KR20180119890A - Method of screening antibiotics targeting bacterial RNA Pyrophosphohydrolase - Google Patents

Abstract

The present invention relates to a method for screening candidate antibiotics targeting RNA pyrophosphohydrolase (RppH) involved in the regulation of cell membrane permeability, wherein the candidate material selected in the screening method of the present invention has a new antibiotic mechanism which increases cell membrane permeability, and thus the candidate material is an effective antibiotic in itself and can also be used as an adjunct to other antibiotics in combination with other antibiotics.

Description

[0001] The present invention relates to a screening antibiotic targeting bacterial RNA Pyrophosphohydrolase

The present invention relates to antibiotic screening targeting RppH of bacteria.

Antibiotics used for bacterial control have been experiencing many difficulties due to the recent increase in resistant bacteria. However, the development of new antibiotics effective against resistant bacteria is insignificant, and in particular, antibiotics that are effective against gram negative bacteria are lacking. Nevertheless, a small number of antibiotic candidates currently under clinical trials are antibiotics that act on Gram-positive bacteria. There are only two antibiotics based on the new mechanism of action developed in the last 40 years: linezolid and daptomycin.

The antibiotic candidate substance or antibiotics act on Gram-positive bacteria mostly because Gram-negative bacteria have difficulties in developing new antibiotics due to difficulty of penetration of antibiotics due to two cell membranes (outer membrane and inner membrane). Indeed, carbapenem, an effective antibiotic for Gram-negative bacteria, is effective against Gram-negative bacteria because of its small size and easy access into bacteria. It is very useful as an adjuvant, but antibiotics with high molecular weight can not pass through the extracellular membrane of Gram-negative bacteria and do not act on Gram-negative bacteria at all. The vancomycin, also known as the 'last antibiotic', can not pass through the cell membrane of Gram-negative bacteria due to its large size, and is mainly used to control Gram-positive bacteria. For this reason, the development of new antibiotics for Gram-negative bacteria has been hampered by the development of antibiotics due to many limitations such as low molecular weight.

Therefore, regulation of cell membrane permeability of Gram-negative bacteria is a very good antibiotic target. Antibiotics with new mechanism of action to increase cell membrane permeability may not only act as good antibiotics in themselves, but also increase the permeability of cell membranes, which may also help other antibiotics. Previously, antibiotics such as colistin, which bind directly to bacterial cell membranes and regulate permeability, are medically important antibiotics used to control Gram negative strains resistant to carbapenem. Recently, Resistant strains have been found to be more difficult to control Gram-negative bacterial infection.

Therefore, it is necessary to develop new target antibiotics that can regulate cell membrane permeability.

RNA pyrophosphohydrolase (RppH) is an enzyme that acts early in intracellular RNA degradation. It is an enzyme that removes two phosphate groups from three phosphate groups at the 5 'end of RNA. However, it is not known at all whether RppH has any effect on bacterial cell membrane permeability.

Korean Patent Application Publication No. 2009-010652 (Published September 29, 2009)

The present invention provides an antibiotic screening method which can be used as a drug that can antibiotically function or enhance the use of other antibiotics by increasing the permeability of bacterial cell membranes by targeting RppH (RNA Pyrophosphohydrolase) of bacteria.

In one embodiment, the present invention provides a method comprising: i) providing RppH (RNA pyrophosphohydrolase); ii) treating the RppH with a test substance expected to inhibit the activity of RppH; iii) measuring the activity of RppH; And iv) selecting the test substance as an antibiotic candidate substance when the activity of the RppH is decreased as compared with the case where the RppH activity measurement is not treated when treated with the test substance. A method for screening a substance that enhances cell membrane permeability is provided.

It is possible that Gram-negative bacteria or Gram-positive bacteria can penetrate the cell membranes, but it is difficult to penetrate antibiotics due to two cell membranes (outer membrane and inner membrane) ≪ / RTI >

In one embodiment, the method according to the present invention is carried out in vitro in the presence of RppH protein and RNA as a substrate, wherein said RppH activity can be measured as an activity of removing phosphate groups from the 5 'end of said RNA of said RppH. As a substrate, the RNA is not particularly limited as long as it contains three phosphate groups at the 5 'terminus.

