in the triple labeling panels indicate the sequence of labeling. White scale bars 2 ⁇ m, red scale bars, 1 ⁇ m.
- FIG. 10A shows time-lapse microscopy of HADA-labeled E. coli and B. subtilis ⁇ dacA cells imaged during growth on LB agarose pads.
- FIG. 10B shows super-resolution microscopy of E. coli after short pulses with HADA (panels (i) and (ii)).
- FIG. 10C show super-resolution microscopy of A. tumefaciens after short pulses with HADA.
- FIG. 10D shows super-resolution microscopy of S. aureus after a short pulse with HADA. Autofluorescence is shown in red.
- FIG. 10E shows triple labeling of A. tumefaciens with HADA (blue), EDA (clicked with red sulfo-Cy3-azide) and NADA (green).
- FIG. 10F shows triple labeling of S. venezuelae with NADA (green), TDL (red) and HADA (blue).
- FIG. 11 shows that short pulses of HADA label distinct modes of growth in diverse bacteria.
- Strains were labeled for ⁇ 2%-8% of the doubling time: E. coli (30 seconds), A. tumefaciens minutes), B. subtilis ⁇ dacA (30 seconds), S. aureus (2 minutes), L. lactis (2 minutes), S. pneumoniae (4 minutes), C. crescentus (5 minutes), Synechocysfis sp. PCC 6803 (1 hour), S. venezuelae (2 minutes), B. conglomeratum (8 minutes), B. phytofirmans (20 minutes), V. Spinosum (10 minutes). Scale bars, 2 ⁇ m.
- FIG. 12 shows a schematic for sequentially incorporating distinct FDAAs, such as NADA, TDL and HADA, into newly synthesized PG in live bacteria.
- distinct FDAAs such as NADA, TDL and HADA
- indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element.
- the indefinite article “a” or “an” thus usually means “at least one.”
- “about” means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.
- compositions and methods described herein take advantage of mechanisms for incorporating labeled DAAs into the stem peptides displayed on a bacterial cell wall surface.
- the work described herein demonstrates how to make derivatized DAA having a suitable label, such as an appropriate fluorophore and how such derivatized compounds can be visualized in live cells by fluorescence microscopy following the incorporation of the derivatized compounds into PG and thus the cell wall.
- the incorporated FDAAs do not appear to be toxic to bacteria.
- the methods described herein do not appear to adversely affect cell morphology.
- the methods described herein enable pulse-chase experiments that cannot be easily executed in the presence of fluorescently-modified cell wall active drugs. Because the disclosed derivatized compounds have low or minimal toxicity to live cells, they are ideal markers to evaluate and screen microbiostatic or microbiotoxic compounds that do adversely affect microorganism growth and viability, such as studies directed to development of novel antibiotics.
- compositions and methods are applicable to a wide array of Gram-positive and Gram-negative bacteria and provides significant utility for probing PG biosynthesis, cell wall morphogenesis and the response of the PG biosynthetic machinery to cell wall-active agents and/or cell wall-disrupting agents.
- present disclosure therefore provides compositions and methods for studying bacterial cell wall PG biosynthesis and for discovering bacterial cell wall-acting and/or cell wall-disrupting agents.
- compositions of the invention include labeled D-amino acids (DAAs), especially fluorescent D-amino acids (FDAAs).
- DAAs labeled D-amino acids
- FDAAs fluorescent D-amino acids
- “ammo acid” or “amino acid residue” are used interchangeably to mean a molecule containing a first, or alpha, carbon attached to an amine group, a carboxylic acid group and a side-chain that is specific to each amino acid.
- a natural amino acid can include conventional elements such as carbon, hydrogen, oxygen, nitrogen and sulfur.
- An amino acid may be a naturally occurring amino acid or artificially-created unnaturally occurring amino acid.
- the amino acid is naturally occurring, and, unless otherwise limited, may encompass known analogues/synthetics of natural amino acids that can function in a similar manner as naturally occurring amino acids.
- the natural amino acids all contain at least one chiral carbon atom. These amino acids therefore exist as pairs of stereoisomers (D- and L-isomers).
- D-isomers or D-amino acids particularly D-Ala, D-Asp, D-Cys, D-Glu and D-Lys, which are frequently found in the stem peptide of the PG unit.
- the following six groups each contain amino acids that are typical but not necessarily exclusive conservative substitutions for one another: 1) Alanine (A), Serine (S) arid Threonine (T); 2) Aspartic acid (D) and Glutamic acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); and 6) Phenylalanine (F), Tyrosine (Y) and Tryptophan (W).
- suitable labels for the DAAs include, but are not limited to, radiolabels, biotin (which may be detected by avidin or streptavidin conjugated to peroxidase), lanthanides, alkaline phosphatase and fluorescent labels (e,g, coumarins, fluoresceins, cyanines, bodipy dyes, green fluorescent protein, quantum dots rhodamine, especially the Alexa Fluor® family of fluorescent dyes available from Invitrogen/Molecular Probes).
- fluorescent labels e,g, coumarins, fluoresceins, cyanines, bodipy dyes, green fluorescent protein, quantum dots rhodamine, especially the Alexa Fluor® family of fluorescent dyes available from Invitrogen/Molecular Probes.
- Other labels amenable for use in the modified D-amino acids disclosed herein include metals and isotopic labels.
- Labeling of DAAs can be carried out by covalently attaching the label to a free amine group, such as free amine groups present on the side-chain that is specific to each amino acid, If the side chain lacks a free amine group, one of skill in the art understands how to add such groups, as is the case of adding such a group to D-Ala to obtain 3-amino-D-Ala.
- Some labels can be detected by using a labeled counter suitable for the detection of the label in question.
- HCC-OH 7-hydroxycoumarin 3-carboxylic acid
- NBD 7-nitrobenzofurazan
- NBD-Cl 4-chloro-7-nitrobenzofurazan
- fluorescein F
- T carboxytetramethylrhodamine
- amino acids having functional groups other than an amine include a functional alcohol group (e.g., serine and tyrosine), thiol group (e.g, cysteine), or carbonyl or carboxylate group (e.g., aspartate and glutamate).
- a functional alcohol group e.g., serine and tyrosine
- thiol group e.g, cysteine
- carbonyl or carboxylate group e.g., aspartate and glutamate.
- Such functional groups can be derivatized or reacted with suitably modified, activated coupling agents having labels of the types disclosed herein.
- FDAA fluorescent-labeled amino acid
- HADA HCC-OH-labeled 3-amino-D-Ala
- NADA NBD-Cl-labeled 3-amino-D-Ala
- FDL F-labeled D-Lys
- TDL T-labeled D-Lys
- HDL HCC-OH-labeled D-Lys
- NDL NBD-Cl-labeled D-Lys
- FADA F-labeled 3-amino-D-Ala.
- TADA which is a T-labeled 3-amino-D-Ala.
- FDAAs can include a D-Glu having its side chain modified to include a free amine group linked to any of the fluorescent labels above (e.g., HADD, NADG, FADG and TADG).
- Compositions of the invention also include clickable D-amino acids (CDAAs).
- the CDAAs have a DAA backbone that includes, for example, an alkyne or azide functional group present on the side-chain that is specific to each amino acid that can be captured in situ by a labeled, detecting agent carrying, a conjugate functional group via click-chemistry.
- Functional groups in a DAA backbone that can be targeted by the labeled, detecting agent include, but are not limited to, primary amines, carboxyls, sulfhydryls, carbohydrates and carboxylic acids.
- Cross-linking and enrichment strategies for separating a cross-linking reaction from enrichment steps have been developed based on bioorthogonal chemistries including the azide-alkyne “click” cycloaddition and Staudinger ligation using alkyne- or azide-labeled cross-linking agents (e.g., fluorescent labels).
- alkyne- or azide-labeled cross-linking agents e.g., fluorescent labels.
- Azides and alkynes are not naturally found in proteins, peptides, nucleic acids or glycans; therefore, these moieties can be engineered onto the DAAs and labeled, detecting agent to generate azide-containing molecules and alkyne-containing molecules that are reactive with one another.
- click chemistry and “clickable” therefore mean a reaction between azide-containing molecules and alkyne-containing molecules to yield a covalent product-1,5-disubstituted 1,2,3-triazole.
- the reaction can be a copper(I)-catalyzed alkyne azide cycloaddition (CuAAC) or, in cases where copper toxicity may be an issue, can be a copper(I)-free-catalyzed alkyne azide cycloaddition.
- CuAAC copper(I)-catalyzed alkyne azide cycloaddition
- Examples of functional groups for use on azide-containing molecules and alkyne-containing molecules include, but are not limited to, hexynyl groups, pentynyl groups, heptynyl groups, azido-propyl groups, azido-butyl groups and azido-pentyl groups.
- the functional group of alkyne-containing molecules e.g., DAAs
- the functional group of azide-containing molecules e.g., labeled, detecting agents
- the functional group of azide-containing molecules e.g., DAAs
- the functional group of alkyne-containing molecules e.g., labeled, detecting agents
- the functional group of alkyne-containing molecules has the corresponding clickable alkynyl group.
- various labeling designs for detecting agents are known including, but not limited to, biotinylated agents, isotope-coded agents, fluorophore-labeled agents, mass-tag-labeled agents and chromophore-labeled agents.
- the addition of functional groups can cause the cross-linker to become very bulky or less cell-permeable, and thus not very effective for in vivo and/or in situ cross-linking.
- separation of the cross-linking step from conjugation of affinity tags can be one effective strategy. See, Trester-Zedlitz et al. (2003) J. Am. Chem. Soc. 125:2416-2425; Tang et al.
- the detecting agents can be fluorophore-labeled.
- fluorophores include, but are not limited to, Alexa Fluor® dyes, BODIPY® dyes, fluorescein, Oregon Green® 488 and Oregon Green® 514 dyes, Rhodamine Green and Rhodamine Green-X dyes, eosin, tetramethylrhodamine, Lissamine Rhodamine B and Rhodamine Red-X dyes, X-Rhodamine, Texas Red® and Texas Red®-X dyes, naphthofluorescein, Carboxyrbodamine 6G, QSY dyes:fluorescence quenchers, nonfluorescent malachite green, coumarin derivatives, Pacific Orange dye, cascade blue and other pyrene derivatives, cascade yellow and other pyridyloxazole derivatives, naphthalenes (e.g., dansyl chlor
- An example of a CDAA includes EDA.
- Another example of a CDAA includes ADA. See, e.g., FIG. 7 .
- FMPUs Fluorescent Muramylpentapeptide Precursor Units
- Compositions of the invention also include fluorescent muramylpentapeptide precursor units (FMPUs) having an NAM moiety with a peptide chain of three to five amino acids in which one or more of the amino acids in the stem peptide are FDAAs and/or CDAAs as described herein. See, e.g., FIG. 9A and 9D .
- FMPUs fluorescent muramylpentapeptide precursor units
- Compositions of the invention also include fluorescent peptidoglycan units (FPGUs).
- FPGUs have a FMPU as described herein linked to a NAG moiety. See, e.g., FIG. 9A and 9D .
- Compositions of the invention also include live bacteria having FDAAs, CDAAs, FMPUs and/or FPGUs as described herein incorporated into PG in a cell wall.
- Gram-positive bacteria tend to have a thicker PG layer
- the bacteria can be Gram-positive bacteria or Gram-negative bacteria.
- suitable Gram-positive bacteria include, but are not limited to, Actinomyces spp., Bacillus spp., Brachybacterium spp., Clostridium spp., Corynebacterium spp. Diplococcus spp., Enterococcus spp., Lactococcus spp., Listeria spp. Nocardia spp., Propionibacterium spp., Staphylococcus spp., Streptococcus spp. Streptomyces spp.
- live B. subtitis, B. conglomeratum, L. lactis, S. aureus, S. pneumoniae, S. venezuelae were grown in the presence of FDAAs and/or CDAAs.
- suitable Gram-negative bacteria include, but are not limited to, Acinetobacter spp., Agrobacterium spp., Bordetella spp., Borrelia spp., Brucella spp., Burkholderia spp., Campylobacter spp., Caulobacter spp., Chlamydia spp., Enterobacter spp., Escherichia spp., Helicobacter spp., Herophilus spp., Klebsiella spp., Legionella spp., Neisseria spp., Proteus spp., Pseudomonas spp, Salmonella spp., Shigella spp., Synechocystis spp., Verrucomicrobia spp., Fibrio spp.
- live A. tuniefriciens, B. phytofirmans, C. crescentus, E. coli, Synechocystis sp. PCC 6803 and V. spinosum were grown in the presence of FDAAs and/or CDAAs.
- compositions of the invention also include kits having one or more FDAA, CDAA, FMPU and/or FPGLI as described herein and optionally one or more labeled detecting agents (if CDAAs are included in the kits) for use in in situ labeling/probing of PG during biosynthesis, as well as for screening for bacterial cell wall-acting and/or cell wall-disrupting agents.
- the kits also can include additional reagents such as unlabeled DAAs, unlabeled L-amino acids (LAAs) and/or labeled LAAs.
- the kits also can include positive and/or negative bacterial controls, where the controls have unlabeled DAAs, CDAAs and LAAs or labeled DAAs and LAAs incorporated into PG in a cell wall.
- kit means any manufacture (e.g., a package or a container) having, for example, at least one FDAA and/or CDAA and a positive and/or negative control.
- the kit may be promoted, distributed, or sold as a unit for performing any of the methods described herein.
- kits preferably include instructions, procedures and/or directions that guide users or ones skilled in the art how to use the agents, reagents, and/or other components for their intended purpose.
- kits can include a package insert describing procedures for carrying out any one of the methods described herein or analytical information for correlating the level of expression measured in live bacteria.
- the package insert can include representative images of positive or negative samples with low or high levels of incorporation as compared to an appropriate control. The kits can be promoted, distributed or sold as units for performing the methods described below.
- kits also can include a receptacle or other means for holding a sample to be evaluated for FDAA and/or CDAA incorporation, and means for determining the presence and/or quantity of FDAA and/or CDAA incorporation in live bacteria.
- the kits also can include at least one buffer.
- buffers include, but are not limited to, cell isolation buffers, fixation buffers, lysis buffers, permeabilization buffers, sonication buffers, separation buffers, stabilization buffers and ash buffers.
- buffers include strong acids in combination with weak bases, strong bases in combination with weak acids, a combination of weak acids and bases, or even a small or low concentration (e.g., within the range from about 0.1 MM to about 10 MM) of an acid or base, in the absence of a conventional conjugate base or acid, respectively; typically, however another component of the mixture may provide such conjugate acid or base function.
- acids and bases both in terms of ionization/dissociation strength (i.e., strong or weak) and type (i.e., inorganic or organic), are well known in the art.
