Antibiotic resistance arises from the maintenance of resistance mutations or genes acquired from the acquisition of adaptive de novo mutations or the transfer of resistance genes. Antibiotic resistance is acquired in response to antibiotic therapy by activating SOS-mediated DNA repair and mutagenesis and horizontal gene transfer pathways. Initiation of the SOS pathway promotes activation of RecA, inactivation of LexA repressor, and induction of SOS genes. Here, we have identified and characterized phthalocyanine tetrasulfonic acid RecA inhibitors that block antibiotic-induced activation of the SOS response. These inhibitors potentiate the activity of bactericidal antibiotics, including members of the quinolone, β-lactam, and aminoglycoside families in both Gram-negative and Gram-positive bacteria. They reduce the ability of bacteria to acquire antibiotic resistance mutations and to transfer mobile genetic elements conferring resistance. This study highlights the advantage of including RecA inhibitors in bactericidal antibiotic therapies and provides a new strategy for prolonging antibiotic shelf life.

Here, we extended these observations by developing phthalocyanine tetrasulfonate (PcTs)-based inhibitors of RecA. These anionic, aromatic molecules tap into the cationic feature of the DNA-binding site of RecA through cation-π () and cation-anion interactions. PcTs inhibitors blocked ATPase, DNA binding, DNA strand-exchange, and LexA proteolysis activities of RecA. They blocked antibiotic-induced activation of the SOS response, reducing the ability of ciprofloxacin (CFX) to induce in vitro filamentation and biofilm formation. PcTs inhibitors potentiated the activity of bactericidal antibiotics, reduced the ability of bacteria to acquire antibiotic resistance mutations in vitro, and decreased the frequency of conjugational transfer of ICEs in E. coli. In addition, PcTs inhibitors potentiated CFX and reduced the acquisition of CFX resistance in a neutrapenic murine bacterial thigh infection model. This study highlights usefulness of including RecA inhibitors in antibacterial therapies and provides a much needed strategy for prolonging antibiotic shelf life.

Cation-pi interactions in aromatics of biological and medicinal interest: electrostatic potential surfaces as a useful qualitative guide.

Previously, attempts have been made to obtain a structure of the post-ATP hydrolysis conformation of the RecA homolog MvRadA by co-crystallizing it in the presence of ADP and the phosphate analog sodium tungstate (NaWO) (). A cluster of 12 tungsten atoms have been located near cationic DNA-binding loops. The highly anionic metatungstate (W) compound inhibited MvRadA ATPase, DNA-binding, and DNA strand-exchange activities (). This study shows that drug-sized anionic molecules competitively block DNA binding by RecA-like protein filaments. However, metatungstate is unable to inhibit RecA activity within bacteria.

Based on these studies, we investigated whether inhibiting RecA would prevent induction of SOS response and block antibiotic-induced DNA repair and mutagenesis and horizontal gene transfer pathways. Several compounds have been discovered that inhibit RecA ATPase activity in vitro, including polysulfonated naphthylurea (), metal cations (), nucleotide analogs (), polysulfated naphthyl compounds (), small organic molecules based on 2-amino-4,6-diarylpyridine, 1,2,4-oxadiazole, quinaxolinone, and benzimidazole diazepinone scaffolds (), and α-helical peptides (). In addition, the natural phenol (curcumin) has been reported to inhibit the SOS response induced by levofloxacin (). However, no studies have been reported that characterize the in vivo activity of RecA inhibitors or their ability to decrease the evolution of antibiotic resistance.

A molecular target for suppression of the evolution of antibiotic resistance: inhibition of the Escherichia coli RecA protein by N(6)-(1-naphthyl)-ADP.

Suramin is a potent and selective inhibitor of Mycobacterium tuberculosis RecA protein and the SOS response: RecA as a potential target for antibacterial drug discovery.