In one embodiment, RppH according to the method of the present invention can be provided in the form of a protein or a gene capable of expressing the protein or a vector containing the gene, and when expressed in a form other than a protein, need.

In another embodiment, the method according to the invention is provided wherein the RppH is provided in the form of a cell, wherein the RppH activity is selected from the group consisting of a change in permeability of the cell, a change in the growth rate of the cell at a low temperature of from 15 캜 to 20 캜, ≪ / RTI >

Substances which enhance bacterial membrane permeability herein are used as antibiotics against gram-positive or gram-negative bacteria, or as adjuvants to antibiotics.

The selected material according to the method of the present invention can be used as an adjunct to conventional antibiotics and antibiotics of any mechanism that can be aided by increased permeability of membranes such as these conventional antibiotics include glycopeptide antibiotics But are not limited to, antibiotics that inhibit cell wall synthesis, antibiotics that affect cell membrane permeability, including polymyxin antibiotics, or antibiotics that inhibit nucleic acid synthesis, including rifampicin or quinolone.

The present invention relates to a method for screening candidate antibiotics targeting RppH (RNA pyrophosphohydrolase) involved in the regulation of cell membrane permeability.

The candidate substance selected in the screening method of the present invention is an antibiotic based on a novel mechanism of increasing cell membrane permeability and is not only an antibiotic substance which is useful in itself, but also can be used in combination with other antibiotics of other antibiotics, It is also possible to use as a material to help penetrate into the furnace.

In particular, the substance screened by the present method can be useful for Gram-negative bacteria, which are difficult to develop effective antibiotics due to difficulty in permeation of cell membranes, and the substance can be useful for antibiotic resistant bacteria due to low antibiotic permeability of bacteria.

Also, the antibiotic developed by the method according to the present invention is an adjuvant for antibiotics such as colistin which directly binds to the cell membrane of a bacterium and regulates the permeability, and can be used for enhancing the sensitivity of the bacteria have.

Therefore, the screening method targeting RppH of the present invention can be usefully used for the development of antibiotics of new mechanism, especially antibiotics effective for resistant bacteria or Gram-negative bacteria.

Figure 1 shows that the rppH mutation has increased sensitivity to antibiotics. (a) to LB medium was sequentially diluted by 10-fold the stationary phase cells of the indicated strains at 10 ⁸ to 10 ⁴ cells / ml in culture was spot on LB plate with antibiotics indicating 2㎕ amount. After incubation at 37 ° C for 14-20 hours, the plates were scanned. (b) Stator cells cultured in LB medium were inoculated into LB medium containing cholestin (2 μg / ml) or nalidixic acid (10 μg / ml) and growth was recorded by measuring optical density at 600 nm . : Diamonds, MG1655; Square, KM101 (? RppH ); KM101 (? RppH ), transformed into pHRppH; Triangle, KM101 (? RppH ) transformed with pHRppH (E56 & 57A). These data demonstrate that the enzymatic activity of RppH is required to overcome many antibiotic resistance.
Figure 2 shows the sensitivity of the rppH mutation to the envelope stress response. (a) Stator cells of the indicated strain cultured in LB medium were harvested at low speed. After washing once with 50 mM NaCl, the second was spun in water and incubated at room temperature for 30 minutes. And viability was measured by plating on an LB agar plate. (b) sequentially diluted with a 10-fold to a stationary phase cells of the indicated strain cultured in LB medium at 10 ⁸ to 10 ⁴ cells / ml, the manner, the amount 2㎕ the LB plate, 5% ethanol was added as indicated LB plate, or LB plate supplemented with 2% SDS. After incubation at 37 ° C for 14-20 hours, the plates were scanned. These results indicate that RppH deficient strains have increased cell membrane permeability.
Figure 3 shows the increase in membrane permeability of rppH mutants. (a) Cells of the indicated strains were patched onto LB plates containing CPRG (20 [mu] g / ml) and 50 [mu] M IPTG. After incubation at room temperature for 20 hours, the plate was scanned. : Lane 1, MG1655; Lane 2, KM101 (? RppH ); Lane 3, KM101 ([ Delta ] rppH ) transformed into pHRppH; Lane 4, KM101 ([Delta] rppH ) transformed with pHRppH (E56 & (b) Stable stromal cells of wild-type and rppH mutants containing pBAD-GFP were inoculated on LB medium containing 0.02% L-arabinose. When A ₆₀₀ was 0.5, the cells were spotted onto a 1.5% agarose pad made of PBS containing 4 / / ml of cholestin. Cells were visualized using a Nikon Eclipse Ni microscope (Nikon, Japan). These results indicate that the RppH-deficient strain is associated with increased cell membrane permeability.
Figure 4 shows the resistance of the rppH mutation to cold stress. (a) sequentially diluted with a 10-fold to a stationary phase cells of the indicated strain cultured in LB medium at 10 ⁸ to 10 ⁴ cells / ml were spot on LB plates. After incubation for 24 hours (37 and 42 ° C) or 120 hours (16 ° C), the plates were scanned. The results show that the membrane permeability of RppH-deficient strains is increased.