- kit components can be provided within containers that protect them from the external envirorrrrrent, such as in sealed containers.
- Methods include assessing bacterial cell wall biosynthesis (and PG recycling) in real time.
- bacterial cell wall biosynthesis typically involves three steps: translocation, transglycosylation and transpeptidation.
- carbohydrate backbone is formed by polymerization via glycosidic bond formation between the C(4)-hydroxyl of a membrane-bound lipid II intermediate and the anomeric center of a membrane-bound glycan strand.
- Bacterial transpeptidases mediate crosslinking of the resulting elongated glycan strand.
- the cross-link is installed via attack of an amino group, either from the Lys residue itself or from a short peptide chain appended to the Lys residue, onto the penultimate D-Ala residue of an adjacent pentapeptide strand and results in cleavage of the terminal D-Ala residue.
- This rigid macromolecular structure essential to both Gram-negative and Gram-positive bacteria, enables bacterial cells to resist lysis and, subsequently, cell death resulting from high internal osmotic pressure.
- the methods typically begin by providing live Gram-positive or Gram-negative bacteria with FDAAs and/or CDAAs as described herein under conditions where the bacteria can covalently incorporate the FDAAs and/or CDAAs into PG of a bacterial cell wall.
- the FDAAs and/or CDAAs can be provided to organisms preferably within a given range of concentrations, for example, from about 0.1 ⁇ M to about 1 mM, as well as in any whole integer or fractional integer concentration thereof within this preferred range.
- the FDAAs and/or CDAAs can also be provided to organisms at preferred concentrations, for example, at about 0.1 ⁇ M and about 1 mM.
- Determination of the optimal concentration (or amount) of FDAAs and/or CDAAs and the preferred ranges thereof for a particular organism is the subject of routine experimentation well within the purview of those skilled in the art.
- a typical route to ascertaining the optimal concentrations and preferred ranges of the FDAAs and/or CDAAs described herein is to perform a dose response experiment, wherein the parallel populations of a given organism are contacted with different concentrations (or amounts) of a given FDAA and/or CDAA, and the extent of incorporation of the compound(s) is assessed by biochemical assay (e.g., extent of compound labeling in PG fractions) and/or by visualization methods (e.g., fluorescence microscopy).
- biochemical assay e.g., extent of compound labeling in PG fractions
- visualization methods e.g., fluorescence microscopy
- the methods also can include detecting the FDAAs and/or CDAAs in the bacterial cell wall to verify that they have been incorporated.
- the FDAAs and/or CDAAs (after being clicked) can be detected via fluorescence microscopy and other methods, depending upon the type of label or reporter used.
- cell wall biosynthetic pathway is unique to bacterial cells; therefore, agents that inhibit steps within this pathway are anticipated to show selective toxicity toward bacterial cells.
- methods of the invention also can include screening for putative cell wall-acting or cell wall-disrupting agents.
- cell wall-acting means an ability of an agent to interfere with PG biosynthesis in a bacterial cell wall, especially at the transglycosylati on step, as this step takes place on the outer leaflet of the cell membrane so cellular penetration is not a prerequisite for the agent to manifest its biological activity.
- cell wall-disrupting means an ability of an agent to disrupt or weaken the integrity of PG in an existing bacterial cell wall.
- the methods can begin by contacting bacteria with a putative cell wall-acting agent or putative cell wall-disrupting agent, where the agent is cell wall-acting if the agent interferes with ongoing peptidoglycan biosynthesis in a bacterial cell wall or is cell wall-disrupting if the agent weakens integrity of peptidoglycan in an existing bacterial cell wall.
- the bacteria can be co-contacted with FDAAs and/or CDAAs as described herein simultaneously with the putative agent.
- the bacteria can have FDAAs and/or CDAAs as described herein covalently incorporated into PG of the cell wall prior to being contacted with the putative agent.
- the methods also can include detecting whether the FDAAs and/or CDAAs have been incorporated in the bacterial cell wail or whether the FDAAs and/or CDAAs remain in the bacterial cell wall.
- the FDAAs and/or CDAAs (after being clicked) can be detected via fluorescence microscopy and other methods, depending upon the type of label or reporter used.
- the pattern and/or location of FDAAs and/or CDAAs incorporation can be used to identify the bacteria (see, e.g., FIGS. 10-12 ).
- the methods also can include comparing the esults from the putative cell wall-acting agent or cell wall-disrupting agent with a known cell wall-acting agent or known cell wall-disrupting agent.
- the compounds of the present disclosure have utility for identifying bacteria. As demonstrated in the Examples set forth herein, certain bacterial species display unique specificity for incorporating certain D-amino acids in PG and the bacteria cell wall. Thus, the use of the disclosed modified D-amino acids of the present disclosure enable identification of bacterial species by virtue of the pattern of labeling observed in the bacteria as a result of incorporation of the modified D-amino acids into PG of the bacterial cell wall.
- a series of pulse-chase experiments were performed in which exponentially growing cells were diluted and treated with one of the D-Ala probes (250 ⁇ M-500 ⁇ M), The cells were incubated until saturation, washed, and placed on LB-containing agar pads and imaged. at 5-minute intervals for 12-18 hours ( FIG. 4 ).
- experiments were performed with the labeled D-Ala derivative in a dual-labeling format.
- A. tumefaciens cells were incubated for a period of 4 minutes in 500 ⁇ D-NBD, followed by washing and incubation for 4 minutes in 500 ⁇ M D-HCC. The excess dye was removed and the cells were pelleted and fixed.
- the fluorescence micrographs reveal distinct patterns of growth, in terms of polar growth and septal synthesis, based on the age of the daughter cell ( FIG. 6 ).
- HADA/HALA 7-hydroxycoumarin-3-carboxylic acid (HCC) was added in anhydrous DMF (14.5 mL, 0.1 M) under an atmosphere of argon. Carbonyidiimidazole (236 mg, 1.455 mmol) was added in one portion and stirred at room temperature (RT) for 2 hours.
- HCC 7-hydroxycoumarin-3-carboxylic acid
- Boc-D-2,3-diaminopropionic acid for HADA
- Boc-L-2,3-diaminopropionic acid for HALA
- 297 mg, 1.455 mmol was added in one portion and the reaction mixture was allowed to stir at RT overnight (17 hours).
- the majority of the solvent was removed in vacuo, and the product was diluted with EtOAc (100 ml) and washed with 1 N HCl (50 ml) and water (100 ml). The water layers were combined and back-extracted with EtOAc (50 ml) to prevent loss of product due to an emulsion.
- NADA/NALA Boc-D-2,3-diaminopropionic acid (for NADA) or Boc-L-2,3-diaminopropionic acid (for NALA) (100 mg, 0.49 mmol) and sodium bicarbonate (123 mg, 1.47 mmol) were dissolved in water (1.8 ml) and heated to 55° C. in water bath. A solution of 4-chloro-7-nitrobenzofurazan (NBD, 108 mg, 0.539 mmol.) in methanol (8.5 ml) was added dropwise over 10 minutes. Care was taken at all times to avoid excessive exposure to light during the reaction and workup. The reaction was allowed to stir at 55° C. for 1 hour.
- the solvent was removed in vacuo and acidified with 1 N HCl.
- the aqueous mixture was extracted with dichloromethane (50 ml per extraction ⁇ 3 extractions) and the organic extracts were washed with brine (50 ml), dried over Na 2 SO 4 , filtered, and the solvent was removed in vacuo.
- the crude product was treated with 4 N HCl/dioxane (10 ml) for 1 hour at RT, and the solvent was removed in vacuo.
- the product was purified via reverse-phase HPLC with 20%-90% MeCN/H 2 O.
- TDL To a flame dried flask was added N ⁇ -Boc-D-Lys-OH (3.3 mg, 0.0134 mmol), 5-(and 6-) carboxytetrarnethylrhodamine succinimidyl ester (5 mg, 0.0095 mmol), and diisopropylethylamine (2.5 ⁇ l, 0.0143 mmol) in dry DMF (0.2 ml). The reaction was stirred under argon at room temperature overnight. The solvent was removed in vacuo, and the crude mixture was treated with trifluoroacetic acid dichloromethane (1:1) for 0.5 hours. The reaction was dried in vacuo, and purified by reverse-phase HPLC with 20%-40% MeCN/H 2 O.
- Excitation and emission spectra of FDAAs 500 ⁇ M in 100 mM Tris pH 7.0 were deters fined in black 96-well polystyrene plates (Corning) using top-read function of a Spectra Max M2 plate reader. The excitation and emission spectra were rneasured in separate runs within a range of 200 nm and with increments of 1 nm.
- EDA and ELA, and Sulfo-Cy3-Azide were gifts from Boaopharma and Lumiprobe, respectively
- AZA and “clickable” Alexa 488 Fluors were purchased from Iris Biotech GmbH and Invitrogen, respectively.
- the Cu(I) catalyzed click chemistry was performed using the chemicals supplied by Invitrogen following their standard protocol once the cells had been fixed with EtOH (70% v/v) and permeabilized with methanol (100% v/v).
- DMSO was added to the growth media to a final concentration of 1% to help solubilize the FDAAs and/or CDAAs. Presence of 1% DMSO did not affect labeling or growth in bacteria tested. When necessary, chloramphenicol or spectinomycin was added to the growth media at 5 ⁇ g/ml or 100 ⁇ g/ml, respectively. Strains were maintained on plates containing growth media with 1.5% agar.
- exponentially growing cells were screened for the minimum concentration of FDAAs or CDAAs (250 ⁇ M-1 mM) and minimum amount of exposure duration to identify the optimal conditions for each bacterium, as shown in FIG. 11 and Table 2.
- OD 600 ⁇ 0.3 exponentially growing cells (OD 600 ⁇ 0.3) were labeled, fixed, washed, “clicked” if appropriate, and imaged.
- Phase and fluorescence microscopy was performed with a Nikon® 90i Fluorescence Microscope equipped with a Plan Apo 100 ⁇ /1.40 Oil Ph3 DM Objective and a Chroma 83700 triple filter cube with corresponding excitation and emission filters (DAPI for HADA/HALA; FITC for NADA/NALA; and Alexa Fluor® 488s and Texas Red® for Sulfo-Cy3 or WGA-594). All images were captured using NIS software from Nikon® and a Photometrics Cascade 1K cooled charge-coupled device camera, and were processed and analyzed using ImageJ. When a comparison was made, cultures were treated in exactly the same manner and the same parameters were applied for collecting and post processing of the microscopy data.
- Exponentially growing cells were diluted to OD 600 0.05 in media containing half of the optimal FDAA concentration used for short labeling pulses and were grown until late exponential phase.
- the cells were fixed, washed and then imaged using the Nikon® 90i as described previously.
- the cells were washed with media arid mounted onto LB+1% (w/v) agarose pads on 25-mm by 75-mm glass slides, sealed with 1:1:1 mixture of vasoline, lanolin and paraffin and imaged with intervals of 4 minutes ( B. subtilis ⁇ dacA ), 5 minutes ( E. coli ) or 10 minutes ( A.
- Structured illumination microscopy was performed using a DeltaVision® OMX Imaging System equipped with an Olympus® UPlanSApo 100 ⁇ /1.40 Oil PSF Objective and a Photometrics Cascade II EMCCD Camera.
- the samples were excited with a laser at 405 nm and the emission was detected through a 419-465 emission filter.
- HADA 500 ⁇ M+1% DMSO
- NADA 500 ⁇ M+1% DMSO
- E.coli cells were labeled with HADA in 0.1% DMSO. Cells were subsequently stained using the LIVE-DEAD BacLight Kit (Invitrogen) according to the manufacturer's standard protocol.
- Sacculi from cells were purified as described in Litzinger et al. (2010) J. Bacteriol. 192:3122-3143 with following modifications. Exponentially growing cells were diluted to OD 600 0.05 in 10 ml LB containing half of the optimum FDAA concentration+1% (v/v) DMSO and grown to late exponential phase. After aliquots were taken for whole cell imaging, cells were collected by centrifugation at 25,000 ⁇ g for 15 minutes at RT and resuspended in 0.8 nil water. The suspension was added to boiling sodium dodecyl sulfate (SDS, 5% w/v) drop-wise and incubated with stirring for 30 minutes.
- SDS sodium dodecyl sulfate
- SDS insoluble material was collected by ultracentrifugation at 39,000 ⁇ g for 10 minutes at 30° C. and was resuspended in 1 ml water and boiled again in SDS (4% w/v)with stirring for 30 minutes. Samples were then washed four times in 1.5 ml water and resuspended in I ml 10 mM Tris-HCl pH 7.0+10 mM NaCl+0.32 M imtnidazole+ ⁇ -amylase (100 ⁇ m/ml)+DNase I (50 ⁇ g/ml)+MgSO 4 (1 mM) and incubated for 2 hours at 37° C.
- PG from FDAA labeled cells was purified by the boiling SDS extraction method and muramidase digestion treatment (Cellosyl) as previously described in Brown et al. (2012) Proc. Natl. Acad. Sci. USA 109:1697-1701. Solubilized muropeptide mixtures were then either directly injected into the HPLC system (native or non-reduced samples) or subjected to BH 4 Na reduction as described in Brown et al. (2012), supra.
- Muropeptides were analyzed using a binary-pump Waters® HPLC System (Waters Corporation) fitted with a reverse phase RP18 Aeris® Peptide Column (250 mm ⁇ 4.6 mm; 3.6 ⁇ m particle size) (Phenomenex) and a dual wavelength absorbance detector. Elution conditions were: flow rate 1 ml/min; temperature 35° C.; 3 minutes isocratic elution in 50 mM sodium phosphate, pH 4.35 followed by a 57 minute linear gradient to 75 mM, sodium phosphate, pH 4.95 in 15% (v/v) methanol (90 mM sodium phosphate, pH 5.2 in 30%(v/v) methanol for B.
- FIG. 8 Retained fluorescence on the purified sacculi ( FIG. 8 ) demonstrated that the labeling of PG by the FDAAs was covalent.
- HPLC analyses of muropeptides isolated from labeled cells revealed that 0.2%-2.8% of total muropeptides were modified ( FIG. 9C ), which is sufficient for detection in various experiments while avoiding possible toxicity issues that could result from abundant incorporation.
- the FDAA-specific peaks which were absent in samples treated with FLAAs, could be distinguished from unlabeled muropeptides at FDAA-specific absorption wavelengths ( FIG. 9B ).
- subtilis indicated that FDAAs were exclusively incorporated in the fifth position of the stem peptide ( FIG. 9D ).
