US20020143163A1 - Gene conferring resistance to the antibacterial 4,5-dihydroxy-2-cyclopenten-1-one (DHCP), the protein encoded by same, and applications thereof - Google Patents

Gene conferring resistance to the antibacterial 4,5-dihydroxy-2-cyclopenten-1-one (DHCP), the protein encoded by same, and applications thereof Download PDF

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US20020143163A1
US20020143163A1 US09/805,681 US80568101A US2002143163A1 US 20020143163 A1 US20020143163 A1 US 20020143163A1 US 80568101 A US80568101 A US 80568101A US 2002143163 A1 US2002143163 A1 US 2002143163A1
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dhcp
protein
gene
resistance
dep
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Sangita Phadtare
Kunitoshi Yamanaka
Ikunoshin Kato
Masayori Inouye
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Takara Bio Inc
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University of Medicine and Dentistry of New Jersey
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Assigned to TAKARA SHUZO CO., LTD. reassignment TAKARA SHUZO CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • DHCP 4,5-dihydroxy-2-cyclopenten-1-one
  • uronic acid is galacturonic acid, glucuronic acid, mannuronic acid or iduronic acid. It is also produced from roasted or parched vegetables, fruits, cereals, mushrooms, sea algae, cortex or cartilage. It has been demonstrated that this compound induces cancer cell differentiation and apoptosis. It has potential application as therapeutic or preventive agent against cancer and also as an antibacterial agent in antiseptics, dentrifices, cosmetics and bathing agents (Koyama et al., 1999).
  • This patent discloses a method of manufacturing 4,5-dihydroxy-2-cyclopenten-1-one (DHCP). It also describes the antibacterial activity of DHCP.
  • the invention disclosed in the present provisional application relates to a gene, dep, that, when present in multiple copies in bacterial cells, confers resistance to the antibacterial activity of DHCP, thus rendering the bacteria resistant to killing by DHCP.
  • the present application also describes the protein encoded by the dep gene.
  • This patent relates to functionally equivalent ether derivatives of DHCP and discloses the biological activity of these derivatives.
  • This patent relates to functionally equivalent ester derivatives of DHCP and discloses the biological activity of these derivatives.
  • MDR multidrug-resistance
  • P-gp P-glycoprotein
  • loperamide is a substrate for the efflux membrane transporter P-glycoprotein.
  • loperamide is a potent opiate drug, it does not opioid central nervous system effects, such as respiratory depression, when given to patients at usual doses.
  • This study tested the hypothesis that inhibition of P-glycoprotein with quinidine would increase the entry of loperamide into the central nervous system, thus causing respiratory depression.
  • the results demonstrated that although loperamide produced no respiratory depression when used alone, respiratory depression was seen when loperamide was administered with quinidine.
  • MDS myelodysplastic syndromes
  • P-gp P-glycoprotein
  • MRP1 multidrug resistance-associated protein
  • Soft tissue leiomyosarcomas and malignant gastrointestinal stromal tumors differences in clinical outcome and expression of multidrug resistance proteins.
  • MDR multidrug resistance
  • the bioluminescent marine bacterium Vibrio harveyi controls light production (lux) by a quorum-sensing circuit.
  • lux response regulator protein
  • ⁇ 54 the response regulator protein
  • LuxO functions as an activator protein via interaction with the alternative sigma factor, ⁇ 54 . Since LuxO is responsible for repression of the luciferase structural operon (luxCDABEGH), these results suggest that LuxO, together with ⁇ 54 , functions to activate a negative regulator of luminescence.
  • FIG. 1 is the chemical structure of 4,5-dihydroxy-2-cyclopenten- 1 -one (DHCP).
  • FIG. 2A is a graphical representation of the effect of DHCP concentration on the growth of E. coli.
  • FIG. 2B is a graphical representation of the effect of DHCP concentration on the survival of E. coli.
  • FIG. 3 is a restriction mapping of the plasmid pSP001 showing the DNA fragments conferring resistance to DHCP.
  • FIG. 4 is a comparison of the amino acid sequence of the polypeptide encoded by dep with the proteins encoded by cmr, cmrA, cmx, cmlv, bcr, bmr3, yjcC, and tet.
  • FIG. 5 is a comparison of the hydropathic profiles of the putative proteins encoded by dep, cmr, and cml.
  • FIG. 6 is a nucleotide sequence showing the DNA sequence of a region of the E. coli genome containing the sequence of the dep gene.
  • FIG. 7 is a nucleotide sequence showing the isolated DNA sequence of the dep gene.
  • the provisional application describes the cloning of a gene encoding a transmembrane protein from E. coli. This protein, when expressed from a multi-copy plasmid, functions to transport 4,5-dihydroxy-2-cyclopenten-1-one (DHCP) out of the cell.
