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H2S: A Universal Defense Against Antibiotics in Bacteria

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Science  18 Nov 2011:
Vol. 334, Issue 6058, pp. 986-990
DOI: 10.1126/science.1209855

Abstract

Many prokaryotic species generate hydrogen sulfide (H2S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine β-synthase, cystathionine γ-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H2S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H2S suppresses this effect. Moreover, in bacteria that normally produce H2S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics.

Until recently H2S has been known merely as a toxic gas. It is now associated with beneficial functions in mammals from vasorelaxation, cardioprotection, and neurotransmission to anti-inflammatory action in the gastrointestinal tract (13). The ability of H2S to function as a signaling molecule parallels the action of another established gasotransmitter, nitric oxide (NO) (35). Like NO, H2S is produced enzymatically in various tissues (13). Three H2S-generating enzymes have been characterized in mammals: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). CBS and CSE produce H2S predominantly from L-cyst(e)ine (Cys). 3MST does so via the intermediate synthesis of 3-mercaptopyruvate produced by cysteine aminotranferase (CAT), which is inhibited by aspartate (Asp) competition for Cys on CAT (1) (fig. S1).

In contrast to mammal-derived H2S, bacteria-derived H2S has been known for centuries but was considered to be only a byproduct of sulfur metabolism, with no particular physiological function in nonsulfur microorganisms. Likewise, little is known about the metabolic pathways involving H2S in mesophilic bacteria. However, analysis of bacterial genomes has revealed that most, if not all, have orthologs of mammalian CBS, CSE, or 3MST (figs. S1 and S2), which suggested an important cellular function(s) that preserved these genes throughout bacterial evolution. We became interested in the role of these enzymes after establishing that endogenous NO protects certain Gram-positive bacteria against antibiotics and oxidative stress (68). Considering some functional similarities between mammalian gasotransmitters (13), we hypothesized that bacterial H2S may, similarly, be cytoprotective.

To determine whether CBS, CSE, or 3MST produces H2S in bacteria, we inactivated each enzyme genetically or chemically in four clinically relevant and evolutionarily distant pathogenic species: Bacillus anthracis (Sterne), Pseudomonas aeruginosa (PA14), Staphylococcus aureus (MSSA RN4220 and MRSA MW2), and Escherichia coli (MG1655). The first three species have the CBS/CSE operon, but not 3MST, whereas E. coli carries 3MST, but not CBS/CSE. The chromosomal organization of H2S genes (fig. S3) and the strategy we used for their replacement prevented any polar effects. We monitored H2S production in wild-type (wt) and mutant cells using lead acetate [Pb(Ac)2], which reacts specifically with H2S to form a brown lead sulfide stain. The rate of change of staining on a Pb(Ac)2-soaked paper strip is directly proportional to the concentration of H2S (9). Deletion of CBS/CSE in B. anthracis and P. aerugenosa or 3MST in E. coli greatly decreased or eliminated PbS staining (Fig. 1A). Similar results were obtained when dl-propargylglycine (PAG), amino-oxyacetate (AOAA), or Asp were used, respectively, as specific inhibitors of CSE, CBS, or 3MST (Fig. 1A). Addition of Cys markedly increased PbS staining for all wt, but not CSE-CBS– or 3MST-deficient bacteria (Fig. 1B). In addition, overexpression of the chromosomal 3MST gene from a strong pLtetO-1 promoter in E. coli resulted in increased production of H2S (Fig. 1A). We conclude that all three enzymes produce H2S endogenously from Cys during exponential growth of bacteria in rich media.

Fig. 1

Endogenous H2S protects bacteria against antibiotic toxicity. (A) H2S production by B. anthracis, S. aureus, P. aeruginosa, and E. coli depends on CBS/CSE and 3MST, respectively. Lead acetate–soaked paper strips show a PbS brown or black stain as a result of reaction with H2S. Strips were affixed to the inner wall of a culture tube, above the level of the liquid culture of wt or mutant bacteria, for 18 hours. CBS/CSE and 3MST inhibitors PAG/AOAA (inh) and aspartate (Asp, 3.2 mM), respectively, were added as indicated. Numbers (%) show the relative decrease in H2S production due to chemical or genetic inhibition of CBS/CSE and 3MST. pMST indicates the E. coli strain that expresses an extra copy of the 3MST gene under a strong pLtetO-1 promoter. (B) Cysteine (Cys) is a substrate for bacterial CBS/CSE and 3MST. Addition of Cys (25 μM for E. coli; 200 μM for other species) greatly stimulated H2S synthesis in wt, but not in CBS/CSE- or 3MST-deficient strains. (C) H2S suppresses antibiotic-mediated bacterial killing. Representative survival curves show the effect of CBS/CSE (B. anthracis) and 3MST (E. coli) deletions or CBS/CSE inhibition (S. aureus and P. aeruginosa) by PAG/AOAA (inh) on Gm-mediated (50 μg/ml) killing. Where indicated, NaHS (0.2 mM) was added before the antibiotic challenge (see Materials and Methods). The percentage of surviving cells was determined by counting colony-forming units (CFU) and is shown as the mean ± SD from three experiments.