- the fraction of labeled muropeptides and the fluorescent signal were substantially higher than in wild-type B. subtilis, which is likely due to the D,D-carboxypeptidase activity of DacA.
- the detectable incorporation was solely at the fourth position in E. coli and A. tumefaciens ( FIG. 9D ).
- FDAAs incorporate mainly through periplasmic exchange reactions with the muropeptides catalyzed either by D,D-transpeptidases (e.g., in B. subtilis ) or by L,D-transpeptidases (e.g., in E. coli and A. tumefaciens ).
- D,D-transpeptidases e.g., in B. subtilis
- L,D-transpeptidases e.g., in E. coli and A. tumefaciens.
- FIG. 11 Labeling of S. venezuelae was predominantly apical, with some weak labeling of vegetative septa and lateral walls suggestive of a low but continuous lateral PG synthesis ( FIG. 11 ). In C. crescentus, labeling occurred at the sites of septal elongation, lateral elongation, and stalk synthesis ( FIG. 11 ). B. phylofirmans exhibited polar and mid-cell PG synthesis, B. conglomeratum exhibited prominent peripheral PG synthesis in addition to seemingly alternating perpendicular division planes, and V. spinosum exhibited strong peripheral PG synthesis and asymmetric septal labeling ( FIG. 11 ).
- FDAAs therefore DAAs
- incorporation is common to the bacterial domain and that FDAAs can thus be used to analyze natural bacterial populations, providing a convenient and quick standard to measure bacterial activity and to probe the diversity of growth modes in complex inicrobiomes.
- labeling times with FDAAs as short as 2 hours revealed diverse modes of growth in saliva and freshwater samples in situ, but did not label dead cells as suggested by the strong correlation with Live-Dead staining.
- CDAAs namely ethynyl-D-alanine (FDA) or azido-D-alanine (ADA) ( FIG. 7 ), that can be specifically captured by any molecule carrying the conjugate functional group via click-chemistry also were used. Similar to FDAAs, these bioorthogonal DAAs, but not the L-enantiomer control ELA, labeled both E. coli and B. subtilis when captured by commercially available azidolalkyne fluorophores.
- FDA ethynyl-D-alanine
- ADA azido-D-alanine
- custom DAAs containing different colored fluorophores can be used sequentially to enable “virtual time-lapse microscopy.” Since addition of each new probe indicates the location and extent of PG synthetic activity during the respective labeling periods, this approach provides a chronological account of shifts in PG synthesis of individual cells over time. Examples of such serial labeling, including a combination with click chemistry, were performed in Gram-negative A. tumefaciens ( FIG. 10E ) and in Gram-positive S. venezuetae ( FIG. 10F ).
- compositions for and methods of covalently labeling PG in live bacterial cells This method works very efficiently in both Gram-positive and Gram-negative organisms (Gram-negative organisms represent a liability for approaches using fluorescently modified vancomycin/ramoplanin), and the probe substrates do not appear to be toxic to cells and show no adverse effects on cell morphology, even at concentrations as high as 1 mM.
- the probes rapidly label sites of active peptidoglycan biosynthesis and can be used in time-lapse and dual labeling experiments.
- compositions and methods have been described herein for in situ labeling/probing of PG synthesis in bacteria with fluorescent D-amino acids (FDAAs) as well as for screening bacterial cell wall-acting and/or cell wall-disrupting agents.
- FDAAs fluorescent D-amino acids
- the FDAAs are based upon D-amino acids (DAAs) derivatized to covalently include a small fluorophore.
- DAAs D-amino acids
- the FDAAs can be directly incorporated into bacterial cell walls during PG biosynthesis, as occurs at sites of cell division in actively dividing cells.
- the compositions include FDAAs.
- the FDAAs have a DAA covalently attached to a fluorophore such as 7-hydroxycoumarin 3-carboxylic acid (HCC-OH), 7-nitrobenzofurazan (NBD), 4-chloro-7-nitrobenzofurazan (NBD-Cl), fluorescein (F) or carboxytetramethylrhodamine (T).
- the DAA can be any of the twenty known, standard amino acids, such as D-Ala, D-Asp, D-Cys, D-Glu or D-Lys.
- compositions also include clickable DAAs (CDAAs).
- CDAA have a DAA backbone including an alkyne or azide functional group that can be captured by any labeled detecting agent carrying a conjugate functional group via click-chemistry, where the label can be a fluorescent molecule.
- compositions also include fluorescent muramylpentapeptide precursor units (FMPUs).
- FMPUs have an N-acetyl muramic acid (NAM) moiety with a stem peptide of three to five amino acids in which one or more of the amino acids in the stem peptides are FDAAs and/or CDAAs as described hererin.
- NAM N-acetyl muramic acid
- compositions also include fluorescent PG units (FPGUs).
- FPGUs have a FMPU as described herein linked to an N-acetyl glucosamine (NAG) moiety.
- NAG N-acetyl glucosamine
- compositions also include live bacteria having one or more FDAA, CDAA, FMPU and/or FPGU as described herein incorporated into PG in a cell wall.
- compositions also include kits having one or more FDAA, CDAA, FMPU and/or FPGU as described herein and optionally one or more labeled, detecting agents for use in in situ labeling/probing of PG synthesis, as well as for screening for bacterial cell wall-acting and/or cell wall-disrupting agents.
- kits also can include additional reagents such as unlabeled DAAs unlabeled L-amino acids (LAAs), fluorescent LAAs (FLAAs) and/or clickable LAAs (CLAAs).
- kits also can include positive and/or negative bacterial controls, where the bacterial controls have unlabeled DAAs, CDAAs, LAAs and CLAAs and/or labeled DAAs, CDAAs, LAAs and CLAAs incorporated into PG in a cell wall.
- the methods have been disclosed herein that include assessing bacterial cell wall synthesis in real time by providing live bacteria with one or more FDAA, CDAA, FMPU and/or FPGU as described herein, where the bacteria covalently incorporate the one or more FDAA, CDAA, FMPU and/or FPGU into PG of a bacterial cell wall.
- the one or more FDAA, CDAA, FMPU and/or FPGU can be provided to live bacteria together or sequentially during cell wall synthesis.
- the methods also include screening for putative cell wall-acting or cell wall-disrupting agents by contacting bacteria with a putative cell wall-acting agent or putative cell wall-disrupting agent, where the agent is cell wall-acting if the agent interferes with ongoing PG biosynthesis in a bacterial cell wall or is cell wall-disrupting if the agent weakens integrity of PG in an existing bacterial cell wall.
- the bacteria can be provided with one or more FDAA, CDAA, FMPU and/or FPGU as described herein simultaneously with the putative agent.
- the bacteria can have one or more FDAA, CDAA, FMPU and/or FPGU as described herein covalently incorporated into PG of the cell wall prior to being contacted with the putative agent.
- the methods also include identifying a bacteria by providing live, unknown bacteria with one or more FDAA, CDAA, FMPU and/or FPGU as described herein under conditions sufficient for bacterial cell wall synthesis, where the bacteria covalently incorporate one or more FDAA, CDAA, FMPU and/or FPGU into a cell wall, and where each of the one or more FDAA CDAA, FMPU and/or FPGU includes a distinct fluorophore.
- the methods also include observing an incorporation pattern of the one or more FDAA, CDAA FMPU and/or FPGU, where the incorporation pattern identifies the bacteria. Such methods are amenable for use in screening platforms to identify novel compounds having bacteriostatic or bacteriotoxic properties.
- the bacteria can be Gram-positive or Gram-negative bacteria.
- the methods also include detecting one or more FDAA, CDAA, FMPU and/or FPGU as described herein that have been incorporated in the bacterial cell wall or that have been disrupted from the bacterial cell wall by, for example, fluorescence microscopy and other methods, depending upon the label used.
- the methods also can include comparing the results of the putative agent with a known cell wall-acting agent or with a known cell wall-disrupting agent.
- compositions and methods described herein therefore find use in the study of bacterial cell wall biosynthesis and in the discovery of bacterial cell wall-acting and/or cell wall-disrupting agents.
- the FDAAs, CDAAs, FMPUs and/or FPGUs as described herein simultaneously are non-toxic and can be tunable to label sites of active PG biosynthesis, enabling fine spatiotemporal tracking of cell wall dynamics in phylogenetically and morphologically diverse bacteria.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
- The present application is a continuation of U.S. patent application Ser. No. 14/395,815, filed Oct. 20, 2014, which is the National Stage of International Application No. PCT/US13/37504, tiled Apr. 21, 2013, and entitled “COMPOSITIONS FOR IN SITU LABELING OF BACTERIAL CELL WALLS WITH FLUOROPHORES AND METHODS OF USE THEREOF,” which claims benefit of priority under 35 U.S.C. 119 to U.S. provisional patent application Ser. No. 61/636,640, filed Apr. 21, 2012, and entitled “COMPOSITIONS FOR COVALENTLY LABELING BACTERIAL CELL WALLS WITH FLUOROPHORES AND METHODS OF USE,” and U.S. provisional patent application Ser. No. 61/718,048, tiled Oct. 24, 2012, and entitled “COMPOSITIONS FOR IN SITU LABELING OF BACTERIAL CELL WALLS WITH FLUOROPHORES AND METHODS OF USE THEREOF,” the contents of both which are herein incorporated by reference in their entireties.
- This invention was made with government support under A1059327 and GM051986 awarded by the National Institutes of Health. The government has certain rights in the invention.
- The invention relates generally to microbiology, and more particularly to compositions and methods for assessing cell wall synthesis in bacteria, for identifying bacteria, and for screening for cell wall-acting/-disrupting agents.
- Bacterial growth is controlled by the domain-specific peptidoglycan (PG) cell wall, a rigid and essential structure composed of glycan strands cross-linked by D-amino acid (DAA)-containing short peptides, whose biosynthesis machinery is a target for antibiotics.
- Despite the importance of PG, knowledge of its dynamics has been severely hampered by lack of a strategy for direct imaging of sites of PG biosynthesis in live cells. Significant limitations of current labeling methods, such as toxic effects and poor membrane permeability of the probes, have limited their applicability to only a small set of bacterial species. Moreover, these methods are labor-intensive and their sensitivity suffers from their indirect and multiple-step nature.
- Methods relying on fluorescently labeled antibiotics to study bacterial cell wall synthesis and to discover new antibiotics to which bacteria remain susceptible have had a profound impact on the field. The current methods, however, have at least two inherent limitations. First, antibiotic concentration needs to be carefully controlled to avoid damage to the cell. Second, because these agents bind to specific sites on cell surfaces, they only will appear at sites of active PG biosynthesis.
- For the foregoing reasons, there is a need for additional compositions and methods for assessing PG biosynthesis in bacteria and for discovering bacterial cell wall-acting/-disrupting agents that can be used to treat infections caused by multidrug-resistant bacteria.
- In a first respect, a modified amino acid is disclosed that includes a D-amino acid covalently attached to a fluorescent label.
- In a second respect, a muramylpentapeptide precursor unit is disclosed that includes an N-acetyl muramic acid (NAM) moiety having a stem peptide of three to five amino acids. One or more of the amino acids in the stem peptide includes a modified amino acid that includes a D-amino acid covalently attached to a fluorescent label and optionally an additional modified amino acid. The additional modified amino acid includes a clickable D-amino acid.
- In a third respect, a peptidoglycan unit is disclosed that includes a muramylpentapeptide precursor unit as described above in the second respect that is covalently linked to an N-acetyl glucosamine (NAG) moiety.
- In a fourth respect, a method of assessing bacterial cell wall synthesis in real time is described. The method includes the step of providing live bacteria with a first amount of at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally a second amount of at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient for bacterial cell wall synthesis. The bacteria covalently incorporate the at least one modified amino acid and optionally the at least one additional modified amino acid into a stem peptide of peptidoglycan of the bacterial cell wall.
- In a fifth respect, a method of screening for a putative cell wall-acting agent is disclosed. The method includes the step of co-contacting bacteria with an effective amount of an agent and an amount of at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally an amount at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient to permit ongoing peptidoglycan biosynthesis in a bacterial cell wall. The agent comprises a cell wall-acting agent if the agent interferes with ongoing peptidoglycan biosynthesis in the bacterial cell wall.
- In a sixth respect, a method of screening for a putative cell wall-disrupting agent is disclosed. The method includes the step contacting modified bacteria with an amount of an agent. The agent is a cell wall-disrupting agent if the agent weakens integrity of peptidoglycan in an existing bacterial cell wall. In this method, the modified bacteria have a modified cell wall containing modified peptidoglycan having at least one stem peptide containing at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally at least one additional modified amino acid comprising a clickable D-amino acid.
- In a seventh respect, a method of identifying bacteria is disclosed. The method includes two steps. The first step includes contacting live bacteria with an amount of at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally an amount of at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient for ongoing bacterial cell wall synthesis. The bacteria covalently incorporate into peptidoglycan of a bacterial cell wall the at least one modified amino acid, and optionally the at least one additional modified amino acid. Each of the least one modified amino acid and optionally the at least one additional modified amino acid comprises a spectrally distinct fluorescent label. The second step includes visualizing the spectrally distinct fluorescent labels to determine an incorporation pattern of the at least one modified amino acid, and optionally the at least one additional modified amino acid, wherein the incorporation pattern identifies the bacteria.
- In an eighth respect, a kit for incorporating labeled D-amino acids into live bacteria is disclosed. The kit includes at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label and a positive bacterial control. The kit can include an optional negative bacterial control. The positive bacterial control has at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label incorporated into a stem peptide of peptidoglycan of the bacterial cell wall. The optional negative bacterial control, if included, does not have the modified amino acid comprising a D-amino acid covalently attached to a fluorescent label incorporated into a stem peptide of peptidoglycan of the bacterial cell wall.
- These and other features, objects and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention.
- The features, objects and advantages other than those set forth above will become more readily apparent when consideration is given to the detailed description below. Such detailed description makes reference to the following drawings.