  • DHCP 4,5-dihydroxy-2-cyclopenten-1-one
  • DHCP and functionally equivalent compounds are represented by the formulas [I] and [II] and include optically active compounds thereof.
  • R 3 -R 6 are independently hydrogen or an alkyl group, preferably a lower alkyl group such as a C 1 -C 6 alkyl.
  • R 3 -R 6 are independently hydrogen or an alkyl group, preferably a lower alkyl group such as a C 1 -C 6 alkyl.
  • DHCP is shown to possess anti-bacterial activity; it inhibits cell growth at a concentration of 350 ⁇ M or higher. At lower concentrations, it causes cells to elongate and grow poorly.
  • a library of E. coli genomic DNA fragments was transformed into strain JM83 and grown on agar plates containing 400 ⁇ M DHCP. Colonies that were capable of growing on this medium were isolated. DNA was isolated from these colonies to identify and sequence the cloned genomic fragment that specified resistance. Four genes were found in the fragment that conferred resistance. Inactivation of various combinations of these four genes led to the conclusion that ORF389 was responsible for conferring resistance. This was confirmed by cloning ORF389 by itself into pUC19 (a multi-copy plasmid) and transforming strain JM83. The resultant cells were resistant to DHCP.
  • ORF389 Comparison of the nucleotide sequence of ORF389 with the E. coli gene database showed that it was similar to known efflux proteins involved in conferring resistance to chloramphenicol and other antibiotics. Further analysis of the predicted structure of the protein encoded by ORF389 suggested that it was a membrane protein; it possesses multiple transmembrane domains and shares structural similarity with the aforementioned chloramphenicol efflux polypeptides.
  • ORF389 was capable of conferring resistance to other antibiotics such as chloramphenicol, spectinomycin, and tetracycline
  • the transformed JM83 cells containing the pUC19/ORF389 plasmid were plated on media containing these antibiotics.
  • the presence of ORF389 failed to confer resistance to any antibiotic other than DHCP, suggesting that the efflux activity of the Dep protein is specific for DHCP.
  • ORF389 confers resistance to DHCP only when it is present in multiple copies in the cell.
  • the gene is naturally found in the genome of E. coli cells, but it is present in single copy. Such cells are susceptible to the antimicrobial activity of DHCP.
  • ORF389 is cloned into pUC19 and introduced into JM83 cells, it is present in multiple copies (up to several hundred copies of the gene per cell), since pUC19 is maintained in up to several hundred copies per cell. Only when the gene dosage is increased, is resistance to DHCP found. The mechanism of resistance is simply increased efflux activity arising from the increased expression of the efflux protein in the transformed cells.
  • nucleotide sequence encoding an efflux protein that is responsible for conferring resistance to DHCP or a compound functionally equivalent to DHCP may vary from the nucleic acid sequence disclosed herein.
  • DHCP is a compound that exhibits antimicrobial and anti-tumor activity. It is made by heating various uronic acids (e.g., glucuronic acid, galacturonic acid, mannuronic acid).
  • uronic acids e.g., glucuronic acid, galacturonic acid, mannuronic acid.
  • the inventors have cloned a gene from E. coli that encodes a protein which is capable of transporting DHCP out of the cell.
  • This transport protein shows sequence similarity with known efflux proteins that function to transport antibiotics such as chloramphenicol out of the cell. It has been shown that organisms which overexpress the transport protein become resistant to DHCP, probably because they are able to efficiently transport DHCP.
  • Overexpression of the transport protein arises from the presence of multiple copies of the gene, rather than increased expression from the endogenous gene in E.
  • E. coli possess a single copy of the transport gene.
  • the level of transport protein expression from a single copy of the gene is insufficient to confer resistance to DHCP.
  • the inventors have cloned the gene into a high copy number plasmid, pUC19, which is maintained in E. coli cells at 200-500 copies per cell.
  • transformed E. coli containing this plasmid construct will possess 200-500 copies of the transport gene, and protein expression from multiple copies is greater than from a single copy. These transformed cells are resistant to DHCP.
  • DHCP The general mode of action of DHCP requires that it enter the target cell. Resistance to DHCP can occur if DHCP is transported out of the cell as fast as or faster than it enters the cell. Given that, the concentration of DHCP within the cell can never accumulate to a toxic dose and the cell is resistant to the antimicrobial effects of the compound. Hence, the transport protein encoded by gene disclosed does not transport DHCP very efficiently, or the amount of transport protein expressed from the endogenous gene is very low. In either case, the presence of more transport protein (arising from many copies of the gene) will result in more efficient transfer of DHCP out of the cell.