This article is based on the discovery that RNA pyrophosphohydrolase, an enzyme involved in the degradation of RNA in bacteria, is involved in the regulation of bacterial membrane permeability and that these enzymes can be a new target for antibiotic development.

Materials targeted by RppH selected by the method according to the present invention increase the permeability of the cell membrane and thus can directly inhibit the growth of bacteria and increase penetration of other antibiotics used in the past. In particular, cell membrane permeability control is useful for the control of resistance to existing antibiotics and is particularly useful for the development of antibiotics that are effective against gram negative bacteria.

Accordingly, in one embodiment, the present invention provides a method of screening a substance that increases the cell membrane permeability of the RppH-targeting bacteria.

The method according to the present invention, in one embodiment, comprises the steps of: i) providing RppH (RNA pyrophosphohydrolase); ii) treating the RppH with a test substance expected to inhibit the activity of RppH; iii) measuring the activity of RppH; And iv) selecting the test substance as an antibiotic candidate substance when the activity of the RppH is decreased as compared with the case where the test substance is treated with the result of the RppH activity measurement.

'RNA pyrophosphohydrolase (RppH)' of the present screening method can be provided in the form of a protein or a gene encoding the protein, or a vector containing the gene. If provided as the latter, protein expression from the gene must precede. In one embodiment, it is provided as a protein.

RppH is an enzyme that acts early on in RNA degradation in bacteria. It is an enzyme that removes two phosphate groups from three phosphate groups at the 5 'end of RNA. RNA with one phosphate group can be easily degraded by attack of RNase such as RNase E.

RppH that can be used in the screening method according to the present invention may be of various lengths, various sequence variations or various origins as long as the function of the enzyme is maintained. In the present application, Gram-negative bacteria are derived, particularly E. coli or Pseudomonas putida But the present invention is not limited thereto. Protein and nucleic acid sequences of RppH are known and include, for example, E. coli RppH (Accession: NP_417307), Pseudomonas putida RppH (Accession: NP_747247).

The RppH protein can be isolated and / or purified after expression in cells based on known sequences. A method of cloning a gene into an appropriate vector through a recombinant method and introducing it into an expression cell to express the protein is known, and a person skilled in the art can use an appropriate method. Or RppH protein is commercially available from New England Biolabs.

The method according to the present invention can be carried out in vitro or using cells.

In one embodiment, it is performed in vitro . In this case, RppH can be provided as a protein, and a ribonucleic acid (Ribonucleic acid) as a substrate can be provided as a substrate at the 5 'terminus, particularly including two or three phosphate groups, especially three phosphate groups. RppH can be referred to above for proteins.