-
FIG. 1 shows the three general stages of PG biosynthesis and general structures of the NAM and NAG units of PG. -
FIG. 2 shows exemplary D-Ala-based FDAAs. D-NBD and D-HCC (based on (R)-diaminopropionic acid) emit in the green and blue regions, respectively. -
FIG. 3 shows results of control experiments in which the cell walls of Agrobacterium tumefaciens (“A. tumefaciens” top row), Bacillus subtilis (“B. subtilis” middle row) and Escherichia coli (“E. coli” bottom row) were fluorescently labeled with fluorescent D-Ala (D-HCC) or fluorescent L-Ala (L-HCC). An exemplary structure for D-Ala (D-HCC) also is shown in the bottom row. -
FIG. 4 shows results of a pulse chase experiment with a fluorescent D-Ala in B. subtilis (top row) or A. tumefaciens (bottom)). -
FIG. 5 shows results of a short pulse experiment with fluorescent D-Ala in B. subtilis -
FIG. 6 shows results of a fluorescent D-Ala derivative in a dual-labeling format. -
FIG. 7 shows exemplary structures for FDAAs, such as HCC-OH-labeled 3-amino-D-Ala (HADA), NBD-Cl-labeled 3-amino-D-Ala (NADA), F-labeled D-Lys (FDL) and T-labeled D-Lys (TDL), as well as exemplary structures for CDAAs, such as EDA and ADA. -
FIG. 8 shows that long labeling pulses with HADA uniformly label PG in live Escherichia coli (left), Bacillus subtilis (center) and Agrobacterium tumefaciens (left). The FDAA fluorescence was retained in isolated sacculi, which also stained with a NAG-specific wheat germ agglutinin (WAG) lectin conjugated to Alexa Fluor® 594 (red). Scale bars, 2 μm. -
FIGS. 9A-D show FDAA incorporation into the stem peptide of the PG unit. -
FIG. 9A shows a schematic representing the muramylpentapeptide precursor as incorporated into a nascent PG unit and a modified D-amino acid (FDAA). -
FIG. 9B shows HPLC detection of modified muropeptides in E. coli incubated with HADA, HALA and NADA, or NALA. Samples were monitored using a dual wavelength UV monitor set fix general muropeptide detection and for FDAA-specific wavelengths. Peaks HEC-1 (panel (i)) and NEC-1 (panel (ii)) correspond to HADA- or NADA-modified muropeptides in E. coli that were further characterized by electrospray ionization MS/MS (ESI-MS/MS). -
FIG. 9C shows percentage of FDAA incorporation into the total muropeptides varies among bacteria as revealed by HPLC analysis. -
FIG. 9D shows a schematic representing MS/MS analyses of FDAAs exclusively incorporated into the 5th position in B. subtilis (panel (i)) and the 4th position of muropeptides in E. coli and A. tumefaciens (panel (ii)). -
FIGS. 10A-F show FDAAs label diverse bacterial growth patterns. Arrows in the triple labeling panels indicate the sequence of labeling.White scale bars 2 μm, red scale bars, 1 μm. -
FIG. 10A shows time-lapse microscopy of HADA-labeled E. coli and B. subtilis ΔdacA cells imaged during growth on LB agarose pads. -
FIG. 10B shows super-resolution microscopy of E. coli after short pulses with HADA (panels (i) and (ii)). -
FIG. 10C show super-resolution microscopy of A. tumefaciens after short pulses with HADA. -
FIG. 10D shows super-resolution microscopy of S. aureus after a short pulse with HADA. Autofluorescence is shown in red. -
FIG. 10E shows triple labeling of A. tumefaciens with HADA (blue), EDA (clicked with red sulfo-Cy3-azide) and NADA (green). -
FIG. 10F shows triple labeling of S. venezuelae with NADA (green), TDL (red) and HADA (blue). -
FIG. 11 shows that short pulses of HADA label distinct modes of growth in diverse bacteria. Strains were labeled for ˜2%-8% of the doubling time: E. coli (30 seconds), A. tumefaciens minutes), B. subtilis ΔdacA (30 seconds), S. aureus (2 minutes), L. lactis (2 minutes), S. pneumoniae (4 minutes), C. crescentus (5 minutes), Synechocysfis sp. PCC 6803 (1 hour), S. venezuelae (2 minutes), B. conglomeratum (8 minutes), B. phytofirmans (20 minutes), V. Spinosum (10 minutes). Scale bars, 2 μm. -
FIG. 12 shows a schematic for sequentially incorporating distinct FDAAs, such as NADA, TDL and HADA, into newly synthesized PG in live bacteria. - While the present invention is amenable to various modifications and alternative forms, exemplary embodiments thereof are shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description of exemplary embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the embodiments above and the claims below. Reference should therefore be made to the embodiments and claims herein for interpreting the scope of the invention.
- The compositions and methods now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all permutations and variations of embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided in sufficient written detail to describe and enable one skilled in the art to make and use the invention, along with disclosure of the best mode for practicing the invention, as defined by the claims and equivalents thereof.
- Likewise, many modifications and other embodiments of the compositions and methods described herein will come to mind to one of skill in the art to which the invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
- Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
- Moreover, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one.”
- As used herein, “about” means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.
- Previous efforts to label PG in live bacteria principally have relied upon cell wall-active antibiotics (e.g., vancomycin, ramoplanin) modified with fluorophores or cell wall precursors/substrates covalently modified with fluorescent reporter groups. The compositions and methods described herein, however, take advantage of mechanisms for incorporating labeled DAAs into the stem peptides displayed on a bacterial cell wall surface.
- The work described herein demonstrates how to make derivatized DAA having a suitable label, such as an appropriate fluorophore and how such derivatized compounds can be visualized in live cells by fluorescence microscopy following the incorporation of the derivatized compounds into PG and thus the cell wall. In the range of physiologically relevant concentrations, the incorporated FDAAs do not appear to be toxic to bacteria. Unlike previous methods that employ covalently modified cell wall precursors, the methods described herein do not appear to adversely affect cell morphology. In addition, the methods described herein enable pulse-chase experiments that cannot be easily executed in the presence of fluorescently-modified cell wall active drugs. Because the disclosed derivatized compounds have low or minimal toxicity to live cells, they are ideal markers to evaluate and screen microbiostatic or microbiotoxic compounds that do adversely affect microorganism growth and viability, such as studies directed to development of novel antibiotics.
- Studies disclosed herein demonstrate that the compositions and methods are applicable to a wide array of Gram-positive and Gram-negative bacteria and provides significant utility for probing PG biosynthesis, cell wall morphogenesis and the response of the PG biosynthetic machinery to cell wall-active agents and/or cell wall-disrupting agents. The present disclosure therefore provides compositions and methods for studying bacterial cell wall PG biosynthesis and for discovering bacterial cell wall-acting and/or cell wall-disrupting agents.
- Fluorescent D-Amino Acids (FDAAs)
- Compositions of the invention include labeled D-amino acids (DAAs), especially fluorescent D-amino acids (FDAAs). As used herein, “ammo acid” or “amino acid residue” are used interchangeably to mean a molecule containing a first, or alpha, carbon attached to an amine group, a carboxylic acid group and a side-chain that is specific to each amino acid. A natural amino acid can include conventional elements such as carbon, hydrogen, oxygen, nitrogen and sulfur. An amino acid may be a naturally occurring amino acid or artificially-created unnaturally occurring amino acid. Preferably, the amino acid is naturally occurring, and, unless otherwise limited, may encompass known analogues/synthetics of natural amino acids that can function in a similar manner as naturally occurring amino acids. With the exception of glycine, the natural amino acids all contain at least one chiral carbon atom. These amino acids therefore exist as pairs of stereoisomers (D- and L-isomers). Of particular interest herein are D-isomers or D-amino acids, particularly D-Ala, D-Asp, D-Cys, D-Glu and D-Lys, which are frequently found in the stem peptide of the PG unit.
- It is well known in the art that amino acids within the same conservative group typically can substitute for one another with substantially affecting the function of a protein. For the purpose of the present disclosure, such conservative groups are set forth in Table 1 and are based preferably on shared properties, as readily appreciated to those skilled in the art. See also, Alberts et al., “Small molecules, energy, and biosynthesis” 56-57 In: Molecular Biology of the Cell (Garland Publishing Inc. 3rded. 1994).
-
TABLE 1 Amino Acids and Their Conservative Substitutions. Side Preferred Chain Side Chain Hydropathy Conservative Residue Polarity pH Index Substitutions Ala (A) Non-polar Neutral 1.8 Ser Arg (R) Polar Basic (strongly) −4.5 Lys, Gln Asn (N) Polar Neutral −3.5 Gln, His Asp (D) Polar Acidic −3.5 Glu Cys (C) Non-polar Neutral 2.5 Ser Gin (Q) Polar Neutral −3.5 Asn, Lys Glu (E) Polar Acidic −3.5 Asp Gly (G) Non-polar Neutral −0.4 Pro His (H) Polar Basic (weakly) −3.2 Asn, Gln Ile (I) Non-polar Neutral 4.5 Leu, Val Leu (L) Non-polar Neutral 3.8 Ile, Val Lys (K) Polar Basic −3.9 Arg, Gln Met (M) Non-polar Neutral 1.9 Leu, Ile Phe (F) Non-polar Neutral 2.8 Met, Leu, Tvr Pro (P) Non-polar Neutral −1.6 Gly Ser (S) Polar Neutral −0.8 Thr Thr (T) Polar Neutral −0.7 Ser Trp (W) Non-polar Neutral −0.9 Tyr Tyr (Y) Polar Neutral −1.3 Trp, Phe Val (V) Non-polar Neutral 4.2 Ile, Leu - The following six groups each contain amino acids that are typical but not necessarily exclusive conservative substitutions for one another: 1) Alanine (A), Serine (S) arid Threonine (T); 2) Aspartic acid (D) and Glutamic acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); and 6) Phenylalanine (F), Tyrosine (Y) and Tryptophan (W).
- Examples of suitable labels for the DAAs include, but are not limited to, radiolabels, biotin (which may be detected by avidin or streptavidin conjugated to peroxidase), lanthanides, alkaline phosphatase and fluorescent labels (e,g, coumarins, fluoresceins, cyanines, bodipy dyes, green fluorescent protein, quantum dots rhodamine, especially the Alexa Fluor® family of fluorescent dyes available from Invitrogen/Molecular Probes). Other labels amenable for use in the modified D-amino acids disclosed herein include metals and isotopic labels.
- Labeling of DAAs can be carried out by covalently attaching the label to a free amine group, such as free amine groups present on the side-chain that is specific to each amino acid, If the side chain lacks a free amine group, one of skill in the art understands how to add such groups, as is the case of adding such a group to D-Ala to obtain 3-amino-D-Ala. Some labels can be detected by using a labeled counter suitable for the detection of the label in question. In the Examples below, 7-hydroxycoumarin 3-carboxylic acid (HCC-OH), 7-nitrobenzofurazan (NBD), 4-chloro-7-nitrobenzofurazan (NBD-Cl), fluorescein (F) and carboxytetramethylrhodamine (T) were covalently attached to DAAs as labels.
- Other coupling chemistries are known in the art that can be used for introducing labels into amino acids having functional groups other than an amine. Such amino acids include a functional alcohol group (e.g., serine and tyrosine), thiol group (e.g, cysteine), or carbonyl or carboxylate group (e.g., aspartate and glutamate). Such functional groups can be derivatized or reacted with suitably modified, activated coupling agents having labels of the types disclosed herein.
- An example of a FDAA includes HADA, which is a HCC-OH-labeled 3-amino-D-Ala. Another example of a FDAA includes NADA, which is a NBD-Cl-labeled 3-amino-D-Ala. Another example includes FDL, which is a F-labeled D-Lys. Another example is TDL, which is a T-labeled D-Lys. Another example includes HDL, which is a HCC-OH-labeled D-Lys. Another example includes NDL, which is a NBD-Cl-labeled D-Lys. Another example includes FADA, which is a F-labeled 3-amino-D-Ala. Another example includes TADA, which is a T-labeled 3-amino-D-Ala. Other FDAAs can include a D-Glu having its side chain modified to include a free amine group linked to any of the fluorescent labels above (e.g., HADD, NADG, FADG and TADG).
- See, e.g.,
FIG. 7 , for other examples of preferred labels and modified FDAAs. - Methods of fluorescently labeling and detecting amino acids are well known in the art. See, Braun & Dittrich (2010) Beilstein J. Org. Chem. 6:69; Katritzky & Narindoshvili (2009) Org. Biomol, Chem. 7:627-634; Merkel et al. (2010) Chembiochem. 11:305-314; Cava et al. (2011) Cell Mol. Life Sci. 68:817-831; Lam et al. (2009) Science 325:1552-1555; Cava et at. (201 1) EMBO J. 30:3442-3453; and Lupoli et al. (2011) J. Am. Chem. Soc. 133:10748-10751.
- Clickable D-Amino Acids (CDAA's)
- Compositions of the invention also include clickable D-amino acids (CDAAs). The CDAAs have a DAA backbone that includes, for example, an alkyne or azide functional group present on the side-chain that is specific to each amino acid that can be captured in situ by a labeled, detecting agent carrying, a conjugate functional group via click-chemistry. Functional groups in a DAA backbone that can be targeted by the labeled, detecting agent include, but are not limited to, primary amines, carboxyls, sulfhydryls, carbohydrates and carboxylic acids.
- One of skill in the art is familiar with “click” chemistry, which utilizes chemical cross-linking agents to add functional groups to molecules. See, Kolb et al. (2001) Angew. Chem. Int. Ed. 40:2004-2021; and Evans (2007) Aust. J. Chem. 60:384-395.
- Cross-linking and enrichment strategies for separating a cross-linking reaction from enrichment steps have been developed based on bioorthogonal chemistries including the azide-alkyne “click” cycloaddition and Staudinger ligation using alkyne- or azide-labeled cross-linking agents (e.g., fluorescent labels). Azides and alkynes are not naturally found in proteins, peptides, nucleic acids or glycans; therefore, these moieties can be engineered onto the DAAs and labeled, detecting agent to generate azide-containing molecules and alkyne-containing molecules that are reactive with one another. As such, the orthogonality of azides and alkynes to biological processes (e.g:, competing reactions) is a significant advantage of these methods. Moreover, “click” cycloadditions can be performed under aqueous conditions, allowing enrichment by conjugation of an appropriate affinity or labeling tag. See, generally, Rostovtsev et al. (2002) Angew. Chem. Int Ed. 41:2596-2599; Tornoe et al. (2002) J. Org. Chem. 67:3057-3064; Baskin et al. (2007) Proc. Natl. Acad. Sci. USA 104:16793-16797; Saxon et al. (2000) Science 287:2007-2010; Chowdhury et al. (2009) Anal. Chem. 81:5524-5532; Trnka & Burlingame (2010) Mol. Cell. Proteomics 9:2306-2317; Nessen et al. (2009) J. Proteome Res. 8:3702-3711; Vellucci et al. (2010) J. Am. Soc. Mass Spectrom. 21:1432-1445; and Jewett & Bertozzi (2010) Chem. Soc. Rev. 39:1272-1279. See also, Int'l Patent Application Publication No. WO 2012/006603. As used herein, “click chemistry” and “clickable” therefore mean a reaction between azide-containing molecules and alkyne-containing molecules to yield a covalent product-1,5-disubstituted 1,2,3-triazole. The reaction can be a copper(I)-catalyzed alkyne azide cycloaddition (CuAAC) or, in cases where copper toxicity may be an issue, can be a copper(I)-free-catalyzed alkyne azide cycloaddition.