To elucidate the physiological role of H2S, we first compared wt and 3MST-deficient E. coli in a phenotype microarray (PMA) (fig. S4 and table S1). Whereas these strains showed little or no growth defects (fig. S5), a large number of antibiotics, highly diverse in structure and function, preferentially suppressed growth of 3MST-deficient cells (Table 1 and table S1). The killing and growth curves obtained for wt and 3MST and CBS/CSE mutant E. coli, P. aerugenosa, S. aureus, and B. anthracis in the presence of several representative antibiotics confirmed the results of the screen and generalized them to both Gram-positive and -negative species (Fig. 1C and figs. S6 and S7). 3MST overexpression resulted in increased resistance to spectinomycin (fig. S6A), whereas chemical inhibition of CBS, CSE, or 3MST rendered bacteria more sensitive to different antibiotics (Fig. 1C and fig. S6B). An H2S donor, NaHS, suppressed the antibiotic sensitivity of CBS-CSE– and 3MST-deficient cells (Fig. 1C and figs. S6C and S7). Taken together, these results establish that endogenously produced H2S confers multidrug resistance.

Table 1

3MST protects E. coli against different classes of antibiotics. A representative list of chemicals from the Phenotype MicroArray that preferentially suppressed the growth of 3MST-deficient cells (ΔsseA). Major classes of antibiotics are indicated by type (column 4). Negative numbers indicate the relative growth inhibition of the 3MST-deficient strain compared with that of the wt strain (as provided by Biolog Inc.) (fig. S4). The minimum inhibitory concentration drop for ΔsseA, as determined by Biolog, for two representative antibiotics, norfloxacin and troleandomycin, is 12- and 7-fold, respectively.

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H2S-mediated cytoprotection resembles that of NO, which defends certain Gram-positive bacteria against some of the same antibiotics as does H2S (8). NO-mediated protection relies, in part, on its ability to defend bacteria against oxidative stress imposed by antibiotics (68). To examine whether H2S acts by a similar mechanism, we performed detailed analyses of its effect on bacterial killing by the representative antibiotics, gentamicin (Gm), ampicillin (Ap), and nalidixic acid (NA) (Fig. 2). All three have been shown to exert their bactericidal effect via oxidative stress (8, 10). Indeed, pretreatment of cells with 2,2′-dipyridyl, an iron chelator that suppresses the damaging Fenton reaction (11), or the hydroxyl radical scavenger thiourea, substantially decreased the toxicity of Gm (Fig. 2A). Note that wt and H2S-deficient cells became equally resistant to Gm in the presence of dipyridyl or thiourea (Fig. 2A). Moreover, the H2S donor added together with Gm was as effective as dipyridyl or thiourea in protecting against antibiotics but failed to further protect cells that had already been pretreated with antioxidants (Fig. 2A). Thus, H2S, like NO, acts by suppressing the oxidative component of antibiotic toxicity. Consistently, H2S-generating enzymes provided protection against antibiotics only under aerobic conditions. Anaerobically grown CBS/CSE-deficient B. anthracis cells were as resistant to NA or pyocyanin as wt bacteria (Fig. 2B and fig. S8).