In another embodiment, RppH in the method according to the invention is provided as a cell expressing it. The cell expressing RppH may be a cell originally used to express RppH, or a cell which induces overexpression or expression of RppH by introducing a recombinant vector containing RppH into the cell or to a cell that does not express RppH.

Such cells are not particularly limited as long as they can achieve the object of the present invention as bacterial cells, and various Gram-positive and Gram-negative cells can be used as long as they express RppH.

In one embodiment, in particular, gram negative bacteria such as E. coli , Pseudomonas aeruginosa , Klebsiella pneumoniae , Bacillus subtilis , cobacterium tuberculosis Etc., may be used, but are not limited thereto.

The test substance used in the method according to the present invention may be a low molecular weight substance which is expected to affect the activity of RppH. For example, compounds having a weight of about 1000 Da such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may constitute a part of a library of compounds, and the number of compounds constituting the library may vary from several tens to several millions. Such library of compounds includes peptides, peptoids and other cyclic or linear oligomeric compounds and small molecule compounds based on a template such as benzodiazepines, hydantoins, biaryls, carbocycles and polycycle compounds such as naphthalene, phenothiazine Carbohydrates and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (such as triazines, indoles, thiazolidines, etc.), but this is merely exemplary and should not be construed as limiting the scope of the present invention. But is not limited thereto.

In the method according to the present invention, RppH is treated with a test substance for the screening of candidate substances. As mentioned above, RppH can be provided as a protein or a cell expressing it. Those skilled in the art will be able to determine the amount of the test substance in consideration of the method to be used, the type of cell to be used, the kind of the specific test substance, and the like.

In one embodiment, a test substance according to the present invention may be treated in vitro in addition to a reagent comprising an RppH protein.

In another embodiment, cells expressing RppH can be cultured on a cell culture plate and then treated with the addition of a test substance thereto.

The activity of RppH is measured after the step of treating with the test substance for screening candidate substances in the method according to the present invention.

The activity of RppH can be measured directly or indirectly.

In a direct manner, for example, the activity of RppH can be determined by using RNA as a substrate containing at least two or three phosphate groups at the 5 'end, for example, in the case of in vitro , to determine whether phosphate groups are removed from the substrate Can be measured. Therefore, RNA molecules of various lengths and / or types can be used as long as these conditions are satisfied. For example, RppH utilizes the activity of making the 5'end of triphosphate of mRNA mono-phosphate as a substrate, and using short-sized nucleotides such as RNA, for example, pppGCA, After 20 minutes, the separated phosphate group or pGCA can be measured by HPLC or the like. A more straightforward direct method is to use RppH to inactivate ATP in api ₅ A (diadenosine penta-phosphate) in vitro . For example, the activity of RppH can be measured by measuring the amount of ATP released after 10 minutes of reaction using 10 μM Ap ₅ A as a substrate using luciferase (Lee CR, et al ., RppH-dependent pyrophosphohydrolysis of mRNAs is regulated by direct interaction with DapF in Escherichia coli. Nucleic Acids Res. 2014 Nov 10; 42 (20): 12746-57.).

In another embodiment, when cells expressing RppH are used in the method according to the present invention, the cell membrane permeability of cells treated with the test substance can be measured indirectly to confirm whether RppH is inhibited by the test substance . Methods for measuring changes in cell permeability are well known in the art, and those skilled in the art will be able to select and use appropriate ones. For example, Gram-negative bacteria such as E. coli ( Escherichia The coli) using β- galactosidase for substrate, chlorophenyl red -β-d- galacto-pyrano side (CPRG) can not pass through the outer membrane can be measured by checking the LacZ activity. CPRG can not be degraded by the cytoplasmic LacZ in the control cells not treated with the test substance, but when the permeability of the test substance is increased by the treatment of the test substance, the CPRG molecule can permeate better or release LacZ into the medium, Cells with a change in permeability are principles that can form red colonies by chlorophenyl red (CPR), a cleavage product of CPRG.