Bactericidal antibiotics are powerful instigators of the SOS response (). They induce a common mechanism of cell death by stimulating the formation of lethal amounts oxidative radicals (), which activates RecA and SOS response (). Escherichia coli strains lacking RecA are more sensitive to bactericidal antibiotics (). Thus, RecA is essential for increased tolerance to antibiotic treatment by enhancing repair of DNA damage that occurs either directly by antibiotic-induced DNA damage or indirectly from metabolic and oxidative stress. RecA-mediated repair also induces a hypermutable state that promotes acquisition of antibiotic resistance. If DNA damage is not successfully repaired, then mutagenic polymerases (PolIV and PolV) are induced, causing mutagenesis to occur and enabling bacteria to develop antibiotic resistance (). Bacteria can also develop antibiotic resistance by obtaining resistance genes from foreign DNA using the SOS response-mediated horizontal gene transfer pathway (). Mobile genetic elements, including conjugative plasmids and integrating conjugative elements (ICEs), are key mediators for obtaining antibiotic resistance genes ().

Bacteria have a remarkable ability to acquire resistance against antibiotics by several mechanisms, including target modification, target overexpression, multidrug transporter expression, and horizontal gene transfer (). New strategies are needed to block development of resistance and to prolong the life of antibiotics. Recent studies suggest that adaptive resistance mutations and acquisition of resistance genes are induced by antibiotic therapy in bacteria (), and caused by activation of RecA () and the SOS DNA repair and mutagenesis pathway (). The SOS pathway is initiated through the activation of RecA, which in turn inactivates the LexA repressor and induces SOS response genes, including SOS error prone polymerases (). RecA is involved in DNA repair, induction of SOS response, horizontal gene transfer, and biofilm formation (), and is a promising target for developing therapeutics to reduce the acquisition of antibiotic resistance.

To establish whether PcTs-based RecA inhibitors can inhibit the acquisition of CFX resistance in vivo, we assayed the activity of Fe-PcTs in a neutrapenic murine bacterial thigh infection model (). Mice were rendered neutrapenic by intraperitoneal (i.p.) injection of cyclophosphamide. Mice thighs were then infected with ATCC25922 cells. Two hours after infection, mice were administered subcutaneous (s.c.) injections of CFX or CFX and Fe-PcTs every 24 hr up to 72 hr. At 48 and 72 hr post infection, three mice from each group were killed and their thighs removed and homogenized to determine the number of viable cells for both CFX-sensitive and CFX-resistant ATCC25922 cells. Approximately 50,000 CFX-resistant cells were observed after 72 hr infection when the mice were only treated with CFX. Remarkably, no CFX-resistant cells were observed when mice were co-treated with CFX and Fe-PcTs ( Figure 5 D). Pretreatment of mice with Fe-PcTs before infection potentiated the activity of CFX more than when mice were only co-treated with CFX and Fe-PcTs shortly after infection ( Figure 5 D). No CFX-resistant colonies were observed in any mice treated with Fe-PcTs.

Neutropenia induced in outbred mice by a simplified low-dose cyclophosphamide regimen: characterization and applicability to diverse experimental models of infectious diseases.

Fe-PcTs and CFX treatment reduced the total number of viable and CFX-resistant ATCC25922 cells relative to CFX treatment alone ( Figures 5 A and 5B ). Since Fe-PcTs potentiated the activity of CFX, there were fewer cells present that could acquire resistance mutations. To account for the decreased viability in cells treated with Fe-PcTs and CFX, we defined the CFX mutation rate as CFX-resistant colonies per viable cell per day ( Figure 5 C) as described previously (). For ATCC25922 cells treated with Fe-PcTs and CFX, no CFX-resistant mutants were observed after day 4. In contrast, ATCC25922 cells treated with only CFX showed an increase in CFX-resistant cells at day 5. These results highlight the ability of Fe-PcTs to inhibit the acquisition of CFX resistance mutation in an in vitro mutagenesis assay.

