The Old Yellow Enzyme OfrA Fosters Staphylococcus aureus Survival via Affecting Thiol-Dependent Redox Homeostasis

Abstract

Old yellow enzymes (OYEs) are widely found in the bacterial, fungal, and plant kingdoms but absent in humans and have been used as biocatalysts for decades. However, OYEs' physiological function in bacterial stress response and infection situations remained enigmatic. As a pathogen, the Gram-positive bacterium Staphylococcus aureus adapts to numerous stress conditions during pathogenesis. Here, we show that in S. aureus genome, two paralogous genes (ofrA and ofrB) encode for two OYEs. We conducted a bioinformatic analysis and found that ofrA is conserved among all publicly available representative staphylococcal genomes and some Firmicutes. Expression of ofrA is induced by electrophilic, oxidative, and hypochlorite stress in S. aureus. Furthermore, ofrA contributes to S. aureus survival against reactive electrophilic, oxygen, and chlorine species (RES, ROS, and RCS) via thiol-dependent redox homeostasis. At the host-pathogen interface, S. aureusΔofrA has defective survival in macrophages and whole human blood and decreased staphyloxanthin production. Overall, our results shed the light onto a novel stress response strategy in the important human pathogen S. aureus.

Keywords: MRSA; ROS; blood; electrophilic stress; phagocytes; quinone; stress response.

FIGURE 1

OfrA conservation in Firmicutes and staphylococci. (A) Phylogenetic analysis of OYE examples in Gram-positive S. aureus (OfrA, OfrB) and B. subtilis (YqiG, YqjM) compared to the Gram-negative E. coli (NemA) and Pseudomonas fluorescens (XenB) with distinctive multiple sequence alignment generated by Clustal Omega. (B) Maximum likelihood tree showing the evolutionary relationship of OfrA in different Firmicutes chromosomes. Multiple sequence alignment was utilized to build the phylogenetic tree using RAxML software and visualized with ggtree. (C) Bar chart shows the presence or absence of OfrA in seven Firmicutes genera (Bacillus, Paenibacillus, Streptococcus, Clostridium, Staphylococcus, Lactobacillus, and Enterococcus). Filtration criteria were based on 35% amino acid identity cutoff and protein length = 375 ± 38 amino acids, refer to section “Materials and Methods”. (D) OfrA conservation across the different staphylococci compared to the conservation of OfrB, MvaA (mevalonate pathway), and CrtM (staphyloxanthin biosynthesis) in the same genomes. Filled and unfilled circles indicate gene presence and absence, respectively. CrtM, squalene desaturase; MvaA, hydroxymethylglutaryl-CoA reductase; OfrA, old yellow enzyme flavin oxidoreductase A; OfrB, old yellow enzyme flavin oxidoreductase B.

FIGURE 2

ofrA is induced in RES, ROS, and RCS in a dose-dependent manner. (A) ofrA induction conditions using the reporter strain EI011, which harbors a chromosomally encoded lacZ under P_ofrA (Supplementary Figure 3). After 2-h incubation with shaking at 37°C, β-galactosidase assay was used to report ofrA transcriptional level. The corresponding concentrations were indicated in the graph. A total of four biological replicates were compared to untreated controls using unpaired two-tailed Student’s t-test. Error bars represent the standard error of the means. (B) Dose dependency of ofrA induction using β-galactosidase assays. The highest concentration is the minimum inhibitory concentration (1 × MIC), the intermediate concentration is 0.5 × MIC, and compared to control (no compounds were added = 0 × MIC). Log2FC was calculated as average from three biological replicates. (C) RT-qPCR shows ofrA induction in S. aureus JE2 background in agreement with the reporter system. JE2 strain was cultivated in RPMI until mid-logarithmic phase (OD₆₀₀ = 0.5). Samples were taken before adding the compounds as a control. After adding the compounds, the bacterial pellets were collected after 15 min of incubation at 37°C with shaking. A total of three biological replicates were compared to untreated controls via unpaired two-tailed Student’s t-test. Error bars represent the standard error of the means. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. CHP, cumene hydroperoxide; FA, formaldehyde; Fosfo, fosfomycin; MG, methylglyoxal; MHQ, methylhydroquinone; MIC, minimum inhibitory concentration; RCS, reactive chlorine species; RES, reactive electrophilic species; ROS, reactive oxygen species.

FIGURE 3

OfrA provides S. aureus resistance against quinone stress, toxic aldehydes, oxidative, and hypochlorite stresses. Bacterial survival assays in RES (MHQ and MG), ROS (H₂O₂), and RCS (NaOCl). The three strains; JE2, ΔofrA, and pofrA were allowed to grow until the logarithmic phase (OD₆₀₀ = 0.4–0.6). Bacterial pellets were washed with sterile 1 × PBS, and the OD₆₀₀ were adjusted to 0.4 in fresh RPMI. We added: (A) 0.5 mM MHQ for 3 h, (B) 2 mM MG for 3 h, (C) 40 mM H₂O₂ for 1 h, or (D) 1.5 mM NaOCl for 30 min. Samples were taken from the untreated control (for normalization) or with the stress conditions after the indicated time points for CFU determination using SP-SDS method on LB agar. Data represent four–five biological replicates. Error bars represent the standard error of the means. Statistical analysis was carried out using one-way ANOVA and pairwise t-test with Bonferroni p-value adjustment; ns, not significant; *p < 0.05; **p < 0.01. MG, methylglyoxal; MHQ, methylhydroquinone; RCS, reactive chlorine species; RES, reactive electrophilic species; ROS, reactive oxygen species.