According to the present invention, when the permeability of the cell membrane is increased, the low temperature resistance and the resistance to the outer membrane stress are lowered. In another embodiment, when cells expressing RppH are used in the method according to the present invention, they may be tested indirectly, for example, by low temperature resistance testing of cells treated with the test substance, or by osmotic pressure, ethanol or SDS (sodium dodecyl sulfate) , Which method is well known in the art and can be selected by one of ordinary skill in the art or refer to the methods described in the Examples herein (Lee J, et al ., &Quot; , Dephosphorylated NPr is involved in an envelope stress response of Escherichia coli. Microbiology. 2015 May; 161 (Pt 5): 1113-23; Lee CR et al ., RppH-dependent pyrophosphohydrolysis of mRNAs is regulated by direct interaction with DapF in Escherichia Nucleic Acids Res., 2014 Nov 10; 42 (20): 12746-57.).

In one embodiment according to the present application, low temperature resistance, i.e., growth at low temperature, is measured. In this study, when membrane fluidity of cells decreased, membrane permeability change by rppH was considered to be helpful for growth at low temperature compared with wild type, and the growth was generally measured at a low temperature of 37 degrees which measures E. coli growth. Low temperature is an easy experimental technique for determining the dysfunction of RppH. Low temperature is selected to allow cell growth. In particular, the growth rate of E. coli rapidly decreases below 21 degrees, and growth below a level below 10 degrees may not be sufficient to measure RppH function. Thus, in one embodiment, it can be performed at a low temperature of 10 to 21 degrees. More particularly 15 to 20 degrees, even more particularly 16 degrees. Methods for measuring growth at low temperature can be found in the examples herein or at http://www.gatewaycoalition.org/files/hidden/react/ch4/4_4f.htm.

As described above, in the method according to the present invention, when the test substance is treated with the test substance, the test substance is selected as an antibiotic candidate substance when the activity of the RppH is decreased as compared with the case where the test substance is not treated do.

In order to screen for candidate substances in the method according to the present invention, the inhibition of the enzymatic activity of RNA pyrophosphorylase (RppH) in the presence of the test substance as compared with the control group not in contact with the test substance was about 99% , Less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70% , About 40% or less, about 30% or less, about 20% or less, about 10% or less of activity, but does not exclude the range of deviation.

Compared with the control (when the test substance is not treated), when the permeability of the cell membrane is increased, it can be judged that the activity of RppH is inhibited. Such a test substance can be selected as a substance that increases cell membrane permeability.

The increase in cell permeability by inhibition of the activity of RNA pyrophosphorylase (RppH) in the presence of the test substance compared to the control not contacted with the test substance, for the selection of the candidate substance in the method according to the present invention, More than 30% increase, more than 40% increase, more than 45% increase, more than 10% increase, more than 15% increase, more than 20% increase, About 75% increase, about 80% increase, about 85% increase, about 90% increase, about 55% increase, about 60% increase, about 65% increase, % Increase, more than about 95% increase, more than about 100% increase, but do not exclude ranges beyond this.

According to the present invention, a substance that increases bacterial membrane permeability increases the permeability of the cell membrane by inhibiting the activity of RppH, thereby inhibiting the growth of bacteria. Therefore, the substance is selected as a candidate substance for antibiotics. In addition, since these substances can be used together with other antibiotics to increase the permeability of the cell membrane to enhance the action of other antibiotics, the substance is selected as an auxiliary substance of antibiotics. These adjuvants are particularly resistant to resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), and penicillin resistant pneumococci (PRSP) It is particularly useful for Gram-negative bacteria which are useful and also have little effective antibiotic.