(D) In vivo analysis of Fe-PcTs activity in neutrapenic mouse bacterial infection model. Mice were infected with ATCC25922 cells and treated with CFX or CFX and Fe-PcTs. Fe-PcTs was either administered 24 hr before CFX treatment (pre-) or co-administered with CFX. Mice were killed at 48 and 72 hr and ATCC25922 cells from mice thighs were cultured on LB plates with or without CFX (40 nM) to determine the number of CFX-sensitive (dashed lines) and CFX-resistant (solid lines) cells. Error bars represent SD from five independent experiments.

(C) The number of CFX-resistant cells per viable cell per day in the presence and absence of Fe-PcTs. Error bars represent the SD from three independent experiments.

(B) Viable ATTC29522 cells present on LB plates containing CFX or CFX and Fe-PcTs per day.

(A) CFX-resistant ATTC29522 CFUs obtained in the presence and absence of Fe-PcTs per day.

ATTC29522 cells (3 × 10 8 ) were cultured on LB plates containing CFX (40 nM) with or without Fe-PcTs (25 μM).

Fe-PcTs Potentiates the Activity of CFX and Reduces the Acquisition of CFX Resistance

Figure 5 Fe-PcTs Potentiates the Activity of CFX and Reduces the Acquisition of CFX Resistance

Using this in vitro mutagenesis and reconstruction assays, we calculated that 29% and 60% of the CFX-resistant ATCC25922 colonies isolated were caused by pre-existing mutations in the CFX-alone and CFX and Fe-PcTs assays, respectively ( Table 1 ). These results are consistent with the ability of Fe-PcTs to reduce the number of acquired resistance mutations in response to CFX exposure.

We used an in vitro mutagenesis assay () to measure the ability of Fe-PcTs to block the acquisition of CFX-induced resistance in ATCC25922 E. coli. ATCC25922 cells were plated on media containing CFX in the presence or absence of Fe-PcTs and cultured for 10 days. Colonies that appeared early in the assay (days 1 and 2) are believed to arise from pre-existing CFX resistance mutations (). Colonies that appear later in the incubation (days 3–8) are due to CFX resistance mutations acquired during exposure to CFX or pre-existing mutations with a slow growth phenotype (). To distinguish between these two types of mutants, we used a reconstruction assay that tests for the time it takes for colonies to appear in the presence of CFX. Colonies that appear at least 2 days faster than they appeared in the original CFX mutagenesis screen were classified as colonies that acquire resistance after exposure to CFX (). Colonies that appeared in the same number of days as in the CFX resistance assay were classified as colonies with pre-existing mutations ().

RecA-mediated recombination can incorporate a foreign DNA segment containing an antibiotic-resistant gene derived from exogenous homologous DNA into the host genome through horizontal gene transfer (). ATPase-dependent activity of RecA is necessary for recombinational DNA repair () and horizontal gene transfer (). To test whether PcTs-based RecA inhibitors reduce the frequency of horizontal transfer in bacteria, we used a conjugation assay with the SXT mobile genetic element () or the R388 plasmid (). Treatment of E. coli strains VB82 (donor) and VB38 (recipient) with CFX and Fe-PcTs caused a 10-fold reduction in SXT transfer ( Figure 4 C). In contrast, Fe-PcTs did not have any effect on the transfer of the R388 plasmid from the donor MG1655 strain to the recipient 7651 strain ( Figure 4 C).

To confirm that Fe-PcTs interfered with bactericidal antibiotic induction of the SOS response, we characterized the ability of Fe-PcTs to reduce CFX-mediated in vitro bacteria filamentation and biofilm formation; two biological processes regulated by the SOS response (). Induction of the SOS response leads to increased levels of SulA, a cell division inhibitor (), and bacteria filamentation. SOS regulators, RecA and LexA, have been shown to be involved in regulating biofilm formation caused by DNA-damaging agents (). Consistent with the ability of Fe-PcTs to block CFX-induced SOS response, Fe-PcTs reduced the ability of CFX to induce in vitro filamentation ( Figure 4 A ) and biofilm formation ( Figure 4 B) in E. coli (ATCC25922).

Error bars in (B, C) represent the SD from three independent experiments where *p < 0.05 and ** p < 0.01.