  • inhibitors of efflux activity will function to block the transport activity.
  • a microbe or a tumor cell that is resistant to DHCP can be made to be more sensitive to the compound by preventing the resistant cell from transporting the compound back out.
  • inhibitors of the transport gene of the invention may also be active in blocking transport of other efflux proteins such as the efflux proteins that transport chloramphenicol, or the P glycoprotein family of multiple drug resistant proteins.
  • the P glycoproteins are expressed in many tumor cells, making these tumors resistant to chemotherapy agents. Abstracts regarding studies of P glycoproteins are referenced above.
  • FIG. 1 is the chemical structure of 4,5-dihydroxy-2-cyclopenten-1-one (DHCP).
  • FIG. 2 Effect of DHCP concentrations on the growth of E. coli.
  • DHCP concentration 0 ⁇ M, open squares; 50 ⁇ M, closed diamonds; 100 ⁇ M, open circles; 250 ⁇ M, open diamonds; 400 ⁇ M closed squares.
  • FIG. 3 Restriction mapping of the plasmid pSP001 conferring resistance to DHCP.
  • ORFs comprising the DNA fragment (5.2 kb) conferring resistance to DHCP and the flanking ORFs are shown. The orientation of each ORF is marked with an arrow. The restriction enzyme sites are also shown. The ORFs are not drawn to scale.
  • the plasmid pSP001 containing the DNA fragment conferring resistance to DHCP was digested with restriction enzymes to disrupt each of four ORFs, religated and transformed into JM83 cells. The transformants were then examined for their sensitivity to DHCP (400 ⁇ M).
  • the enzymes used for digestion were: forpurR: MluI for ydhB; NruI-Eco47III, for ORF389, purR , and ydhB: NruI and SmaI, for ORF389: Aval and forpurR and ydhB: MluI and NruI.
  • for construction of plasmid with ORF389 (dep) the plasmid pSP001 was digested with SmaI and MscI, the fragment was purified and cloned into pUC19 to yield plasmid pSP007.
  • FIG. 4 The sequence homology between Dep, Cmr, CmrA, Cmx, CmlV, BcR, Bmr3, YjcC and Tet. Identical and similar sequences are marked with black and gray boxes, respectively. The consensus sequences for transmembrane proteins are marked with dotted lines and are represented as I, II, and III stretches.
  • FIG. 5 Hydropathic profiles of Dep (A), Cmr from Rhodococcusfaciens (B) (6) and Cml from Streptomyces lividans (C) (8). Horizontal bars indicate predicted transmembrane regions.
  • FIG. 6 is a nucleotide sequence showing the DNA sequence of a region of the E. coli genome containing the sequence of the dep gene. This region of the E. coli genome is available at Accession No. AE000261 U00096. The sequence shown is that of nucleotides 4381-8280. The dep gene is encoded by nucleotides 4627-5838. The dep sequence is shown in brackets.
  • FIG. 7 is a nucleotide sequence showing the isolated DNA sequence of the dep gene.
  • the plasmid pSP007 was confirmed to contain the dep gene by obtaining DNA sequence data from one end of the 1.7 kb insert. Sequence data obtained in this manner matched the first.
  • E. coli wild-type strain JM83 [F ⁇ ara ⁇ (lac-proAB) rpsL(str′)](Yanisch-Perron et al., 1985) was grown in Luria broth (LB). Media were supplemented with ampicillin (final concentration of 50 ⁇ g/ml) whenever required.
  • ampicillin final concentration of 50 ⁇ g/ml
  • DHCP was added at various concentrations (0-400 ⁇ M) and growth was further monitored. After it reached to the Klett unit of 90-100, it was diluted 10-fold into media containing respective concentrations of DHCP.
  • 2A shows the effect of different concentrations of DHCP on E. coli.
  • the growth was slowed after 3 h of incubation in the presence of 50 ⁇ M DHCP, but it reached the maximum density after 8 h, similar to that without DHCP.
  • the cells grew more slowly after 3 h incubation with 100 ⁇ M of DHCP and the maximum cell density was lower than that without DHCP.
  • growth was severely impaired after 3 h of incubation and cells stopped growing after 5 h.
  • 400 ⁇ M DHCP cell growth stopped after 4 h of incubation.
  • E. coli genomic library was screened.
  • the construction of E. coli genomic library was described previously (Lu and Inouye, 1998).
  • the partially digested Sau3AI chromosomal DNA fragments from E. coli JM83 were cloned into the BamHI site of pUC19.
  • the JM83 cells were transformed with the genomic library. Transformants were isolated for their ability to grow on DHCP (400 ⁇ M) containing LB plates at 37° C. Plasmid DNA was isolated from the resistant colonies, purified and retransformed into JM83 cells to confirm its ability to confer resistance to DHCP.