Other antibiotics that can be used in combination with substances that increase the cell membrane permeability of the present bacteria include antibiotics such as beta-lactam antibiotics and glycopeptide antibiotics such as vancomycin, antibiotics that inhibit cell wall synthesis, antibiotics such as amphotericin antibiotics such as amphotericin, Antibiotics that alter cell membrane permeability such as, rifampicin, quinolone, etc., and antibiotics that inhibit the synthesis of nucleic acids, and protamine.

In addition, the RppH-deficient strains are rapidly increased in sensitivity to cholestin, and can be used as an antibiotic adjuvant that can work together with cholestin. Previously, antibiotics such as colistin, which bind directly to bacterial cell membranes and regulate permeability, are medically important antibiotics used to control Gram negative strains resistant to carbapenem. Recently, Resistant strains have appeared, and antibiotics developed by the method according to the present invention can be usefully used for this purpose.

Therefore, the substance screened by the method of the present invention is effective as an antibiotic having a novel action mechanism, and is particularly effective for Gram-negative bacteria and can be used for Gram-negative resistant bacteria.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Materials and methods

Bacterial strains, plasmids and culture conditions

The bacterial strains and plasmids used in the present invention are listed in Table 1. Bacterial cells were cultured as described in the prior art (Lee J, Park YH, Kim YR, Seok YJ & Lee CR (2015) Dephosphorylated NPr is involved in an envelope stress response of Escherichia coli. Microbiology 161 : 1113-1123). Unless otherwise indicated, LB (Luria broth) was used as a composite culture medium for normal culture of bacteria. To construct pBAD-GFP, a vector for expression of green fluorescent protein (GRP), a forward primer (shown underlined) for synthesizing the ATG start codon of the gfp gene (shown in bold) to amplify the gfp gene (5'-GCAGGTCGAC TCTAGA TTA CGGGCCCAATTTGTATA-3 ') (in which the stop codon is indicated in bold) present in the synthetic Xhol site (underlined) was used as the primer (5'-CTAGCAGGAG GAATTC ATG AGTAAAGGAGAAGAACT-3' After digestion, an EcoRI-XhoI fragment was inserted into the corresponding site of pBAD24.

[Table 1]

Low salt  Low salt survival

Cells cultured for 20 hours were pelleted at low speed and washed with 50 mM NaCl. Cells were spun in water and incubated at room temperature for 30 minutes. Plates were plated on LB agar plates to determine viability.

In live cells  Visualization of fluorescent fusion protein

For DIC and fluorescence imaging, the cells were cultured in LB medium containing 0.02% L-arabinose until OD ₆₀₀ = 0.5, resulting in the presence of PBS (phosphate buffered saline) containing 4 μg / ml of cholestin And spotted on a 1.5% agarose pad. Cells were visualized using a Nikon Eclipse Ni microscope (Nikon, Japan).

Chlorophenyl  Red-β-D- Galactopyranoside ( CPRG ) Measurement of phenotype

To evaluate the CPRG phenotype in various strains, cells cultured overnight in LB medium were patched in an X-shape on LB medium containing 20 / / ml of CPRG and 50 M of IPTG. Plates were cultured overnight at room temperature and then photographed.

Example  One. RppH  Phenotype of mutation MicroEray  analysis

It has been reported in previous studies that the RNA pyrophosphorohydrolase activity of RppH is activated by intimate interaction with DapF, but the exact physiological function in the interaction has not yet been fully understood. Although RppH has an RNA pyrophosphorylase activity that catalyzes the removal of pyrophosphate at the 5 'end of the triple oxidation mRNA, a relatively small mRNA has been identified as an invivo target of RppH. Thus, in order to obtain a greater degree of RppH, the present inventors analyzed the phenotype of the rppH mutation using a phenotype microarray. Growth of MG1655 and RppH mutant cells was measured in 96-well microplates containing different nutrients or inhibitors, as measured by redox indicator of cellular respiration. Through nearly 2,000 cell-phenotype assays, rppH mutations have been shown to be sensitive to a variety of inhibitors including antibiotics, nucleic acid metabolism inhibitors, folic acid antagonists, membrane-destroying agents, and toxic ions (Tables 2-1 to 2-3)

[Table 2-1]

[Table 2-2]

[Table 2-3]

Example  2. To overcome antibiotic resistance RppH  Identify the need for activity

To confirm the results of phenotypic microarrays, the inventors investigated the sensitivity of rppH mutations to several inhibitory chemicals tested in phenotypic microarrays.