(C) Inhibition of conjugational gene transfer by Fe-PcTs. (i) Conjugational transfer of mobile genetic elements (SXT) between donor (VB82) and recipient (VB38) strains. (ii) Conjugational transfer of R388 plasmid, between donor (MG1655) and recipient (7651) strains. Conjugation experiments were carried out on LB agar plates containing 10 g/l NaCl with or without 50 μM Fe-PcTs. Conjugation rate was calculated as conjugants observed per recipient cell.

(B) Inhibition of CFX-induced biofilm formation of ATCC25922 on LB plates by Fe-PcTs (25 μM) with or without CFX (40 nM).

(A) Inhibition of CFX-induced E. coli filamentation by Fe-PcTs. ATCC25922 cells were treated with CFX (40 nM) and/or Fe-PcTs (25 μM) for 3 hr. Cells were then imaged at 100× magnification after Gram staining.

Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation.

Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation.

Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation.

Bactericidal antibiotics increase hydroxyl radical formation (), which causes damage to proteins, lipids, and DNA (), and induces the SOS response (). In contrast, bacteriostatic antibiotics do not induce hydroxyl radical production or the SOS response (). To confirm that the bactericidal-potentiating activity of PcTs-based RecA inhibitors was correlated with reduced SOS response, we monitored antibiotic-mediated SOS induction in the E. coli strain SS996. This strain contains a GFP gene regulated by the sulAp SOS response promoter (). Fe-PcTs reduced the ability of CFX and AMP to induce the SOS response ( Figure 3 B). KAN treatment did not induce the SOS GFP reporter ( Figure 3 B). Similar results have been reported previously and are attributed to the inhibition of translation by KAN (), which would block GFP expression. In contract, bacteriostatic antibiotics CAM, TET, and SPECT only induced very low level of SOS response, which was blocked by Fe-PcTs ( Figure 3 C).

To determine the range of antibiotic potentiation activities of PcTs-based RecA inhibitors, we assayed the activity of Fe-PcTs with bactericidal and bacteriostatic antibiotics. We co-treated ATCC25922 with Fe-PcTs and bactericidal antibiotics CFX, ampicillin (AMP), and kanamycin (KAN), which are members of the quinolone, β-lactam, and aminoglycoside families, respectively, and monitored cell viability. Fe-PcTs potentiated the activity of CFX, KAN, and AMP, eliminating all CFUs after 24 hr ( Figure 3 A ). In contrast, Fe-PcTs had a very slight effect on the activity of the bacteriostatic antibiotics chloramphenicol (CAM), tetracycline (TET), and spectinomycin (SPECT) ( Figure 3 A), which are members of the cephalosporins, tetracycline, and aminocyclitol families, respectively.

(C) SOS response induction in E. coli treated with Fe-PcTs and bacteriostatic antibiotics. GFP expression was measured using flow cytometry 3 hr after addition of TET (21 μM), CAM (46 μM), or SPECT (808 μM) in the presence or absence of Fe-PcTs (25 μM).

(B) SOS response induction in E. coli treated with Fe-PcTs and bactericidal antibiotics. SOS response was monitored using an E. coli strain (SS996) engineered to express GFP under the control of the LexA-regulated sulAp promoter. GFP expression was measured using flow cytometry 3 hr after addition of CFX (2.5 μM), AMP (40 μM), or KAN (43 μM) in the presence or absence of Fe-PcTs (25 μM).

(A) Fe-PcTs activity with bactericidal and bacteriostatic antibiotics. Survival of E. coli ATCC25922 cells treated with Fe-PcTs (25 μM) and the following bactericidal antibiotics: (i) CFX (40 nM), (ii) KAN (43 μM), and (iii) AMP (40 μM), or bacteriostatic antibiotics: (iv) CAM (46 μM), (v) TET (21 μM), and (vi) SPECT (808 μM). Cells were untreated (No treatment), treated with Fe-PcTs (25 μM), treated with indicated antibiotic, or treated with Fe-PcTs (25 μM) and indicated antibiotic. CFUs/ml were determined at indicated time points. Errors bars represent the SD from three independent experiments.