  • the plasmid was designated as pSP001 and was found to contain a 5.2-kb DNA fragment. This fragment was sequenced from both ends using Sequenase and BLAST search was carried out for the analysis of homology of this fragment with the entire E. coli genome. It was found that this DNA fragment is located at 37.5 min on the E. coli chromosome and contains four ORFs (FIG. 3): ORF389, purR encoding purine synthesis repressor, ydhB encoding a homologue of the cyn operon transcriptional activator and ydhC encoding a homologue of bicyclomycin resistance protein (Berlyn et al., 1996).
  • ORF389 is responsible for resistance to DHCP when cloned in a multicopy plasmid and further work was carried out using the plasmid pSP007.
  • the ORF389 was named as dep- D HCP e fflux p rotein (see below).
  • FIG. 4 shows nine proteins showing significantly high homology with Dep. Half of these proteins confer resistance to chloramphenicol.
  • the proteins showing the highest degree of homology include: Cmr from Rhodococcus fasciens (Desomer et al., 1992), CmrA from R. erythropolis (Nagy et al., 1997), Cml from Streptomyces lividans 1326 (Dittrich et al., 1991), Cmx from Corynebacterium striatum (Accession no. U72639), and CmlV from S.
  • Dep has the highest degree of homology with Cmr, product of chloramphenicol resistant gene (cmr) as compared to other proteins.
  • Cmr protein was shown to contain three consensus sequences defined by Rouch et al. (1990) for transmembrane proteins. These sequences are at similar positions with respect to the predicted transmembrane domains. These are marked in FIG. 6 with dotted lines and are designated as I, II, III.
  • the first stretch (I) comprising of LP is completely homologous with the stretch defined by these authors.
  • the second stretch (II) shows 50% similarity with that of Cmr protein and the third stretch (III) is homologous between these two proteins except for one residue.
  • the stretches I and III are located on the outside of the cytoplasmic membrane and the stretch II is located on the inside of the membrane.
  • the positions of the membrane loops for the putative protein encoded by qacA were ascertained by inspecting the antigenic index profile and turn prediction. Such regions have a high antigenic index and turn probability (Rouch et al., 1990).
  • FIG. 5A the hydropathic profile of Dep (FIG. 5A) is significantly similar to those of Cmr of R. faciens (Desomer et al., 1992) (FIG. 5B) and Cml of S. lividans (Dittrich et al., 1991) (FIG. 5C).
  • Dep is predominantly hydrophobic and probably contains 12 predicted transmembrane ⁇ -helices (FIG. 5A).
  • the other proteins homologous to Dep include BcR (bicyclomycin- resistance protein) from E. coli (Bentley et al., 1993), Bmr3 from B. subtilis involved in the multiple drug efflux pump conferring resistance to puromycin, tosufloxacin, norfloxacin (Ohki and Murata, 1997), Tet from Staphylococcus hyicus conferring tetracycline resistance (Schwarz et al., 1992) and YjcC conferring tetracenomycin-resistance (Accession no. D90826) (FIG. 4). All of these are efflux proteins, which is one of the most common mechanisms for drug resistance.
  • dep encodes a putative efflux protein that forms a cytoplasmic channel specific for DHCP.
  • the homologies are more prominent towards the N-terminal end of the proteins, which also is a common feature for efflux proteins (Desomer et al., 1992).
  • Dep shows homology to efflux proteins for multiple drug resistance, we checked if it confers resistance to other antibiotics as well.
  • the E. coli wild-type cells harboring pUC 19 or pSP007 plasmid were grown overnight in LB medium containing ampicillin. The cells were diluted 10- and 1000- times, and 5 ⁇ l of each dilution (corresponding to 3.5 ⁇ 10 5 cells and 3.5 ⁇ 10 3 cells, respectively) was spotted on LB plates containing serial dilutions of kanamycin, chloramphenicol, spectinomycin, tetracycline and DHCP. Plates were incubated at 37° C. for 20 h.
  • pSP007 did not confer significant cross-resistance to any of the antibiotics tested.
  • the MIC values for cells harboring pUC19 and pSP007 were same for spectinomycin, chloramphenicol and tetracycline. The MIC value was two times higher for kanamycin for the cells harboring pSP007 than the cells with pUC19.
  • the MIC value for DHCP on the other hand was 8 times higher for the cells harboring pSP007 than that for the cells with pUC 19. It is interesting that Dep did not confer resistance to chloramphenicol, in spite of the high homology to cmr. TABLE 1 Minimum inhibitory concentrations (MICs) of various antibiotics for E.

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