First, a strain of RppH, which is an RNA pyrophosphohydrolase, was prepared from E. coli, and a phenotype microarray was performed to examine 1000 different phenotypes of the defective strain. As a result, RppH-deficient strains showed phenotypes sensitive to antibiotics such as cholestin, vancomycin, rifampicin, and protamine (Table 3).

[Table 3] Phenotype microarray results of RppH-deficient strain

^a wild type and size differences in growth curves between RppH deficient strain. Units are based on arbitrary values. Negative numbers indicate that growth was inhibited in RppH deficient strains.

These results showed the same results in the spotting experiment, and particularly the sensitivity to the cholestin was strong (Fig. 1). The phenotype of RppH deficient strains with the pRE1 vector expressing RppH was restored to the wild type level but the phenotype of the RppH deficient strain with the pRE1 vector expressing the mutant protein RppH (E56 & 57A) with no enzyme activity was not recovered at all 1).

This phenotype of the RppH mutation was restored by plasmid-derived expression of RppH, confirming the role of RppH in these phenotypes. In conventional results, RppH (E56 & 57A), in which the 56th and 57th glutamates of RppH were mutated to alanine, was unable to remove pyrophosphate from the RNA. As expected, plasmid-derived expression of RppH (E56 & 57A) could not alter the sensitivity of rppH mutations to antibiotics (Fig. 1). Thus, these data demonstrate that the enzymatic activity of RppH is required to overcome many antibiotic resistance.

Example  3. RppH  Influence on envelope stresses such as osmosis, ethanol, and SDS stress

This phenotype of the rppH mutation can be caused by increased permeability of the chemical through enhanced permeability of the membrane, as the rppH mutation is sensitive to a number of inhibitory chemicals. To demonstrate this hypothesis, the present inventors have tested the phenotype of the rppH mutation under various outer membrane stresses. Survival testing of the rppH mutation under low salt conditions showed that the rppH mutation showed reduced resistance to low salt stress (Fig. 2a).

In addition to osmotic stress, the effects of other external stresses such as ethanol and SDS were investigated. The rppH mutation was highly sensitive to ethanol stress and also very sensitive to SDS stress (Fig. 2b). These results indicate that the rppH mutation is sensitive to various outer membrane stresses. This phenotype of the rppH mutation was also due to the loss of its enzymatic activity (Figure 2).

Previous studies have shown that the concentration of osmY transcript is increased in rppH mutant cells and the salt sensitivity of cells with increased RppH activity is inhibited by overexpression of the osmY gene. To see if this phenotype of the rppH mutation is caused by an increase in the expression level of the osmY gene, the present inventors analyzed the phenotype of the rppH osmY mutation. Additional deletion of the osmY gene did not alter the phenotype of the rppH mutation, but the sensitivity of the rppH mutation to rifampicin or SDS was somewhat increased. Thus, these results indicate that phenotype identification of the rppH mutation is independent of OsmY.

Example  4. rppH  Increased permeability of the outer membrane in the mutation

This phenotype of the rppH mutation implies that RppH may affect the permeability of the outer membrane. In order to test the permeability of the outer membrane, the present inventors used chlorophenyl red-β-d-galactopyranoside (CPRG), which is a substrate of β-galactosidase which can not pass through E. coli outer membrane, . Although CPRG could not be degraded by cytoplasmic LacZ in wild-type cells, mutants with increased permeability of the outer membrane were able to release LacZ with better penetration or persistence of CPRG molecules, and these cells were therefore able to release chlorphenyl Red (CPR) to form red colonies. In the outer membrane permeability test using CPRG, rppH mutant cells showed a significant red color compared to wild type cells, indicating increased permeability of the outer membrane in rppH mutants (Fig. 3a). Similar to other phenotypes, this CPRG-positive phenotype was altered by plasmid-derived expression of RppH, but not RppH (E56 & 57A).