To confirm that the activity of Fe-PcTs was not specific to the Gram-negative ATCC25922 E. coli, we evaluated the ability of Fe-PcTs to potentiate the activity of CFX in another Gram-negative strain, Pseudomonas aeruginosa (ATCC27853), and two Gram-positive strains, Staphylococcus aureus (ATCC29213) and Enterococcus faecalis (ATCC29212). Fe-PcTs potentiated the activity of CFX in Gram-negative and Gram-positive bacteria strains ( Figure 2 C). ATCC27853 was less sensitive to CFX than the two Gram-positive strains and thus we did not see complete killing of these bacteria when treated with CFX and Fe-PcTs.

To determine the importance of the metal ion chelated to PcTs, we evaluated the CFX potentiating activity of the following PcTs molecules: phthalocyanine tetrasulfonic acid, aluminum(III) phthalocyanine tetrasulfonic acid, zinc(II) phthalocyanine tetrasulfonic acid, nickel(II) phthalocyanine tetrasulfonic acid, and copper phthalocyanine tetrasulfonic acid. These PcTs molecules consist of mixtures of different sulfonic acid regioisomers. All of these PcTs molecules potentiated the activity of CFX at similar levels, decreasing CFUs by ∼100-fold ( Figure 2 B). This result showed that CFX potentiating activities of PcTs molecules, which contain a mixture of sulfonic acid regioisomers, were not influenced by the identity of the chelated metal ion ( Figure 2 B). Since PcTs molecules containing sulfonic acid at fixed positions showed the highest CFX potentiating activity, we choose to further characterize Fe-PcTs.

To determine the importance of the sulfonic acid position on the activity of 3,4-Cu-PcTs, we compared CFX potentiating activities of 3,4-Cu-PcTs to Cu-PcTs, which contains a mixture of sulfonic acid regioisomers. Treatment of ATCC25922 cells with Cu-PcTs and CFX caused an ∼100-fold decrease in CFUs relative to CFX treatment alone, which was substantially lower than 3,4-Cu-PcTs, where no CFUs were observed ( Figure 2 B). This highlighted the importance of the sulfonic acid position on the CFX potentiating activity of PcTs-based RecA inhibitors.

We evaluated the ability of Fe-PcTs and 3,4-Cu-PcTs to potentiate the activity of CFX using the pathogenic E. coli strain ATCC25922, which has been used previously byto characterize the role of SOS response proteins in antibiotic resistance. CFX inhibits DNA gyrase in bacteria, causing accumulation of dsDNA breaks and inducing the SOS response (). Fe-PcTs and 3,4-Cu-PcTs potentiated the activity of CFX and no colony-forming units (CFUs) were observed when ATCC25922 cells were co-treated with CFX and Fe-PcTs or 3,4-Cu-PcTs at concentrations above 25 μM ( Figure 2 A ).

(C) Fe-PcTs activity against Gram-negative and Gram-positive bacteria. (i) Potentiation of CFX activity by Fe-PcTs in Pseudomonas aeruginosa ATCC27853 (Gram-negative). Cells were untreated (No treatment), treated with Fe-PcTs (25 μM), treated with CFX (6.5 μM), or treated with CFX (6.5 μM) and Fe-PcTs (25 μM). CFUs/ml was determined at indicated time points. (ii) Potentiation of CFX activity by Fe-PcTs in E. faecalis ATCC29212 (Gram-positive). Treatments were the same as in (i). (iii) Potentiation of CFX activity by Fe-PcTs in S. aureus ATCC29213 (Gram-positive). Treatments were the same as in (i). Errors bars represent the SD from three independent experiments.

(B) Comparison of PcTs-chelated metal ions on CFX potentiation. ATCC25922 cells were treated with CFX (40 nM) or CFX plus indicated PcTs molecules (25 μM). CFUs/ml was determined at indicated time points.