- Examples of functional groups for use on azide-containing molecules and alkyne-containing molecules include, but are not limited to, hexynyl groups, pentynyl groups, heptynyl groups, azido-propyl groups, azido-butyl groups and azido-pentyl groups. When the functional group of alkyne-containing molecules (e.g., DAAs) is an alkynyl group (e.g., hexynyl, pentynyl or heptynyl), the functional group of azide-containing molecules (e.g., labeled, detecting agents) has the corresponding clickable zido group. Likewise, when the functional group of azide-containing molecules (e.g., DAAs) is an azide group (e.g., azido-propyl, azido-butyl, or azido-pentyl), the functional group of alkyne-containing molecules (e.g., labeled, detecting agents) has the corresponding clickable alkynyl group.
- As such, various labeling designs for detecting agents are known including, but not limited to, biotinylated agents, isotope-coded agents, fluorophore-labeled agents, mass-tag-labeled agents and chromophore-labeled agents. It is also known that the addition of functional groups can cause the cross-linker to become very bulky or less cell-permeable, and thus not very effective for in vivo and/or in situ cross-linking. To reduce the total size of the cross-linker, separation of the cross-linking step from conjugation of affinity tags can be one effective strategy. See, Trester-Zedlitz et al. (2003) J. Am. Chem. Soc. 125:2416-2425; Tang et al. (2005) Anal. Chem. 77:311; Kang et al. (2009) Rapid Commun. Mass Spectrum. 23:1719-1726; Chu et al. (2006) J. Am. Chem. Soc. 128:10362-10636; Muller et al. (2001) Anal. Chem. 73:1927-1934; Collins et al, (2003) Bioorg. Med. Chem. Lett. 13:4023-4026; Petrotchenko et al. (2005) Mol. Cell. Proteomics 4:1167-1179; Wine et a/. (2002) Anal. Chem. 74:1939-1945; Sinz et al. (2001) Biochemistry 40:7903-7913; and Sinz & Wang (2004) Anal. Biochem. 331:27-32.
- In some instances, the detecting agents, whether having an alkynyl or azido functional group, can be fluorophore-labeled. Examples of fluorophores include, but are not limited to, Alexa Fluor® dyes, BODIPY® dyes, fluorescein, Oregon Green® 488 and Oregon Green® 514 dyes, Rhodamine Green and Rhodamine Green-X dyes, eosin, tetramethylrhodamine, Lissamine Rhodamine B and Rhodamine Red-X dyes, X-Rhodamine, Texas Red® and Texas Red®-X dyes, naphthofluorescein, Carboxyrbodamine 6G, QSY dyes:fluorescence quenchers, nonfluorescent malachite green, coumarin derivatives, Pacific Orange dye, cascade blue and other pyrene derivatives, cascade yellow and other pyridyloxazole derivatives, naphthalenes (e.g., dansyl chloride), dapoxyl dye, bimane, 1-dimethylamine-N(2-azido-ethyl) naphthalene-5-sulfonamide, 6-(6-amino-2-(2-azidoethyl)1,3-dioxo-1H-benzo(de)-2(3H)isoquinoline, 6-(6-amino-2-(2-propinyl)1,3-dioxo-1H-benzo(de)-2(3H)isoquinoline, 8-(4-azidoethyloxyphenyl)-2,6-diethyl-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, 8-(4-propynyloxyphenyl)-2,6-diethyl-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, 1-(3-azido-propoxy)-7-methylamino-phenoxazin-3-one, 1-(2-propynyI)-7-methylamino-phenoxazin-3-one, N-(5-(3-azidopropylamino)-9H-berizo(a)-phenoxazin-9-ylidene)-N-methyl-methanarninium chloride, N-(5-(3-propynyl-amin))-9H-benzo(a)-phenoxazin-9-ylidene)-N-methyl-methanaminium chloride, (9-(3-azido-propoxy)-7-piperidin-1-yl-phenoxazin-3-ylidene)-dimethyl-ammonium perchlorate. See also, Kele et al. (2009) Org. Biomol. Chem. 7:3486-3490; Nagy et al. (2010) Chem. Asian J. 5:773-777; Filnov et al. (2011) Nat. Biotechnol. 29:757-761; Subach et al. (2011) Nature Methods 8:771-777; Yang et al. (2011) J. Am. Chem. Soc. 133:9964-9967; Zin (2011) Nature Methods 8:726-728; and “Fluorophores and their amine- reactive derivatives,”
Chapter 1 and “Click Chemistry and other functional group modifications,”Chapter 3 in The Molecular Probes® Handbook (available on the World Wide Web at invitrogen.com/site/us/en/home/References/Molecular-Probes-The- Handbook.html). A variety of clickable fluorophores are commercially available from, for example Sigma Aldrich, Active Motif Chromeon and Invitrogen/Molecular Probes. In the examples below, CDAAs were clicked with red sulfo-Cy3-azide. - An example of a CDAA includes EDA. Another example of a CDAA includes ADA. See, e.g.,
FIG. 7 . - Fluorescent Muramylpentapeptide Precursor Units (FMPUs)
- Compositions of the invention also include fluorescent muramylpentapeptide precursor units (FMPUs) having an NAM moiety with a peptide chain of three to five amino acids in which one or more of the amino acids in the stem peptide are FDAAs and/or CDAAs as described herein. See, e.g.,
FIG. 9A and 9D . - Fluorescent Peptidoglycan Units (FPGUs)
- Compositions of the invention also include fluorescent peptidoglycan units (FPGUs). The FPGUs have a FMPU as described herein linked to a NAG moiety. See, e.g.,
FIG. 9A and 9D . - Bacterial Cells Having Fluorescent D-Amino Acids
- Compositions of the invention also include live bacteria having FDAAs, CDAAs, FMPUs and/or FPGUs as described herein incorporated into PG in a cell wall.
- While Gram-positive bacteria tend to have a thicker PG layer, it is intended that the bacteria can be Gram-positive bacteria or Gram-negative bacteria. Examples of suitable Gram-positive bacteria include, but are not limited to, Actinomyces spp., Bacillus spp., Brachybacterium spp., Clostridium spp., Corynebacterium spp. Diplococcus spp., Enterococcus spp., Lactococcus spp., Listeria spp. Nocardia spp., Propionibacterium spp., Staphylococcus spp., Streptococcus spp. Streptomyces spp. In the examples below, live B. subtitis, B. conglomeratum, L. lactis, S. aureus, S. pneumoniae, S. venezuelae, were grown in the presence of FDAAs and/or CDAAs.
- Examples of suitable Gram-negative bacteria include, but are not limited to, Acinetobacter spp., Agrobacterium spp., Bordetella spp., Borrelia spp., Brucella spp., Burkholderia spp., Campylobacter spp., Caulobacter spp., Chlamydia spp., Enterobacter spp., Escherichia spp., Helicobacter spp., Herophilus spp., Klebsiella spp., Legionella spp., Neisseria spp., Proteus spp., Pseudomonas spp, Salmonella spp., Shigella spp., Synechocystis spp., Verrucomicrobia spp., Fibrio spp. and Yersina spp. In the examples below, live A. tuniefriciens, B. phytofirmans, C. crescentus, E. coli, Synechocystis sp.
PCC 6803 and V. spinosum were grown in the presence of FDAAs and/or CDAAs. - Compositions of the invention also include kits having one or more FDAA, CDAA, FMPU and/or FPGLI as described herein and optionally one or more labeled detecting agents (if CDAAs are included in the kits) for use in in situ labeling/probing of PG during biosynthesis, as well as for screening for bacterial cell wall-acting and/or cell wall-disrupting agents. The kits also can include additional reagents such as unlabeled DAAs, unlabeled L-amino acids (LAAs) and/or labeled LAAs. The kits also can include positive and/or negative bacterial controls, where the controls have unlabeled DAAs, CDAAs and LAAs or labeled DAAs and LAAs incorporated into PG in a cell wall.
- As used herein, “kit” means any manufacture (e.g., a package or a container) having, for example, at least one FDAA and/or CDAA and a positive and/or negative control. The kit may be promoted, distributed, or sold as a unit for performing any of the methods described herein.
- Though not necessarily required, kits preferably include instructions, procedures and/or directions that guide users or ones skilled in the art how to use the agents, reagents, and/or other components for their intended purpose. For example, kits can include a package insert describing procedures for carrying out any one of the methods described herein or analytical information for correlating the level of expression measured in live bacteria. Likewise, the package insert can include representative images of positive or negative samples with low or high levels of incorporation as compared to an appropriate control. The kits can be promoted, distributed or sold as units for performing the methods described below.
- The kits also can include a receptacle or other means for holding a sample to be evaluated for FDAA and/or CDAA incorporation, and means for determining the presence and/or quantity of FDAA and/or CDAA incorporation in live bacteria.
- The kits also can include at least one buffer. Examples of buffers include, but are not limited to, cell isolation buffers, fixation buffers, lysis buffers, permeabilization buffers, sonication buffers, separation buffers, stabilization buffers and ash buffers. Though not limited, buffers include strong acids in combination with weak bases, strong bases in combination with weak acids, a combination of weak acids and bases, or even a small or low concentration (e.g., within the range from about 0.1 MM to about 10 MM) of an acid or base, in the absence of a conventional conjugate base or acid, respectively; typically, however another component of the mixture may provide such conjugate acid or base function. Examples of acids and bases, both in terms of ionization/dissociation strength (i.e., strong or weak) and type (i.e., inorganic or organic), are well known in the art.
- Any or all of the kit components can be provided within containers that protect them from the external envirorrrrrent, such as in sealed containers.
- Methods include assessing bacterial cell wall biosynthesis (and PG recycling) in real time. As shown in
FIG. 1 , bacterial cell wall biosynthesis typically involves three steps: translocation, transglycosylation and transpeptidation. In the translocation and transglycosylation steps, carbohydrate backbone is formed by polymerization via glycosidic bond formation between the C(4)-hydroxyl of a membrane-bound lipid II intermediate and the anomeric center of a membrane-bound glycan strand. Bacterial transpeptidases mediate crosslinking of the resulting elongated glycan strand. The cross-link is installed via attack of an amino group, either from the Lys residue itself or from a short peptide chain appended to the Lys residue, onto the penultimate D-Ala residue of an adjacent pentapeptide strand and results in cleavage of the terminal D-Ala residue. This rigid macromolecular structure, essential to both Gram-negative and Gram-positive bacteria, enables bacterial cells to resist lysis and, subsequently, cell death resulting from high internal osmotic pressure. - These methods typically begin by providing live Gram-positive or Gram-negative bacteria with FDAAs and/or CDAAs as described herein under conditions where the bacteria can covalently incorporate the FDAAs and/or CDAAs into PG of a bacterial cell wall. The FDAAs and/or CDAAs can be provided to organisms preferably within a given range of concentrations, for example, from about 0.1 μM to about 1 mM, as well as in any whole integer or fractional integer concentration thereof within this preferred range. The FDAAs and/or CDAAs can also be provided to organisms at preferred concentrations, for example, at about 0.1 μM and about 1 mM. Other ranges are also possible besides this preferred range and fall within the scope of this disclosure, the specific identification of which depends upon the particular biological oraganism or system under study, as well as upon the nature of the FDAAs and/or CDAAs used, their physiochemical properties and uptake by the particular biological organism or system under study, as well as the experimental set-up and purpose of the study at hand, as one of skill in the art would understand.
- Determination of the optimal concentration (or amount) of FDAAs and/or CDAAs and the preferred ranges thereof for a particular organism is the subject of routine experimentation well within the purview of those skilled in the art. A typical route to ascertaining the optimal concentrations and preferred ranges of the FDAAs and/or CDAAs described herein is to perform a dose response experiment, wherein the parallel populations of a given organism are contacted with different concentrations (or amounts) of a given FDAA and/or CDAA, and the extent of incorporation of the compound(s) is assessed by biochemical assay (e.g., extent of compound labeling in PG fractions) and/or by visualization methods (e.g., fluorescence microscopy). Other approaches to selecting the optimal concentration (of amount) of FDAAs and/or CDAAs and the preferred ranges thereof for a particular organism are viable as well, as one skilled in the art would readily appreciate based upon this disclosure.
- The methods also can include detecting the FDAAs and/or CDAAs in the bacterial cell wall to verify that they have been incorporated. The FDAAs and/or CDAAs (after being clicked) can be detected via fluorescence microscopy and other methods, depending upon the type of label or reporter used.
- The cell wall biosynthetic pathway is unique to bacterial cells; therefore, agents that inhibit steps within this pathway are anticipated to show selective toxicity toward bacterial cells. As such, methods of the invention also can include screening for putative cell wall-acting or cell wall-disrupting agents. As used herein, “cell wall-acting” means an ability of an agent to interfere with PG biosynthesis in a bacterial cell wall, especially at the transglycosylati on step, as this step takes place on the outer leaflet of the cell membrane so cellular penetration is not a prerequisite for the agent to manifest its biological activity. As used herein, “cell wall-disrupting” means an ability of an agent to disrupt or weaken the integrity of PG in an existing bacterial cell wall.
- The methods can begin by contacting bacteria with a putative cell wall-acting agent or putative cell wall-disrupting agent, where the agent is cell wall-acting if the agent interferes with ongoing peptidoglycan biosynthesis in a bacterial cell wall or is cell wall-disrupting if the agent weakens integrity of peptidoglycan in an existing bacterial cell wall. When screening for putative cell wall-acting agents, the bacteria can be co-contacted with FDAAs and/or CDAAs as described herein simultaneously with the putative agent. When screening for putative cell wall-disrupting agents, the bacteria can have FDAAs and/or CDAAs as described herein covalently incorporated into PG of the cell wall prior to being contacted with the putative agent.
- The methods also can include detecting whether the FDAAs and/or CDAAs have been incorporated in the bacterial cell wail or whether the FDAAs and/or CDAAs remain in the bacterial cell wall. As noted above, the FDAAs and/or CDAAs (after being clicked) can be detected via fluorescence microscopy and other methods, depending upon the type of label or reporter used. The pattern and/or location of FDAAs and/or CDAAs incorporation can be used to identify the bacteria (see, e.g.,
FIGS. 10-12 ). - The methods also can include comparing the esults from the putative cell wall-acting agent or cell wall-disrupting agent with a known cell wall-acting agent or known cell wall-disrupting agent.