Fig. 2

H2S protects against antibiotic-inflicted oxidative damage (A) H2S acts by diminishing reactive oxygen species (ROS)–mediated antibiotic toxicity. E. coli cells were pretreated with the iron chelator, 2,2′-dipyridyl (0.05 mM) or the ROS scavenger thiourea (15 mM) for 3 min, followed by treatment with Gm. Cells were grown in triplicate at 37°C with aeration using a Bioscreen C automated growth analysis system. The curves represent averaged values from three parallel experiments with a margin of error of less than 5%. (B) Endogenous H2S renders cells more resistant to NA in aerobic conditions, but fails to do so in anaerobic conditions. A paper disk saturated with 20 μg/ml NA was placed on wt or CBS/CSE-deficient B. anthracis lawns that were grown aerobically or anaerobically for the next 18 hours. Zone borders are marked with dashed lines. (C) Endogenous H2S renders bacteria resistant to hydrogen peroxide. Agar plates seeded with the indicated bacteria were incubated overnight with a filter paper disk saturated with 0.125 or 0.45 M H2O2 placed atop the bacterial lawn. CBS/CSE- or 3MST-deficient cells formed a clear 5- to 10-mm zone around the disk, whereas wt cells grew a complete lawn and so demonstrated strong H2S-dependent resistance to hydrogen peroxide. (D) Pulsed-field gel analysis of chromosomal DSBs. Lane 1: 4.6 Mb linearized E. coli chromosomes (I-SceI); lanes 2 and 3: DNA from wt and ΔMST cells; lanes 6 to 8: DNA from wt, 3MST-deficient, and 3MST-overproducing cells after treatment with 10 μg/ml Amp; lanes 9 and 10: DNA from NaHS-treated cells after Amp treatment; and lane 11: concatemers from 0.05 to 1.0 Mb. “% linear” indicates the relative increase in linearized chromosomal DNA. The values are the average of three independent experiments (P < 0.1). (E) Stimulating effect of H2S on H2O2 degrading activity and SOD activity in crude extracts of wt and 3MST-deficient E. coli cells. Total H2O2 degrading activity was measured as described in (7). Catalase activity at 100% is 30 mM H2O2 min–1•mg–1. Values shown are the means ± SEM from three experiments. SOD activity was measured using a tetrazolium-based assay kit. (F) Dual protective effect of H2S against oxidative stress: Catalase and SOD are required for prolonged defense against H2O2 toxicity mediated by NaHS but not for immediate protection. Wt, katE, and sodA E. coli cells were grown in Luria-Bertani broth (LB) to absorbance (optical density) OD600 of ~1.0, treated with NaHS (200 μM) for the indicated time intervals (min), followed by the addition of H2O2 (2 mM) for 10 min. Cell survival was determined by counting CFU and is shown as the mean ±SD from three independent experiments.

The above results suggested that H2S bolsters the antioxidant capacity of bacterial cells. Indeed, H2S-deficient B. anthracis, E. coli, S. aureus, and P. aeruginosa displayed higher susceptibility to peroxide than their wt counterparts, whereas NaHS rendered them more resistant to the peroxide (Fig. 2C and figs. S9 to S11).

Formation of double-strand DNA breaks (DSBs) is the primary cause of bacterial death from peroxide (12, 13). These DSBs result from the Fenton reaction (14), which can also be triggered by antibiotics (8, 10, 15, 16). To examine whether H2S protects bacteria from the damaging Fenton reaction, we monitored chromosomal DNA integrity by pulsed-field gel electrophoresis (PFGE) (Fig. 2D). The intact E. coli chromosome does not migrate into the agarose gel but remains at the origin (17), whereas linear chromosomes containing a single DSB migrate as a 4.6-Mb species (Fig. 2D, lane 1). Absent antibiotic or H2O2, DNA isolated from wt or H2S-deficient cells was retained almost entirely at the origin (lanes 2 and 3). However, treatment of cells with a sublethal dose of H2O2 or ampicillin resulted in a greater linearization (DSBs) of the chromosome in 3MST-deficient cells (lanes 4 and 6). Overexpression of 3MST suppressed this linearization (lane 8), as did treatment with NaHS (lanes 9 and 10). These results were corroborated by polymerase chain reaction (PCR) analysis of B. anthracis, E. coli, and P. aeruginosa genomic DSBs as a function of H2S production (fig. S12) and further supported by the ability of H2S to suppress the Gm-induced SOS response (fig. S13). Taken together, these results directly implicate endogenous H2S in the mitigation of chromosomal damage inflicted by antibiotics.

The antioxidant effect of endogenous H2S can also be explained, in part, by its ability to augment the activities of catalase and superoxide dismutase (SOD) (Fig. 2E). The rate of H2O2 degradation in crude extracts of wt E. coli cells was >1.5 times that of 3MST-deficient cells and was increased further in cells that overexpressed 3MST (Fig. 2E). SOD activity was also proportional to the level of 3MST expression (Fig. 2E).

Thus, H2S increases bacteria resistance to oxidative stress and antibiotics by a dual mechanism (fig. S14) of suppressing the DNA-damaging Fenton reaction via Fe2+ sequestration (Fig. 2A and figs. S10 and S15) and stimulating the major antioxidant enzymes catalase and SOD (Fig. 2E). The latter is essential for long-term protection but is less important during the first moments of oxidative stress. Indeed, katE and sodA E. coli mutants are well protected by NaHS during the first minutes of H2O2 exposure but then quickly loose viability (Fig. 2F).

