Fig 1.
CBS is the key enzyme for H2S production in V. cholerae.
(A). Schematic illustration of the proposed pathways for H2S biogenesis in V. cholerae. CBS, CSE and 3MST are key enzymes in cysteine degradation that are conserved from bacteria to mammals, while NADPH-dependent sulfite reductase (CysJI) catalyzes the last step in assimilatory sulfite reduction. (B). Cysteine degrading enzyme CBS is essential for H2S production. To investigate the contribution of cysteine degradation (cbs, cse, and 3mst) and assimilatory sulfite reduction (cysI and cysJ) to H2S synthesis in V. cholerae, H2S production was detected with Pb(Ac)2 paper strips with (upper) or without (bottom) supplementation of 200 μM L-cysteine hydrochloride in the culture medium. Stained paper strips were scanned and quantified with ImageJ. Average H2S level of the wild-type (WT) was set to 100% for subsequent normalization. Three replicates were sampled for each strain. Asterisks indicate statistically significant differences compared to WT as found by t-test (***, p-value < 0.001). (C). Real-time detection of intracellular H2S signals. H2S signal was detected by WSP-5 (working concentration 15 μM, green signal) for bacteria that were cultivated in LB. Picture captured in the 488–524 channel was merged with the bright field image. Scale bar indicates length of 10 μm. Bottom chart exhibits end-point detection of H2S production for the corresponding strains in the top chart.
Fig 2.
H2S is the determinant of H2O2 resistance caused by cbs expression in V. cholerae.
(A). CBS-derived H2S renders V. cholerae cells less susceptible to H2O2. Bacteria were exposed to 1 mM of H2O2 for 30 min at their exponential-phase or left untreated in M9 minimal medium (M9 salts plus 2 mM MgSO4, 0.1 mM CaCl2, and 0.2% casein acid hydrolysate as sole carbon source) containing 200 μM IPTG. Viability was determined by comparing the CFU in the H2O2-challenged and the unchallenged samples. Top chart exhibited end-point detection of H2S production for the corresponding strains in the bottom chart. Three replicates were sampled for each strain. Significance was determined by one-way ANOVA; p-value: **, <0.01, ***, <0.001. (B). H2S scavenging via the expression of S. aureus sqr gene impairs the protective effect of cbs expression on the survival of V. cholerae. Left panel: survival under H2O2 stress were tested for Δcbs strains containing vectors only (cbs-sqr-), either Ptac-cbs (cbs+sqr-) or PBAD-sqr (cbs-sqr+), or both of Ptac-cbs and PBAD-sqr (cbs+sqr+). Bacteria were grown in M9 minimal medium containing 200 μM of IPTG and 0.02% arabinose and exposed to 1 mM of H2O2 for 30 min at their mid-log phase. Viability was determined by comparing the CFU in the H2O2-challenged and the unchallenged samples. Values expressed are means ± S.D. from three experiments. Significance was determined by one-way ANOVA; p-value: ***, <0.001. Top chart exhibits end-point detection of H2S production for the corresponding strains in the bottom chart in M9 minimal medium. Right panel: design of H2S production and elimination system in V. cholerae cells. In the system, S. aureus sqr gene was introduced to remove the CBS-derived H2S by H2S oxidation in order to establish a system with only CBS protein produced.
Fig 3.
CBS-derived H2S stimulates the expression of enzymatic ROS scavengers.
(A). Illustration for enzymatic ROS scavengers in V. cholerae. Up to date, there are three SODs, VC2694 (SodA), VC2045 (SodB), and VC1583 (SodC), two catalases, VC1585 (KatB) and VC1560 (KatG), and three peroxidases, VC0731 (AhpC), VC2637 (PrxA), and VCA1006 (OhrA), reported for V. cholerae [6–9]. Expression of katG and prxA are positively regulated by transcription factor VC2636 (OxyR1), while ahpC expression is activated by VC0732 (OxyR2), and in turn inhibits OxyR1 expression [9]. SODs detoxify superoxide and generate H2O2. OhrA specifically degrades organic peroxide species. Peroxidases are the primary scavengers confronting low micromolar (μM) level of H2O2, while catalase activity predominates at millimolar (mM) levels of H2O2 [4]. (B). cbs expression and enzymatic antioxidants expression. The relative expression of SODs, catalases, and peroxidases in exponentially grown cbs deletion mutant (Δcbs/vector), and cbs overexpression strain (Δcbs/Ptac-cbs), with or without H2O2 treatment (1 mM, 10 min), was examined by real-time qPCR. cbs expression was induced by 200 μM of IPTG. Values expressed are means ± S.D. from three experiments. Significance was determined by t-test; p-value: *, <0.05, **, <0.01, ***, <0.001.
Fig 4.
CBS-derived H2S enhances catalase activity in V. cholerae, which relates to the facilitation of cellular iron content.