(A) Potentiation of CFX activity by (i) Fe-PcTs and (ii) 3,4-Cu-PcTs. ATCC25922 Gram-negative cells were treated with CFX (40 nM) or CFX plus indicated concentrations of Fe-PcTs or 3,4-Cu-PcTs. CFUs/ml were determined at indicated time points.

In principle, RecA can be inhibited by targeting three functionally important processes: recruitment and polymerization, ATP binding, and DNA binding. These processes are connected by allosteric regulatory mechanisms and are difficult to differentiate biochemically from each other. To confirm that Fe-PcTs and 3,4-Cu-PcTs do not solely inhibit RecA ATPase activity, we evaluated the ability of these PcTs molecules to inhibit RecA DNA-binding, DNA stand-exchange, and LexA cleavage activities. Fe-PcTs and 3,4-Cu-PcTs inhibited double-stranded DNA (dsDNA)-binding and single-stranded DNA (ssDNA)-binding activity in the micromolar range, with Fe-PcTs showing slightly higher activity ( Figure 1 C). Fe-PcTs and 3,4-Cu-PcTs also inhibited DNA strand-exchange ( Figure 1 D) and LexA cleavage assays ( Figure 1 E). Both DNA strand-exchange and LexA cleavage assays were carried out in the presence of ATPγS, an analog known to stabilize the RecA-DNA complex better than ATP (). In agreement with this, we observed that higher concentrations (80–100 μM) of Fe-PcTs or 3,4-Cu-PcTs were needed to inhibit DNA strand-exchange and LexA cleavage activities of RecA compared with RecA dsDNA- and ssDNA-binding activities. These observations suggest that Fe-PcTs and 3,4-Cu-PcTs competed with DNA binding in a manner similar to the metatungstate MvRadA inhibitor ().

Previously it was shown that metatungstate, a drug-sized, anionic molecule, competitively blocks DNA-binding by RecA-like protein filaments (). The hexa-anionic charge on metatungstate in solution shows the importance of anionic charge for interacting with the cationic surface of the DNA-binding region of RecA-like recombinases. Based on this observation, we used an ATPase assay () to screen a set of commercially available anionic, aromatic molecules for inhibitors of RecA ATPase activity. From this screen, we identified two PcTs molecules, copper phthalocyanine-3,4′,4″,4‴-tetrasulfonic acid (3,4-Cu-PcTs) and iron(III) phthalocyanine-4,4′,4″,4‴-tetrasulfonic acid (Fe-PcTs) ( Figure 1 A ) that inhibited RecA ATPase activity ( Figure 1 B).

(E) Fe-PcTs and 3,4-Cu-PcTs inhibit RecA-stimulated LexA autoproteolysis. Reaction substrate and product, LexA and cleaved LexA (LexA-C), respectively were resolved using SDS-PAGE and protein stained with Coomassie blue.

(D) Fe-PcTs and 3,4-Cu-PcTs inhibit RecA-mediated DNA strand-exchange reaction. Strand-exchange activity was measured by assaying the formation of the slower migrating heteroduplex DNA species (hdDNA) in the presence of increasing concentration of X-PcTs, where X is 3,4-Cu-PcTs or Fe-PcTs. Reaction products were resolved using an agarose gel.

(C) Fe-PcTs and 3,4-Cu-PcTs inhibit RecA (i) dsDNA-binding and (ii) ssDNA-binding. RecA-bound dsDNA or ssDNA (slower migrating DNA) and free dsDNA and ssDNA were resolved using an agarose gel.

(B) Fe-PcTs and 3,4-Cu-PcTs inhibition of poly-(dT)-36-stimulated RecA ATPase activity. ATPase activity was measured by monitoring the release of inorganic phosphate using a malachite green phosphate detection assay. Percentage of ATPase activity is reported relative to the reaction in the absence of RecA inhibitor. DltA was used as a negative control to show that the 3,4-Cu-PcTs and Fe-PcTs were specific for inhibiting RecA ATPase activity. Errors bars represent the SD from three independent experiments.

(A) Structures of PcTs-based RecA inhibitors: Fe-PcTs, 3,4-Cu-PcTs, and X-PcTs molecules, consisting of mixtures of different sulfonic acid regioisomers (X represents Al, Zn, Ni, Cu, or H).