- The compounds of the present disclosure have utility for identifying bacteria. As demonstrated in the Examples set forth herein, certain bacterial species display unique specificity for incorporating certain D-amino acids in PG and the bacteria cell wall. Thus, the use of the disclosed modified D-amino acids of the present disclosure enable identification of bacterial species by virtue of the pattern of labeling observed in the bacteria as a result of incorporation of the modified D-amino acids into PG of the bacterial cell wall.
- The invention will be more fully understood upon consideration of the following non-limiting examples, which are offered for purposes of illustration, not limitation.
- Control experiments were carried out in A. tumefaciens, B. subtilis and E. coli with fluorescent D-Ala (D-HCC) and fluorescent L-Ala (L-HCC). For example, experiments in A. tumefaciens revealed that only D-HCC was incorporated into the cell wall. This observation was true for all strains tested. Likewise, experiments with B. subtilis revealed predominant labeling at the septum, a result consistent with this being the site of active cell wall synthesis. Subsequent isolation of peptidoglycan from these cells also revealed that isolated sacculi retained the fluorescent label (
FIG. 3 ). - In addition, experiments with a B. subtilis dacA mutant (DacA is a D,D-carboxypeptidase that cleaves the terminal D-Ala from the peptide stem) resulted in uniform labeling of the cell wall, which suggested the dominant mode of labeling in B. subtilis is at the terminal position of the peptide stem.
- A series of pulse-chase experiments were performed in which exponentially growing cells were diluted and treated with one of the D-Ala probes (250 μM-500 μM), The cells were incubated until saturation, washed, and placed on LB-containing agar pads and imaged. at 5-minute intervals for 12-18 hours (
FIG. 4 ). - An experiment in B. subtilis revealed that fluorescence persisted at the cell poles, an observation that is consistent with the notion that there is not active cell wall synthesis taking place in these regions (
FIG. 4 ). Interestingly, the labeling pattern observed with A. tumefaciens provides supporting evidence for a mode of growth that involves budding as no signal dilution from the mother cell is observed as recently shown. - More significantly, short exposures to FDAA derivatives have proven to be optimal for imaging the sites of active cell wall biosynthesis. For example, when a culture of exponentially growing cells is contacted with either D-NBD or D-HCC, and the cells were pulsed for 2%-8% of their usual generation time and immediately fixed, sites of active synthesis were clearly visible in evolutionarily distinct bacteria such as E. coli, B. subtilis, A. tumefaciens, L. lactis, M. conglotneratus, C. crescentus and S. aureus. Significantly, with experiments conducted in B. subtilis, these short pulses result in a staining pattern that appears to be consistent with the helical pattern that has been observed with fluorescently modified vancomycin/ramoplanin (
FIG. 5 ). - Finally, experiments were performed with the labeled D-Ala derivative in a dual-labeling format. For example, A. tumefaciens cells were incubated for a period of 4 minutes in 500 μD-NBD, followed by washing and incubation for 4 minutes in 500 μM D-HCC. The excess dye was removed and the cells were pelleted and fixed. The fluorescence micrographs reveal distinct patterns of growth, in terms of polar growth and septal synthesis, based on the age of the daughter cell (
FIG. 6 ). - Methods
- Synthesis of Fluorescent D-Amino Amino Acids (FDAAs)
- HADA/HALA. To a flame-dried flask, 7-hydroxycoumarin-3-carboxylic acid (HCC) was added in anhydrous DMF (14.5 mL, 0.1 M) under an atmosphere of argon. Carbonyidiimidazole (236 mg, 1.455 mmol) was added in one portion and stirred at room temperature (RT) for 2 hours.
- Boc-D-2,3-diaminopropionic acid (for HADA) Boc-L-2,3-diaminopropionic acid (for HALA) (297 mg, 1.455 mmol) was added in one portion and the reaction mixture was allowed to stir at RT overnight (17 hours). The majority of the solvent was removed in vacuo, and the product was diluted with EtOAc (100 ml) and washed with 1 N HCl (50 ml) and water (100 ml). The water layers were combined and back-extracted with EtOAc (50 ml) to prevent loss of product due to an emulsion. The organic layers were combined, washed with brine (50 ml), dried over Na2SO4, filtered and the solvent was removed in vacuo. Without further purification the crude product was treated with trifluoroacetic acid/dichloromethane (50:50, 10 ml) for 30 minutes at RT, and the solvent was removed in vacuo. The product was purified via reverse-phase HPLC with 10%-90% MeCN/H2O. The pure fractions were concentrated in vacuo, and the product was redissolved in 1 N HCl/MeCN and lyophilized to yield the desired product as a pale yellow solid (297 mg, 62% for HADA and 277 mg, 58% for HALA) [α]20 D=−21.8° (c 2.2, DMSO-d6); HRMS-ESI-TOF m/z cal'd for C13H12O6N2 ([M+H]+): 293.0774, Found 293.0774; HPLC: tR=5.96 min (10-90% MeCN/H2O over 10 minutes);
- 1H NMR (400 MHz, DMSO-d6) δ=2.46 (s, 1H), 3.69-3.77 (m, 1H), 3.79-3.87 (m, 1H), 4.07 (t, J=5.6 Hz, 1H), 6.85 (d, J=2.0 Hz, 1H), 6.89 (dd, J=2.0, 8.4 Hz, LH), 7.89 (d, J=8.8 Hz, 1H), 8.43 (br s, 3H), 8.76 (s, 1H), 8.86 (t, J=6.2 Hz, 1H), 11.34 (br s, 1H); 13C NMR (100 MHz, DMSO-d6): δ=52.1, 102.3, 111.3, 113.5, 115.0, 132.5, 148.8, 156.8, 161.2, 163.1, 164.6, 169.7; the signal for one carbon was overlapping with the solvent peak.
- NADA/NALA: Boc-D-2,3-diaminopropionic acid (for NADA) or Boc-L-2,3-diaminopropionic acid (for NALA) (100 mg, 0.49 mmol) and sodium bicarbonate (123 mg, 1.47 mmol) were dissolved in water (1.8 ml) and heated to 55° C. in water bath. A solution of 4-chloro-7-nitrobenzofurazan (NBD, 108 mg, 0.539 mmol.) in methanol (8.5 ml) was added dropwise over 10 minutes. Care was taken at all times to avoid excessive exposure to light during the reaction and workup. The reaction was allowed to stir at 55° C. for 1 hour. The solvent was removed in vacuo and acidified with 1 N HCl. The aqueous mixture was extracted with dichloromethane (50 ml per extraction×3 extractions) and the organic extracts were washed with brine (50 ml), dried over Na2SO4, filtered, and the solvent was removed in vacuo. Without further purification the crude product was treated with 4 N HCl/dioxane (10 ml) for 1 hour at RT, and the solvent was removed in vacuo. The product was purified via reverse-phase HPLC with 20%-90% MeCN/H2O. The pure fractions were concentrated in vacuo, and the product was redissolved in 1N HCl/MeCN and lyophilized to yield the desired product as a bright orange solid (105 mg, 71% for both NADA and NALA). [α]20 D=−32° (c 1.1 DMSO-d6) HRMS-ESI-TOF calc'd for C9H9O9O5N5 (M+H]+): 268.0682, Found 268.0680; HPLC: tR−5.02 minutes (20-90% MeCN/H2O over 10 minutes).
- 1H NMR (400 MHz, DMSO-d6): δ=4.06 (m, 2H), 4.29 (m, 1H), 6.61 (d, J=8.0 Hz, 1H), 8.56 (d, J=8.0 Hz, 1H), 8.66 (br s, 3H), 9.32 (br s, 1H); 13C NMR (100 MHz, DMSO-d6): δ=43.4, 51.5, 100.5, 122.5, 138.1, 144.4, 144.9, 145.2, 169.2.
- FDL: To a flame dried flask was added Nα-Boc-D-Lys-OH (19.3 mg, 0.078 mmol) and fluorescein isothiocyanate (25 mg, 0.065 mmol) in dry DMF (0.65 ml). The reaction was stirred under argon at room temperature for 4 hours. The solvent was removed in vacuo. The residue was redissolved in ethyl acetate (10 ml), washed with 1 N HCl (10 ml) and brine (10 ml), and dried over anhydrous sodium sulfate. The solvent was again removed in vacuo, and the crude product was treated with trifluoroacetic acid/dichloromethane (1:1) for 0.5 hours. The acid was removed in vacuo, and the product was purified by reverse phase HPLC with 30%-45% MeCN/H2O. The pure fractions were lyophilized to yield the product as a dark yellow solid (25.0 mg, 72%). [α]20 D=−7.1° (c 0.72, MeOH-d4); HMRS-ESI-TOF m/z calc'd for C27H26N3O7S ([M+H]+): 536.1492, Found 536.1470; HPLC: tR=5.08 min (30%-45% MeCN/H2O over 10 minutes);
- 1H NMR (400 MHz, MeOD-d4): δ=1.50-1,63 (m, 2H), 1.78 (quintet, J=7.0 Hz, 2H), 1.90-1.99 (m 1H), 2.00-2.10 (m, 1H), 3.66 (br s, 2H), 4.00 (t, J=6.3 Hz, 1H), 6.59 (dd, J=8.8 Hz, 2H), 6.73 (s, 2H), 6.74 (d, J=8.90 Hz, 2H), 7.18 (d, J=8.2 Hz, 1H), 7.76 (d, J=8.2 Hz, 1H), 8.17 (s, 1H), 13C NMR (100 MHz, MeOD-d4): δ=23.3, 29.5, 31.3, 45.0, 53.9, 103.5, 111.9, 114.1, 120.5, 126.1, 129,2, 130.5, 131.8, 142,5, 154.6, 162.1 171.0, 171.9, 182.9.
- TDL: To a flame dried flask was added Nα-Boc-D-Lys-OH (3.3 mg, 0.0134 mmol), 5-(and 6-) carboxytetrarnethylrhodamine succinimidyl ester (5 mg, 0.0095 mmol), and diisopropylethylamine (2.5 μl, 0.0143 mmol) in dry DMF (0.2 ml). The reaction was stirred under argon at room temperature overnight. The solvent was removed in vacuo, and the crude mixture was treated with trifluoroacetic acid dichloromethane (1:1) for 0.5 hours. The reaction was dried in vacuo, and purified by reverse-phase HPLC with 20%-40% MeCN/H2O. The pure fractions were lyophilized to yield the product as a deep red solid (4.6 mg, 61%). [α]20 D−−210° (c 0.20, MeOH-d4); HRMS-EST-TOF mlz calc'd for C13H35N4O6 ([M+H]30): 599.2557, Found 599.2559; HPLC: tR=7.86 and 9.06 minutes (2 isomers isolated results from mixed isomer starting material) (20%-40% MeCN/H2O over 10 minutes);
- 1H NMR (400 MHz, MeOD-d4): δ=1.25-1.35 (m, 2H), 1.46-1.62 (m, 2H), 1.68 (quintet, J=7.2 Hz, 2H), 1.75-2.05 (m, 2H), 3.41 (t, J=7 Hz, 1H), 3.92 (t, J=Hz, 1H), 7.01 (d, J=2 Hz, 2H), 7.05 (dd, J=2.0 Hz, 9.4 Hz, 2H), 7.13 (d, J=9.4 Hz, 2H), 7.81 (d, J=1.5 Hz, 1H), 8.19 (dd, J=1.5 Hz, 8.6 Hz, 1H), 8.39 (d, J=8.6 Hz, 1H).
- Spectral Characteristics of FDAAs
- Excitation and emission spectra of FDAAs (500 μM in 100 mM Tris pH 7.0) were deters fined in black 96-well polystyrene plates (Corning) using top-read function of a Spectra Max M2 plate reader. The excitation and emission spectra were rneasured in separate runs within a range of 200 nm and with increments of 1 nm.
- Click Chemistry
- EDA and ELA, and Sulfo-Cy3-Azide were gifts from Boaopharma and Lumiprobe, respectively AZA and “clickable” Alexa 488 Fluors were purchased from Iris Biotech GmbH and Invitrogen, respectively. The Cu(I) catalyzed click chemistry was performed using the chemicals supplied by Invitrogen following their standard protocol once the cells had been fixed with EtOH (70% v/v) and permeabilized with methanol (100% v/v).
- Growth Conditions
- Strain characteristics and growth conditions are described in Table 2.
-
TABLE 2 Strains, their predicted PG chemotypes and conditions for growth and labeling. PG [HADA] Temperature Species Strain Source Gram Chemotype[ ] Media (mM)* (° C.) Aeration Bacillus subtilis PY76 Daniel Kearns + A1γb[ ] LB N/A 37 Y IUB Bacillus subtilis PY76, Daniel Kearns + A1γb LB 1 37 Y dacA::cam IUB Bacillus subtilis CU1065 John Helmann + A1γb LB N/A 37 Y CU Bacillus subtilis HB0048- John Helmann + A1γb LB N/A 37 Y CU1065, CU dltA::spc Brachybacterium IUB culture + A4γ LB 1 30 Y conglomeratum collection Lactococcus loctis IUB culture + A4α LB 1 37 Y collection Staphylococcus IUB culture + A3α LB 1 37 N aureus collection Streptocuccus IU1945 Malcolin + A1α & A3α[4] BHI 0.5 37 N penumaniae Winkles IUB Streptomyces Justin Nodwell + A3γ LB 0.5 30 Y venezuelae MU Agrobacterium Yves Brun − A1γ LB 1 20 Y tumeficiens IUB Burkholderia Ann Hirsch − A1γ LB 0.5 30 Y phytofirmans UCLA Caulobacter YB5630- Yves Brun − A1γ PYE 0.5 30 Y crescentus CB15 IUB hfsA Escherichia coli MG1655 Particia Foster − A1γ LB 1 37 Y IUB Synechocyans sp. David Kehoe − A1γ BG- 1 26 CO2 + PCC 6503 IUB 11 artificial sanlight Vernucumicrobrum Nanmi Ward − A1γ VM 0.25 30 Y spinosum UW Key: IUB = Indiana University Bloomington CU = Cornell University MU = McMaster University UCLA = University of California, Los Angeles UW = University of Wyoming *Short pulse concentrations. By default, a normalized 1% (v/v) DMSO concentration was used for all labeling experiments. indicates data missing or illegible when filed - For any experiment involving FDAAs and/or CDAAs, DMSO was added to the growth media to a final concentration of 1% to help solubilize the FDAAs and/or CDAAs. Presence of 1% DMSO did not affect labeling or growth in bacteria tested. When necessary, chloramphenicol or spectinomycin was added to the growth media at 5 μg/ml or 100 μg/ml, respectively. Strains were maintained on plates containing growth media with 1.5% agar.