To further confirm the increased permeability of the outer membrane in the rppH mutant, the present inventors used GFP to visualize the outer membrane permeability as fluorescence. Cholestin is a multimeric cationic peptide with both hydrophilic and lipophilic moieties that interact with lipopolysaccharide (LPS) in the outer membrane (outer membrane) to produce a sterilizing effect that causes the outer membrane to break. GFP connections with wild-type and rppH mutant cells were visualized by time-lapse microscopy. rppH mutant cells have somewhat longer cell sizes than wild-type cells and are broadly linked to GFP proteins in the presence of cholestin (Figure 3b). Thus, this result means that the permeability of the outer membrane is increased in the absence of RppH.

Example  5. rppH  Cold stress resistance of mutation

Although increased permeability increases sensitivity to a variety of outer membrane stresses and toxicants, it has a positive effect in reducing membrane fluidity, such as at low temperatures. To test this hypothesis, the inventors checked the sensitivity of rppH mutations to cold stress. At high temperature conditions, rppH mutant cells showed no growth differences with wild-type cells, whereas at low temperature mutant cells grew considerably faster than wild-type cells (Fig. 4A). On the other hand, when RppH was overexpressed, bacterial growth was inhibited at low temperature condition (FIG. 4B). However, overexpression of RppH (E56 & 57A) without enzyme activity had little effect on bacterial growth at low temperatures, suggesting that increased enzymatic activity of RppH leads to a decrease in cold stress tolerance. Thus, these results support the conclusion that rppH mutant cells increase the permeability of the outer membrane.

In a conventional study, the present inventors have found that DAP (diaminopimelate) epimerase DapF and RppH, which catalyze stereonversion of 1,1-DAP with meso-DAP, strongly interact with each other, Thereby increasing the phosphorylase activity. Meso-DAP is not only a second-stage element in the end-to-end lysine biosynthetic pathway, but also as an element of the pentapeptide in the biosynthesis of peptidoglycan in most gram-negative bacteria. The dapF mutation accumulates 1,1-DAP, and its peptidoglycan contains 1,1-DAP instead of meso-DAP. The cross-linking of peptidoglycan with 1,1-DAP appears weak because the dapF mutation significantly increases membrane permeability. In the present invention, it has been found that the rppH mutation also increases membrane permeability. Thus, this result implies that in membrane integrity regulation there is a functional relationship between RppH and DapF.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (5)

i) providing RppH (RNA pyrophosphohydrolase);
ii) treating the RppH with a test substance expected to inhibit the activity of RppH;
iii) measuring the activity of RppH;
iv) the step of selecting the test substance as an antibiotic candidate substance when the activity of RppH is decreased as compared with the case where the RppH activity is measured, when treated with the test substance, Lt; / RTI >
The method according to claim 1,
Wherein said method is performed in vitro in the presence of RNA as a substrate and said RppH activity is measured as activity to remove phosphate groups from the 5 ' end of said RNA of said RppH.
The method according to claim 1,
The RppH is provided in a form expressed in a cell,
Wherein the RppH activity is measured as a change in permeability of the cell, a change in the growth rate of the cell at a low temperature of 15 캜 to 20 캜, or resistance to stress.
The method according to claim 1,
Wherein the substance that increases the cell membrane permeability of the bacteria acts as an antibiotic, or as an adjuvant to antibiotics, against gram negative or Gram-positive bacteria.
The method according to claim 1,
In the adjuvant of the antibiotic, the antibiotic may be an antibiotic that inhibits cell wall synthesis, including a glycopeptide antibiotic, an antibiotic that affects cell membrane permeability, including polymyxin antibiotics, or a nucleic acid that inhibits rifampicin or quinolone- Antibiotics.

Images