This cytoprotective mechanism of H2S parallels that of NO (8), which suggests that bacteria that produce both gases may benefit from their synergistic action. To test this hypothesis, we examined the effect of simultaneously inhibiting H2S and NO on B. anthracis growth. We were unable to generate a strain of B. anthracis in which both bacterial nitric oxide synthase (bNOS) and CBS/CSE were genetically inactivated, which suggested that the absence of both gases is incompatible with B. anthracis survival. Indeed, B. anthracis Δnos cells containing an isopropyl-β-d-thiogalactopyranoside (IPTG)–inducible CBS/CSE conditional knockout could grow only in the presence of IPTG (fig. S16). Notably, the amount of NO produced in H2S-deficient cells or the amount of H2S produced in NO-deficient cells was greater than that produced in wt cells (Fig. 3A), which indicated that one gas compensates for the lack of the other. Also, the activity of both CBS/CSE and bNOS was stimulated in response to antibiotics (Fig. 3A). Moreover, H2O2 and antibiotics (e.g., erythromycin) substantially induced CBS/CSE gene expression (Fig. 3B) and H2S production (Fig. 3B and fig. S17). Furthermore, chemical inhibition of CBS/CSE in bNOS-deficient cells or inhibition of bNOS in CBS/CSE-deficient cells sensitized B. anthracis to antibiotics to a much greater extent than did each mutation alone (Fig. 3C). These results in concert with our previous study (8) demonstrate the synergistic and specific protective effects of H2S and NO against antibiotics. Notably, in contrast to bNOS, which is present in only a small number of Gram-positive species (18), H2S enzymes are essentially universal (fig. S1). Because H2S equilibrates rapidly across cell membranes, a fraction of cells that generate this gas in culture or in biofilms could, in principle, defend the entire population. Indeed, wt E. coli cells effectively protect 3MST-deficient cells from Gm toxicity in exponentially growing coculture (fig. S18).

Fig. 3

Synergistic action of H2S and NO in B. anthracis. (A) Compensatory induction of endogenous H2S and NO. In vivo production of NO in response to deletion of CBS/CSE or cefuroxime (Cef) (20 μg/ml) challenge was detected using the Cu(II)-based NO fluorescent sensor (CuFL) (19) (left bars). Cells were grown in LB to OD600 of ~0.5 followed by addition of freshly prepared CuFL (20 μM) and Cef. Fluorescence was measured in the total culture after 18 hours of incubation using a real-time fluorometer (PerkinElmer LS-55). H2S was measured using Pb(Ac)2 as in Fig. 1A (right bars). (B) H2S induction in response to antibiotic (erythromycin) or H2O2 challenge. The plot shows β-galactosidase activity (Miller units) expressed by B. anthracis cells harboring a chromosomal transcriptional fusion of the cbs/cse promoter and leader region to a promoterless lacZ gene. Bacteria were grown in LB medium until OD600 of ~0.6 followed by the addition of 0.5 μg/ml erythromycin or 2 mM H2O2. The bottom panel shows PbS brown or black stain, which is proportional to the amount of H2S produced. B. anthracis cells were grown in 96-well plates in LB + Cys (200 μM) covered with lead acetate–soaked paper by using a Bioscreen C automated growth analysis system. (C) Representative OD growth curves of wt (black curves), CBS/CSE-deficient (red) or bNOS-deficient (green) B. anthracis (Sterne) cells. Acriflavine, PAG/AOAA, NaSH, and the NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) were added as indicated. Cells were grown in triplicate at 37°C with aeration using a Bioscreen C automated growth analysis system. The curves represent the averaged values (P < 0.05).

Because endogenous H2S diminishes the effectiveness of many clinically used antibiotics, the inhibition of this “gaskeeper” should be considered as an augmentation therapy against a broad range of pathogens. Bacterial CBS, CSE, and 3MST have diverged substantially from their mammalian counterparts (fig. S2), which suggest that it is possible to design specific inhibitors targeting these enzymes.

Supporting Online Material

www.sciencemag.org/cgi/content/full/334/6058/986/DC1

Materials and Methods

SOM Text

Figs. S1 to S20

Table S1

References (2034)

References and Notes

  1. Acknowledgments: We thank E. Avetissova for technical assistance, S. Mashko for materials, and members of the Nudler laboratory for valuable comments and discussion. This research was supported by the NIH Director’s Pioneer Award, Biogerontology Research Foundation, and Dynasty Foundation (E.N.). Provisional patent application has been filed: U.S. Patent No. 61/438,524 “Methods for treating infections by targeting bacterial H2S-producing enzymes.” by K.S. and E.N.
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