Discussion

Cirz et al., 2005 Cirz R.T.

Chin J.K.

Andes D.R.

de Crécy-Lagard V.

Craig W.A.

Romesberg F.E. Inhibition of mutation and combating the evolution of antibiotic resistance. Riesenfeld et al., 1997 Riesenfeld C.

Everett M.

Piddock L.J.

Hall B.G. Adaptive mutations produce resistance to ciprofloxacin. Smith and Romesberg, 2007 Smith P.A.

Romesberg F.E. Combating bacteria and drug resistance by inhibiting mechanisms of persistence and adaptation. McKenzie et al., 2000 McKenzie G.J.

Harris R.S.

Lee P.L.

Rosenberg S.M. The SOS response regulates adaptive mutation. The main classes of antibiotics used in the clinic target a limited number of proteins that are involved in bacterial cell wall biosynthesis, protein synthesis, and DNA replication and repair (). The inevitability of resistance against these antibiotics is well accepted and has been shown to occur by a variety of mechanisms (). Strategies to overcome resistance commonly involve chemical modifications to antibiotics; however, many antibiotics are in their third or fourth modification cycle and it is not clear how many more modifications will be permitted before these antibiotics are rendered ineffective (). Clearly there is a need to implement new strategies to prevent antibiotic resistance and prolong the life of new antibiotics.

Cirz et al., 2005 Cirz R.T.

Chin J.K.

Andes D.R.

de Crécy-Lagard V.

Craig W.A.

Romesberg F.E. Inhibition of mutation and combating the evolution of antibiotic resistance. Cirz et al., 2006 Cirz R.T.

Gingles N.

Romesberg F.E. Side effects may include evolution. Kohanski et al., 2007 Kohanski M.A.

Dwyer D.J.

Hayete B.

Lawrence C.A.

Collins J.J. A common mechanism of cellular death induced by bactericidal antibiotics. Kohanski et al., 2007 Kohanski M.A.

Dwyer D.J.

Hayete B.

Lawrence C.A.

Collins J.J. A common mechanism of cellular death induced by bactericidal antibiotics. Bactericidal antibiotic treatment is a powerful investigator of RecA activity (). Furthermore, E. coli strains lacking RecA are much more sensitive to bactericidal antibiotics, confirming the importance of RecA for protecting against effects of antibiotics (). Thus, RecA is essential for increased tolerance to antibiotic treatment by enhancing DNA repair that occurs either directly by antibiotic-induced DNA damage or indirectly from metabolic and oxidative stress. Together, the above studies highlight RecA as a therapeutic target potentiating the activity of antibiotics and for combating the emergence of antibiotic resistance.

Based on previous studies showing that the highly anionic metatungstate inhibits RecA, we identified hydrophobic, anionic PcTs molecules that inhibited RecA ATPase activity. Consistent with this, PcTs-based RecA inhibitors blocked RecA-mediated DNA strand exchange, DNA binding, LexA autoproteolysis, and RecA filament formation. PcTs-based RecA inhibitors also reduced antibiotic-mediated induction of SOS response genes in E. coli and in vitro filamentation and biofilm formation.

Cirz et al., 2006 Cirz R.T.

Gingles N.

Romesberg F.E. Side effects may include evolution. Rymond-Denise and Guillen, 1991 Rymond-Denise A.

Guillen N. Identification of dinR, a DNA damage-inducible regulator gene of Bacillus subtilis. Karlin and Brocchieri, 1996 Karlin S.

Brocchieri L. Evolutionary conservation of RecA genes in relation to protein structure and function. Schlacher et al., 2006 Schlacher K.

Pham P.

Cox M.M.