FIGURE 4

OfrA promotes S. aureus fitness at the host-pathogen interface by enhancing survival in RAW 264.7 macrophages and whole human blood. (A) Macrophage survival assay. JE2, ΔofrA, and pofrA were added to RAW 264.7 macrophage cell lines in 1:10 MOI. After 1 h of infection, gentamicin (150 μg/ml) was used to kill extracellular bacteria for 1 h. Fresh RPMI + 10% FCS was added (t = 0). At (t = 4 h), viable intracellular bacteria were determined and the CFU/ml was used as a normalization factor. Samples were taken at (t = 24 and 48 h). The assay was repeated for three independent experiments. Data represent five biological replicates from one of the three experiments. (B) Whole human blood killing assay. A total of 5 × 10⁶ CFU/ml of each strain were incubated in whole human blood for 60 min at 37°C with continuous shaking. The number of viable bacteria was enumerated after serial dilutions in sterile 1 × PBS using SP-SDS method on LB agar and normalized to the viable cells without incubation. The experiment was repeated in blood taken from four different blood donors. Data represent four biological replicates from one of the four experiments. Error bars represent the standard error of the means. Statistical analysis was carried out using one-way ANOVA and pairwise t-test with Bonferroni correction p-value adjustment; ns, not significant; *p < 0.05; **p < 0.01.

FIGURE 5

ofrA mutation decreases STX production via the upper mevalonate pathway but cannot solely explain ROS hypersensitivity. Staphyloxanthin assay showing STX levels in TSB medium (A,C), B-medium (B), and RPMI (D). The strains were grown in overnight culture in the respective medium without any supplementation. Then, we diluted the overnight cultures 1:100 in fresh medium without or with supplementation; 0.5% glucose (B) or 1 mM mevalonate (D). After 24 h, the bacteria were collected and washed with sterile water. OD₆₀₀ were recorded for normalization. STX was extracted using methanol (refer to section “Materials and Methods”). A₄₆₅ were used for measuring the extracted STX. Error bars represent the standard error of the means (A,D) and standard deviation (B) of four biological replicates. Statistical analysis was carried out using unpaired two-tailed Student’s t-test (B,D) or one-way ANOVA and pairwise t-test with Bonferroni p-value adjustment (A); ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. (E) Bacterial survival assays showing crtM mutation additive effect to ofrA mutation in ROS hypersensitivity. The strains were grown in overnight culture in RPMI medium. We diluted the overnight cultures 1:100 in fresh RPMI until mid-logarithmic phase. Cells were harvested by centrifugation and washed with sterile PBS. OD₆₀₀ were adjusted to 0.4. Bacteria were challenged with 30 mM H₂O₂. After 1 h of exposure to 30 mM H₂O₂, viable cells were diluted in PBS after catalase treatment for residual H₂O₂. Samples were taken from the untreated control (for normalization) or with the stress condition after 1 h for CFU determination using SP-SDS method on LB agar. STX, staphyloxanthin.

FIGURE 6

ofrA mutation increases ROS-mediated killing via disturbing thiol-dependent redox homeostasis. (A) Bacterial survival assay in 40 mM H₂O₂ with or without 120 mM thiourea. The strains were grown in overnight culture in RPMI medium. We diluted the overnight cultures 1:100 in fresh RPMI until mid-logarithmic phase. Cells were harvested by centrifugation and washed with sterile PBS. OD₆₀₀ were adjusted to 0.4. Bacteria were challenged with H₂O₂ with or without 120 mM thiourea. Bacterial survival assay in 0.5 mM MHQ with or without 120 mM thiourea (B) or 1.25 mM NAC (C). Data represent four biological replicates. Error bars represent the standard error of the means. Statistical analysis was carried out using one-way ANOVA and pairwise t-test with Bonferroni p-value adjustment; *p < 0.05. MHQ, methylhydroquinone; NAC, N-acetyl cysteine.

FIGURE 7

Cartoon representation shows our current understanding of ofrA function in S. aureus. OfrA protein 3D structure was predicted via AlphaFold. For 3D visualization, refer to alphafold.ebi.ac.uk/entry/Q2FZU7. ofrA is induced in ROS, RES, and RCS conditions which are available at the host-S. aureus interface. We showed that ofrA is an important factor in S. aureus resistance to the aforementioned stress conditions. ofrA contributes to S. aureus virulence via human blood and macrophage survival. ofrA mutation is involved in decreased STX production via MVA pathway. Both STX and ofrA protects S. aureus against oxidative stress via different mechanisms. ofrA supports the thiol-dependent redox homeostasis. FPP, farnesyl pyrophosphate; IPP, isopentenyl pyrophosphate; MVA, mevalonate; STX, staphyloxanthin.