- Growth Curves
- For growth curves, exponentially growing E coli, A. tumefaciens and A. tumefaciens and B. subtilis ΔdacA were diluted to OD600 0.05 into wells of polystyrene 24-well plates (Falcon) containing 750 μl LB with 1% DMSO or 1% DMSO+FDAAs (250 μM-1 mM). The absorbance at 600 nm was read every 5 minutes for 18 hours in a BIO-TEK Synergy HT Plate Reader (30° C., static).
- Short Labeling Pulses and Fluorescence Microscopy
- For short labeling pulses, exponentially growing cells were screened for the minimum concentration of FDAAs or CDAAs (250 μM-1 mM) and minimum amount of exposure duration to identify the optimal conditions for each bacterium, as shown in
FIG. 11 and Table 2. To image growth patterns in different species, exponentially growing cells (OD600˜0.3) were labeled, fixed, washed, “clicked” if appropriate, and imaged. - For most strains, excess dye was removed by washing the cells three to four times with 1
ml 1×PBS (NaCl 8 g/L, KCl0.2 g/L, Na2HPO4-2H2O 1.78 g/L, KH2PO4 0.27 g/L, pH 7.4) and pelleting for 2-5 minutes at 10,000-16,000×g in a microfuge. When required, cells were fixed with EtOH (70%, ice-cold, 20-minute incubation). Exceptions included S. pneumoniae (1% gluteraldehyde, 20 minutes incubation) and B. subtilis (cold. PBS treatment). - For dual labeling, the same procedure was followed with the addition of a second round of a short labeling pulse using the second FDAA prior to fixation. Cells were washed before, between and after each FDAA treatment with pre-warmed medium in order ensure similar labeling conditions. For triple labeling, a third round of labeling involving CDAAs was added. For dual and triple labeling of A. tumefaciens, the incubation times with each label were 5 minutes and 7 minutes, respectively.
- Phase and fluorescence microscopy was performed with a Nikon® 90i Fluorescence Microscope equipped with a Plan Apo 100×/1.40 Oil Ph3 DM Objective and a Chroma 83700 triple filter cube with corresponding excitation and emission filters (DAPI for HADA/HALA; FITC for NADA/NALA; and Alexa Fluor® 488s and Texas Red® for Sulfo-Cy3 or WGA-594). All images were captured using NIS software from Nikon® and a Photometrics Cascade 1K cooled charge-coupled device camera, and were processed and analyzed using ImageJ. When a comparison was made, cultures were treated in exactly the same manner and the same parameters were applied for collecting and post processing of the microscopy data.
- Long Labeling Pulses and Time-Lapse Microscopy
- Exponentially growing cells were diluted to OD600 0.05 in media containing half of the optimal FDAA concentration used for short labeling pulses and were grown until late exponential phase. The cells were fixed, washed and then imaged using the Nikon® 90i as described previously. For time-lapse microscopy the cells were washed with media arid mounted onto LB+1% (w/v) agarose pads on 25-mm by 75-mm glass slides, sealed with 1:1:1 mixture of vasoline, lanolin and paraffin and imaged with intervals of 4 minutes (B. subtilis ΔdacA), 5 minutes (E. coli) or 10 minutes (A. tumefacien) using a Nikon® Ti-E Inverted Fluorescence Microscope equipped with a
Plan Apo 60×/1.40 Oil Ph3 DM Objective and a CFP/YFP filter cube and an Andor DU885 EMCCD Camera using CFP settings for detection of HADA. - Super-Resolution Microscopy
- Structured illumination microscopy was performed using a DeltaVision® OMX Imaging System equipped with an Olympus® UPlanSApo 100×/1.40 Oil PSF Objective and a Photometrics Cascade II EMCCD Camera. The samples were excited with a laser at 405 nm and the emission was detected through a 419-465 emission filter.
- Environmental Samples
- HADA (500 μM+1% DMSO) and/or NADA (500 μM+1% DMSO) was added to either a 1.5 ml saliva sample from a 26-year old male or to a 1.5 ml concentrated (˜10 times) fresh water sample collected from Indiana University and incubated for 2 hours (HADA) at 37° C. or for 2 hours (HADA) and 2 hours (N.ADA) at 26° C., respectively. The samples were then fixed, washed and imaged.
- Live-Dead Staining
- E.coli cells were labeled with HADA in 0.1% DMSO. Cells were subsequently stained using the LIVE-DEAD BacLight Kit (Invitrogen) according to the manufacturer's standard protocol.
- Sacculi Purification
- Sacculi from cells were purified as described in Litzinger et al. (2010) J. Bacteriol. 192:3122-3143 with following modifications. Exponentially growing cells were diluted to OD600 0.05 in 10 ml LB containing half of the optimum FDAA concentration+1% (v/v) DMSO and grown to late exponential phase. After aliquots were taken for whole cell imaging, cells were collected by centrifugation at 25,000×g for 15 minutes at RT and resuspended in 0.8 nil water. The suspension was added to boiling sodium dodecyl sulfate (SDS, 5% w/v) drop-wise and incubated with stirring for 30 minutes. SDS insoluble material was collected by ultracentrifugation at 39,000×g for 10 minutes at 30° C. and was resuspended in 1 ml water and boiled again in SDS (4% w/v)with stirring for 30 minutes. Samples were then washed four times in 1.5 ml water and resuspended in
I ml 10 mM Tris-HCl pH 7.0+10 mM NaCl+0.32 M imtnidazole+α-amylase (100 μm/ml)+DNase I (50 μg/ml)+MgSO4 (1 mM) and incubated for 2 hours at 37° C. Samples were pelleted and resuspended in 0.05 M Tris-HCl pH 7.8+1.4 mg/ml pronase (type XXV from Streptomyces griseus) and incubated 2 hours at 60° C. Samples were again pelleted and resuspended in 1 ml water and boiled in SDS (1% w/v) with stirring for 30 min. The sacculus preparations were washed a final time and resuspended in a minimal amount of water. When needed, sacculi were further stained with Wheat Germ Agglutinin,Alexa Fluor® 594 Conjugate (WGA-594, 15 μg/ml). - HPLC Analysis of PG and Muropeptide Identification
- PG from FDAA labeled cells was purified by the boiling SDS extraction method and muramidase digestion treatment (Cellosyl) as previously described in Brown et al. (2012) Proc. Natl. Acad. Sci. USA 109:1697-1701. Solubilized muropeptide mixtures were then either directly injected into the HPLC system (native or non-reduced samples) or subjected to BH4Na reduction as described in Brown et al. (2012), supra. Muropeptides were analyzed using a binary-pump Waters® HPLC System (Waters Corporation) fitted with a reverse phase RP18 Aeris® Peptide Column (250 mm×4.6 mm; 3.6 μm particle size) (Phenomenex) and a dual wavelength absorbance detector. Elution conditions were: flow
rate 1 ml/min; temperature 35° C.; 3 minutes isocratic elution in 50 mM sodium phosphate, pH 4.35 followed by a 57 minute linear gradient to 75 mM, sodium phosphate, pH 4.95 in 15% (v/v) methanol (90 mM sodium phosphate, pH 5.2 in 30%(v/v) methanol for B. suhtilis analyses), and 10 minute isocratic elution under the gradient final conditions. Elution was monitored setting one channel to 204 nm and the second to a wavelength appropriate for detection of corresponding FDAA. Muropeptides of interest were collected following HPLC separation; vacuum dried, and subjected to MALDI-mass spectrometry and electrospray ionization MS/MS as described in Brown et al. (2012), supra. - Results
- Growth of the phylogenetically diverse model species E. coli, A. tumefaciens and B. subtilis in the presence of FDAAs for as little as one generation resulted in strong peripheral and septal labeling of entire cell populations (
FIG. 8 ) without affecting growth rate. Neither of the FLAAs prepared from 3-amino-L-alanine resulted in significant labeling, indicating that labeling is specific to the D-enantiomers. The labeling was exclusive to viable cells treated with the FDAAs and was not the result of non-specific interaction of FDAAs with the PG. Additionally, incorporation did not occur into teichoic acids for B. subtilis as indicated by identical labeling of wild-type and a mutant that does not D-alanylate its teichoic acids. - Retained fluorescence on the purified sacculi (
FIG. 8 ) demonstrated that the labeling of PG by the FDAAs was covalent. HPLC analyses of muropeptides isolated from labeled cells (FIGS. 9B-C ) revealed that 0.2%-2.8% of total muropeptides were modified (FIG. 9C ), which is sufficient for detection in various experiments while avoiding possible toxicity issues that could result from abundant incorporation. Significantly, the FDAA-specific peaks, which were absent in samples treated with FLAAs, could be distinguished from unlabeled muropeptides at FDAA-specific absorption wavelengths (FIG. 9B ). MS/MS analyses of FDAA-modified muropeptides in B. subtilis indicated that FDAAs were exclusively incorporated in the fifth position of the stem peptide (FIG. 9D ). Interestingly, in a ΔdacA mutant of B. subtilis, the fraction of labeled muropeptides and the fluorescent signal were substantially higher than in wild-type B. subtilis, which is likely due to the D,D-carboxypeptidase activity of DacA. In contrast, the detectable incorporation was solely at the fourth position in E. coli and A. tumefaciens (FIG. 9D ). These results are in agreement with the known sites of incorporation of various natural DAAs in these species and suggest that, similar to DAAs, FDAAs incorporate mainly through periplasmic exchange reactions with the muropeptides catalyzed either by D,D-transpeptidases (e.g., in B. subtilis) or by L,D-transpeptidases (e.g., in E. coli and A. tumefaciens). This DAA-like behavior together with the ease of fluorescent detection make FDAAs a novel alternative to radioactive probes for studying in vitro and in vivo activities of PG synthesis enzymes. - Pulse-chase experiments with HADA allowed the real-time tracking of new PG incorporation during growth via, time-lapse microscopy. In E. coli and B. subtilis ΔdacA (
FIG. 10A ), the polar caps retained the HADA signal, but the signal from the lateral walls dispersed as the cells grew, in agreement with previous reports of cell wall growth along the length of the lateral walls. - Strikingly, short labeling times (2%-8% of doubling time) using E. coli and B. subtilis ΔdacA with HADA resulted in preferential localization of the signal at the septal plane of predivisional cells and in punctate patterns on the lateral walls of elongating cells (
FIG. 11 ), Super-resolution microscopy of E. coli revealed reticulated hoop-like patterns of HADA labeling around the lateral wall (FIG. 10B ), supportive of bursts of PG incorporation in the side-walls. This ability of FDAAs to resolve insertion of new PG provides the first direct detection of zones of PG synthesis in a structured rather than a random pattern in E coli, consistent with recent results following the movement of the cell wall elongation machinery. Short labeling times with A. tumefaciens, whose growth occurs predominantly from a single pole and the site of cell division while the mother cell remains inert, resulted in polar and septal labeling. Super-resolution fluorescence microscopy of labeled cells further enhanced the spatial resolution of the site of active PG synthesis (FIG. 10C ). - PG labeling in three evolutionarily distant species suggested that FDAAs could specifically label the active site of PG synthesis across the entire bacterial domain. When species representing diverse phyla and modes of growth were briefly incubated with FDAAs, we observed strong labeling at the sites of cell division in actively dividing cells (
FIG. 11 ). This septal probe incorporation was the sole mode in Synechoeystis sp.PCC 6803, L. lactis and S. aureus. Super-resolution microscopy of S. aureus further highlighted the different stages of these constricting septal rings (FIG. 10D ). Labeling of S. pneumoniae occurred in single or split equatorial rings depending on the length of the cell, with peripheral labeling between the split rings (FIG. 11 ) Labeling of S. venezuelae was predominantly apical, with some weak labeling of vegetative septa and lateral walls suggestive of a low but continuous lateral PG synthesis (FIG. 11 ). In C. crescentus, labeling occurred at the sites of septal elongation, lateral elongation, and stalk synthesis (FIG. 11 ). B. phylofirmans exhibited polar and mid-cell PG synthesis, B. conglomeratum exhibited prominent peripheral PG synthesis in addition to seemingly alternating perpendicular division planes, and V. spinosum exhibited strong peripheral PG synthesis and asymmetric septal labeling (FIG. 11 ). - The efficient label incorporation in all the bacteria indicates that FDAAs, therefore DAAs, incorporation is common to the bacterial domain and that FDAAs can thus be used to analyze natural bacterial populations, providing a convenient and quick standard to measure bacterial activity and to probe the diversity of growth modes in complex inicrobiomes. Indeed, labeling times with FDAAs as short as 2 hours revealed diverse modes of growth in saliva and freshwater samples in situ, but did not label dead cells as suggested by the strong correlation with Live-Dead staining.
- Encouraged by the efficiency of FDAAs, additional and differently functionalized unnatural DAAs were prepared and used. Following a similar approach, a brighter and more versatile core fluorophore, fluorescein (emission maximum ˜515 nm, green), and its analogue, carboxytetramethylrhodamine (TAMRA, emission maximum ˜565 nm, red) were derivatized and linked with D-Lys to separate the bulky fluorophore from the DAA backbone, generating FDL and TDL (see, for example, structures in
FIG. 7 ). Incubation of both E. coli and B. subtilis with FDL showed patterns similar to NADA, although labeling of B. subtilis was stronger than E. coli. In contrast, the larger TDL did not label E. coli cells, but labeling of B. subtilis was prominent and showed patterns similar to other FDAAs. - CDAAs, namely ethynyl-D-alanine (FDA) or azido-D-alanine (ADA) (
FIG. 7 ), that can be specifically captured by any molecule carrying the conjugate functional group via click-chemistry also were used. Similar to FDAAs, these bioorthogonal DAAs, but not the L-enantiomer control ELA, labeled both E. coli and B. subtilis when captured by commercially available azidolalkyne fluorophores. - Furthermore, custom DAAs containing different colored fluorophores can be used sequentially to enable “virtual time-lapse microscopy.” Since addition of each new probe indicates the location and extent of PG synthetic activity during the respective labeling periods, this approach provides a chronological account of shifts in PG synthesis of individual cells over time. Examples of such serial labeling, including a combination with click chemistry, were performed in Gram-negative A. tumefaciens (
FIG. 10E ) and in Gram-positive S. venezuetae (FIG. 10F ). - In view of the foregoing, disclosed herein are compositions for and methods of covalently labeling PG in live bacterial cells. This method works very efficiently in both Gram-positive and Gram-negative organisms (Gram-negative organisms represent a liability for approaches using fluorescently modified vancomycin/ramoplanin), and the probe substrates do not appear to be toxic to cells and show no adverse effects on cell morphology, even at concentrations as high as 1 mM. The probes rapidly label sites of active peptidoglycan biosynthesis and can be used in time-lapse and dual labeling experiments.