Goodman M.F. Roles of DNA polymerase V and RecA protein in SOS damage-induced mutation. PcTs-based RecA inhibitors specifically potentiated the activity of bactericidal antibiotics and not bacteriostatic antibiotics, which correlates with the differential ability of these classes of antibiotics to activate the SOS response (). PcTs inhibitors potentiated the activity of the bactericidal antibiotic CFX in both Gram-negative and Gram-positive bacteria, which is consistent with active SOS response pathways in both species (). RecA-like proteins are nearly ubiquitous and highly conserved in bacterial species (), with the only known exceptions occurring in bacterial species undergoing genome degeneration as part of an adaptation to an endosymbiotic lifestyle (). Our results suggest the PcTs-based RecA inhibitors should be useful with a broad spectrum of bactericidal antibiotics in Gram-negative or Gram-positive bacteria.

Cirz et al., 2005 Cirz R.T.

Chin J.K.

Andes D.R.

de Crécy-Lagard V.

Craig W.A.

Romesberg F.E. Inhibition of mutation and combating the evolution of antibiotic resistance. Cirz et al. (2005) Cirz R.T.

Chin J.K.

Andes D.R.

de Crécy-Lagard V.

Craig W.A.

Romesberg F.E. Inhibition of mutation and combating the evolution of antibiotic resistance. Beaber et al., 2004 Beaber J.W.

Hochhut B.

Waldor M.K. SOS response promotes horizontal dissemination of antibiotic resistance genes. Prudhomme et al., 2006 Prudhomme M.

Attaiech L.

Sanchez G.

Martin B.

Clayerys J.P. Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae. The repair of antibiotic-induced DNA damage by the SOS response promotes mutagenesis and transmission of antibiotic resistance genes. The acquisition of resistance-conferring mutations requires the formation of an RecA/ssDNA filament and the cleavage of LexA (SOS repressor), which eventually leads to derepression of error prone polymerases (PolII, PolIV, and PolV) ().showed that E. coli containing a mutant LexA, which does not undergo autoproteolytic cleavage, or a deletion of RecA reduces the emergence of CFX resistance in vitro and in a murine thigh infection model. SOS response also promotes antibiotic resistance by enhancing horizontal gene transfer. Antibiotic-induced competence factors enhance uptake of exogenous DNA, which are integrated into the bacterial genome by the recombinational activity of RecA ().

Cirz et al., 2005 Cirz R.T.

Chin J.K.

Andes D.R.

de Crécy-Lagard V.

Craig W.A.

Romesberg F.E. Inhibition of mutation and combating the evolution of antibiotic resistance. Using an in vitro resistance assay, we showed that Fe-PcTs suppressed the acquisition of CFX resistance in the pathogenic E. coli ATCC25922 treated with CFX. These results are comparable with the decrease in CFX-induced resistance observed in genetic mutations of SOS response and DNA repair genes (). The in vivo resistance assay confirmed that Fe-PcTs attenuated the acquisition of CFX resistance in a neutrapenic murine thigh bacterial infection, where no CFX-resistant cells were observed when mice were co-treated with CFX and Fe-PcTs.

Capaldo and Barbour, 1975 Capaldo F.N.

Barbour S.D. DNA content, synthesis and integrity in dividing and nondividing cells of rec- strains of Escherichia coli K12. Mushegian and Koonin, 1996 Mushegian A.R.

Koonin E.V. A minimal gene set for cellular life derived by comparison of complete bacterial genomes. In contrast to existing antibiotic combinations aimed at blocking resistance such as Augmentin, which consists of a β-lactam and a β-lactamase inhibitor, PcTs-based RecA inhibitors can be combined with a wide range of bactericidal antibiotics, providing a general strategy for constructing anti-resistance antibiotic combinations. PcTs-based RecA inhibitors may also function as a primary antibiotic under specific growth conditions. On rich media, growth of bacteria lacking RecA is not inhibited. However, the absence of the RecA gene significantly impairs the fitness of bacteria () and it has been identified as an essential gene ().

In summary, PcTs-based RecA inhibitors can function as an adjuvant to a range of bactericidal antibiotics, which will potentiate their activity and prolong their lifespan by reducing acquisition of adaptive de novo resistance mutations, or transfer of DNA encoding antibiotic resistance genes between bacterial species.