(A). Contribution of superoxide dismutases, catalases and peroxidases expression to enzymatic activity in H2O2 detoxification. Superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity in crude extracts of cbs-deficient (Δcbs/vector) and overexpressed (Δcbs/Ptac-cbs) cells. Cells were grown in M9 minimal medium (M9 salts plus 2 mM MgSO4, 0.1 mM CaCl2, and 0.2% casein acid hydrolysate as sole carbon source), containing 200 μM of IPTG, and challenged with or without H2O2 (1 mM, 20 min) at their mid-log phase. Values expressed are means ± S.D. from three experiments. Significance was determined separately for the H2O2- and H2O2+ samples by t-test; p-value: *, <0.05, **, <0.01. (B). Impact of cbs expression on the catalase activity of KatB and KatG, respectively. katB and katG was separately expressed under PBAD inducible promoter in the triple-deletion mutant of cbs, katB and katG. Catalase activity in crude extracts of samples with induced cbs expression (Ptac-cbs) was compared with that of cbs-deficient cells (pMal-c2x vector). Bacterial cultures were treated with or without H2O2 (1 mM, 20 min) before collection. Values are means ± S.D. from three experiments. Significance was determined by t-test; p-value: ns, not significant, *, <0.05, ***, <0.001. (C). CBS-derived H2S helps to retain the catalase activity of KatB under iron-deficient conditions. Cells were grown in M9 medium containing 0.2% casein acid hydrolysate as the sole carbon source and induced with 0.5 mM IPTG and 0.02% arabinose. Iron chelator 2,2’-bipyridine or FeCl3 was added to monitor the iron content of the medium as indicated. Catalase activity in crude extracts of ΔcbsΔkatBΔkatG cells, containing PBAD-katB, and having either Ptac-cbs (cbs+) or vector control (cbs-), was examined. Significance was determined by two-way ANOVA from the data of three independent experiments. Significant differences in the mean rank of the catalase activity of each strain at different iron levels are shown in alphabetical order (cbs-, blue letters; cbs+, red letters), with the same letter indicating a p-value > 0.05. The significant differences between cbs- and cbs+ strains at certain 2,2’-bipyridine concentrations were also indicated; p-value, **, <0.01, ***, <0.001. (D). Impact of cbs expression on the iron content in KatB proteins. Hig-tagged V. cholerae KatB was expressed under pBAD promoter in triple-deletion mutant of cbs, katB and katG, with (Ptac-cbs) or without (vector) additional cbs expression. Strains were cultured and induced in M9 minimal medium (M9 salts plus 2 mM MgSO4, 0.1 mM CaCl2, and 0.2% casein acid hydrolysate as sole carbon source). Purified but not desalted KatB protein samples were subjected to quantification of iron content based on chromogenic reaction with ferrozine (cat. number R22185, Shanghai yuanye Bio-Technology). Values expressed are means ± S.D. from three experiments. Significance was determined by t-test; p-value: **, <0.01.
Fig 5.
CBS-dependent ROS resistance works through promoting iron storage.
(A). cbs expression promotes cellular iron storage. Cells were obtained in M9 minimal medium with 0.2% casein containing 0.5 mM IPTG and 0.02% arabinose. Iron content was assessed with ICP-MS and normalized with cell numbers. Values expressed are means ± S.D. from three experiments. Significance was determined by t-test; p-value: *, <0.05. (B). cbs expression protects cells from H2O2 in iron-deficient conditions. Bacteria were exposed to 1 mM of H2O2 for 30 min at their exponential-phase or left untreated in M9 minimal medium (M9 salts plus 2 mM MgSO4, 0.1 mM CaCl2, and 0.2% casein acid hydrolysate as sole carbon source). Viability was determined by comparing the CFU in the H2O2-challenged and the unchallenged samples. Three replicates were sampled for each strain. Significance was determined by one-way ANOVA; p-value: *, <0.05; ***, <0.001; ns, not significant.
Fig 6.
CBS-derived H2S promotes the fitness of V. cholerae in adult mouse via fighting against oxidative stress.
(A). Adult CD1 mice were treated, without or with 1% N-acetyl cysteine (NAC), in drinking water for 7 days, then administrated with wild-type (WT) and either Δcbs or Δcbs complemented by single copy Plac-cbs (Δcbs lacZ::Plac-cbs) mutants after streptomycin treatment. Fecal pellets were collected at 1, 3, and 5 days post inoculation. Bacterial loads were quantified by plating. Competition index (CI) was calculated as the ratio of mutant to wild-type colonies normalized with the input ratio. Red line indicated for the average CI. Significance was determined by t-test; p-value: ns, not significant, *, <0.05, **, <0.01. (B). Streptomycin-treated adult CD1 mice were administrated with Δdps and ΔcbsΔdps. Fecal pellets were collected at 1, 3, and 5 days post inoculation. Bacterial loads were quantified by plating. Competition index (CI) was calculated as the ratio of ΔcbsΔdps to Δdps colonies normalized with the input ratio. Red line indicated for the average CI. (C). A proposed mechanism for the cytoprotective effects of CBS-derived H2S against oxidative stress in V. cholerae. Iron availability is limited for V. cholerae during infection of host. The cbs-dependent generation of H2S from cysteine stimulates the expression of several antioxidants and facilitates the acquisition and storage of iron in V. cholerae. Enriched intracellular iron pool promotes the activity of heme-catalase KatB, by contributing to the assembly of active catalases. The illustration is created with BioRender.com.