- Thus, compositions and methods have been described herein for in situ labeling/probing of PG synthesis in bacteria with fluorescent D-amino acids (FDAAs) as well as for screening bacterial cell wall-acting and/or cell wall-disrupting agents. The FDAAs are based upon D-amino acids (DAAs) derivatized to covalently include a small fluorophore. As such, the FDAAs can be directly incorporated into bacterial cell walls during PG biosynthesis, as occurs at sites of cell division in actively dividing cells.
- The compositions include FDAAs. The FDAAs have a DAA covalently attached to a fluorophore such as 7-hydroxycoumarin 3-carboxylic acid (HCC-OH), 7-nitrobenzofurazan (NBD), 4-chloro-7-nitrobenzofurazan (NBD-Cl), fluorescein (F) or carboxytetramethylrhodamine (T). The DAA can be any of the twenty known, standard amino acids, such as D-Ala, D-Asp, D-Cys, D-Glu or D-Lys.
- The compositions also include clickable DAAs (CDAAs). The CDAA have a DAA backbone including an alkyne or azide functional group that can be captured by any labeled detecting agent carrying a conjugate functional group via click-chemistry, where the label can be a fluorescent molecule.
- The compositions also include fluorescent muramylpentapeptide precursor units (FMPUs). The FMPUs have an N-acetyl muramic acid (NAM) moiety with a stem peptide of three to five amino acids in which one or more of the amino acids in the stem peptides are FDAAs and/or CDAAs as described hererin.
- The compositions also include fluorescent PG units (FPGUs). The FPGUs have a FMPU as described herein linked to an N-acetyl glucosamine (NAG) moiety.
- The compositions also include live bacteria having one or more FDAA, CDAA, FMPU and/or FPGU as described herein incorporated into PG in a cell wall.
- The compositions also include kits having one or more FDAA, CDAA, FMPU and/or FPGU as described herein and optionally one or more labeled, detecting agents for use in in situ labeling/probing of PG synthesis, as well as for screening for bacterial cell wall-acting and/or cell wall-disrupting agents. The kits also can include additional reagents such as unlabeled DAAs unlabeled L-amino acids (LAAs), fluorescent LAAs (FLAAs) and/or clickable LAAs (CLAAs). The kits also can include positive and/or negative bacterial controls, where the bacterial controls have unlabeled DAAs, CDAAs, LAAs and CLAAs and/or labeled DAAs, CDAAs, LAAs and CLAAs incorporated into PG in a cell wall.
- In view of the foregoing, the methods have been disclosed herein that include assessing bacterial cell wall synthesis in real time by providing live bacteria with one or more FDAA, CDAA, FMPU and/or FPGU as described herein, where the bacteria covalently incorporate the one or more FDAA, CDAA, FMPU and/or FPGU into PG of a bacterial cell wall. The one or more FDAA, CDAA, FMPU and/or FPGU can be provided to live bacteria together or sequentially during cell wall synthesis.
- The methods also include screening for putative cell wall-acting or cell wall-disrupting agents by contacting bacteria with a putative cell wall-acting agent or putative cell wall-disrupting agent, where the agent is cell wall-acting if the agent interferes with ongoing PG biosynthesis in a bacterial cell wall or is cell wall-disrupting if the agent weakens integrity of PG in an existing bacterial cell wall. When screening for putative cell wall-acting agents, the bacteria can be provided with one or more FDAA, CDAA, FMPU and/or FPGU as described herein simultaneously with the putative agent. When screening for putative cell wall-disrupting agents, the bacteria can have one or more FDAA, CDAA, FMPU and/or FPGU as described herein covalently incorporated into PG of the cell wall prior to being contacted with the putative agent.
- The methods also include identifying a bacteria by providing live, unknown bacteria with one or more FDAA, CDAA, FMPU and/or FPGU as described herein under conditions sufficient for bacterial cell wall synthesis, where the bacteria covalently incorporate one or more FDAA, CDAA, FMPU and/or FPGU into a cell wall, and where each of the one or more FDAA CDAA, FMPU and/or FPGU includes a distinct fluorophore. The methods also include observing an incorporation pattern of the one or more FDAA, CDAA FMPU and/or FPGU, where the incorporation pattern identifies the bacteria. Such methods are amenable for use in screening platforms to identify novel compounds having bacteriostatic or bacteriotoxic properties.
- In the methods, the bacteria can be Gram-positive or Gram-negative bacteria.
- The methods also include detecting one or more FDAA, CDAA, FMPU and/or FPGU as described herein that have been incorporated in the bacterial cell wall or that have been disrupted from the bacterial cell wall by, for example, fluorescence microscopy and other methods, depending upon the label used.
- The methods also can include comparing the results of the putative agent with a known cell wall-acting agent or with a known cell wall-disrupting agent.
- The compositions and methods described herein therefore find use in the study of bacterial cell wall biosynthesis and in the discovery of bacterial cell wall-acting and/or cell wall-disrupting agents. Advantageously, the FDAAs, CDAAs, FMPUs and/or FPGUs as described herein simultaneously are non-toxic and can be tunable to label sites of active PG biosynthesis, enabling fine spatiotemporal tracking of cell wall dynamics in phylogenetically and morphologically diverse bacteria.
- All of the patents, patent applications, patent application publications and other publications recited herein are hereby incorporated by reference as if set forth in their entirety.
- The present invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, one of skill in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.
- Typas et al. (2, 12) Nat. Rev. Microbiol. 10:123-36.
- van Dam et al. (2009) Chembiochem 10:617-24.
- Daniel & Errington (2003) Cell 113:767-76.
- Tiyanont et al. (2006) Proc Natl Acad. Sci USA 103:11033-38.
- Olrichs et al. (2011) Chembiochem 12:1124-33.
- Sadamoto et al. (2004) J. Am. Chem. Soc. 126:3755-61.
- de Pedro et al. (1997) J. Bacteriol. 179:2823-34.
- Lam et al. (2009) Science 325:1552-55.
- Cava et al. (2011) EMBO J. 30:3442-53.
- Lupoli et al. (2011) J. Am. Chem. Soc. 133:10748-51.
- Brown et al. (2012) Proc. Natl. Acad. Sci. USA 109:1697-1701.
- Mobley et al. (1984) J. Bacteria 158:169-79.
- Furchtgott et al. (2011) Mol. Microbiol. 81:340-353.
- Dominguez-Escobar et al. (2011) Science 333:225-28.
- Garner et al. (2011) Science 333:222-225.
- Rippka & Herdman (1985) Ann. Inst. Pasteur/Microbiol. 136:33-39.
- Zapun et al. (2008) FEMS Microbiol. Rev. 32:345-360.
- Flärdh (2003) Curr. Opin. Microbiol. 6:564-571.
- Aaron et al. (2007) Mol. Microbiol. 64:938-952.
- Young (2006) Microbiol. Mol. Biol. Rev. 70:660-703.
- Lavis & Raines (2008) ACS Chem. Biol. 3:142-155.
- Decad & Nikaido (1976) J. Bacteriol 128:325-336.
- Prescher & Bertozzi (2005) Nat. Chem. Biol. 1:13-21.
- Neumann et al. (2010) Nature 464:441-44.
- Wang et al. (2001) Science 292:498-500.
- Schneider et al. (2012) Nat. Meth. 9:671-675.
- Litzinger et al. (2010) J. Bacteriol. 192:3132-3143.
- Brown et al. (2012) Proc. Natl. Acad. Sci. USA 109:1697-1701.
- Cava et al. (2011) Embo. J. 30:3442-3453.
- Lupoli et al. (2011) J. Am. Chem. Soc. 133:10748-10751.
- Schleifer & Kandler (1972) Bacteriological Reviews 36:407-477.
- Atrih et al. (1999) J. Bacteriol. 181:3956-3966.
- Filipe et al. (2011) J. Biol. Chem. 276:39618-39628.
- Sham et al. (2011) Proc. Natl. Acad. Sci. USA 108:E1061-1069.
- Shapiro & Agabian-Keshishian (1970) Proc. Natl. Acad. Sci. USA 67:200-203.
- Stanier et al. (1971) Bacteriol. Rev. 35:171-205.
- Staley & Mandel (1973) Intl. J. System. Bacteriol. 23:271-273.
Claims (20)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/706,592 US20200109430A1 (en) | 2012-04-21 | 2019-12-06 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261636640P | 2012-04-21 | 2012-04-21 | |
US201261718048P | 2012-10-24 | 2012-10-24 | |
PCT/US2013/037504 WO2013159078A1 (en) | 2012-04-21 | 2013-04-21 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
US201414395815A | 2014-10-20 | 2014-10-20 | |
US16/048,000 US10544444B2 (en) | 2012-04-21 | 2018-07-27 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
US16/706,592 US20200109430A1 (en) | 2012-04-21 | 2019-12-06 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/048,000 Division US10544444B2 (en) | 2012-04-21 | 2018-07-27 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
US20200109430A1 true US20200109430A1 (en) | 2020-04-09 |
Family
ID=48191043
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/395,815 Abandoned US20150191763A1 (en) | 2012-04-21 | 2013-04-21 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
US16/048,000 Active US10544444B2 (en) | 2012-04-21 | 2018-07-27 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
US16/706,592 Pending US20200109430A1 (en) | 2012-04-21 | 2019-12-06 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/395,815 Abandoned US20150191763A1 (en) | 2012-04-21 | 2013-04-21 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
US16/048,000 Active US10544444B2 (en) | 2012-04-21 | 2018-07-27 | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof |
Country Status (2)
Country | Link |
---|---|
US (3) | US20150191763A1 (en) |
WO (1) | WO2013159078A1 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20160222430A1 (en) * | 2013-09-11 | 2016-08-04 | Indiana University Research And Technology Corporation | D-ala-d-ala-based dipeptides as tools for imaging peptidoglycan biosynthesis |
GB201420222D0 (en) * | 2014-11-13 | 2014-12-31 | Univ Edinburgh | Molecular probes and methods using the same |
CN106279125B (en) * | 2015-06-03 | 2018-11-30 | 首都医科大学 | Coumarin derivative and its preparation method |
US11285490B2 (en) | 2015-06-26 | 2022-03-29 | Ancera, Llc | Background defocusing and clearing in ferrofluid-based capture assays |
US11371941B2 (en) * | 2016-07-04 | 2022-06-28 | Celltool Gmbh | Device and method for the determination of transfection |
WO2019157233A1 (en) * | 2018-02-07 | 2019-08-15 | Indiana University Research And Technology Corporation | Molecular rotor-based d-amino acids as tools for imaging peptidoglycan biosynthesis |
CN111458313A (en) * | 2020-04-07 | 2020-07-28 | 上海交通大学医学院附属仁济医院 | Antibacterial drug sensitivity test detection method based on fluorescent D-type amino acid metabolism marker |
CN111733102B (en) * | 2020-06-30 | 2023-08-18 | 东南大学 | Tyrosinase catalysis-based gram-positive bacterium surface modification method and application thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8778626B2 (en) | 2010-07-08 | 2014-07-15 | California Institute Of Technology | Clickable cross-linker |
-
2013
- 2013-04-21 US US14/395,815 patent/US20150191763A1/en not_active Abandoned
- 2013-04-21 WO PCT/US2013/037504 patent/WO2013159078A1/en active Application Filing
-
2018
- 2018-07-27 US US16/048,000 patent/US10544444B2/en active Active
-
2019
- 2019-12-06 US US16/706,592 patent/US20200109430A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
US20190024135A1 (en) | 2019-01-24 |
WO2013159078A1 (en) | 2013-10-24 |
US20150191763A1 (en) | 2015-07-09 |
US10544444B2 (en) | 2020-01-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10544444B2 (en) | Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof | |
US20190024132A1 (en) | D-ala-d-ala-based dipeptides as tools for imaging peptidoglycan biosynthesis | |
Hsu et al. | Full color palette of fluorescent d-amino acids for in situ labeling of bacterial cell walls | |
Schlafer et al. | Confocal microscopy imaging of the biofilm matrix | |
Liechti et al. | A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis | |
CA2841940A1 (en) | Method for identifying antimicrobial compounds and determining antibiotic sensitivity | |
Tague et al. | Cationic biaryl 1, 2, 3-triazolyl peptidomimetic amphiphiles: synthesis, antibacterial evaluation and preliminary mechanism of action studies | |
Zhang et al. | Synthesis and application of the blue fluorescent amino acid l-4-cyanotryptophan to assess peptide–membrane interactions | |
EP3406731A1 (en) | Metabolic labeling of bacterial teichoic acids cell wall | |
Ignacio et al. | Metabolic labeling probes for interrogation of the host–pathogen interaction | |
Sharifzadeh et al. | Chemical tools for selective activity profiling of bacterial penicillin-binding proteins | |
Yang et al. | A fluorescent probe for detecting mycobacterium tuberculosis and identifying genes critical for cell entry | |
Kado et al. | A cell wall synthase accelerates plasma membrane partitioning in mycobacteria | |
US11168077B2 (en) | Molecular rotor-based D-amino acids as tools for imaging peptidoglycan biosynthesis | |
Noden et al. | Enantioselective Synthesis and Application of Small and Environmentally Sensitive Fluorescent Amino Acids for Probing Biological Interactions | |
Sharma et al. | Rapid detection of major Gram-positive pathogens in ocular specimens using a novel fluorescent vancomycin-based probe | |
Kuru | Novel ways for decorating bacterial cell walls advance fundamental knowledge about bacterial growth | |
Labana | Deciphering the mechanism of action of armeniaspirol: A polyketide Gram-positive antibiotic | |
Xu et al. | Metabolic labeling of Gram-negative bacterial peptidoglycan via tetrazine ligation and its application for studying outer membrane permeability | |
Sminia et al. | Probing Peptidoglycan Synthesis in the Gut Commensal Akkermansia muciniphila with Bioorthogonal Chemical Reporters | |
US11414403B2 (en) | Antibacterial compound and uses of same | |
Yablonowski | Fluorescent Tools for the Study of Peptidoglycan in Life Threatening Bacteria | |
Hsu | Dissecting Bacterial Cell Wall Biosynthesis during Cytokinesis and the Development of Novel Probes for Visualizing Peptidoglycan Formation | |
Nagy | MdfA-a model multidrug efflux pump for single-molecule investigations of transport | |
Lund | Peptidoglycan dynamics in Staphylococcus aureus using super-resolution microscopy |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: INDIANA UNIVERSITY RESEARCH AND TECHNOLOGY CORPORATION, INDIANA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:VANNIEUWENHZE, MICHAEL S.;KURU, ERKIN;BROWN, PAMELA J.;AND OTHERS;SIGNING DATES FROM 20141021 TO 20150303;REEL/FRAME:051208/0343 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |