A Small Membrane Stabilizing Protein Critical to the Pathogenicity of Staphylococcus aureus

Abstract

Staphylococcus aureus is a major human pathogen, and the emergence of antibiotic-resistant strains is making all types of S. aureus infections more challenging to treat. With a pressing need to develop alternative control strategies to use alongside or in place of conventional antibiotics, one approach is the targeting of established virulence factors. However, attempts at this have had little success to date, suggesting that we need to better understand how this pathogen causes disease if effective targets are to be identified. To address this, using a functional genomics approach, we have identified a small membrane-bound protein that we have called MspA. Inactivation of this protein results in the loss of the ability of S. aureus to secrete cytolytic toxins, protect itself from several aspects of the human innate immune system, and control its iron homeostasis. These changes appear to be mediated through a change in the stability of the bacterial membrane as a consequence of iron toxicity. These pleiotropic effects on the ability of the pathogen to interact with its host result in significant impairment in the ability of S. aureus to cause infection in both a subcutaneous and sepsis model of infection. Given the scale of the effect the inactivation of MspA causes, it represents a unique and promising target for the development of a novel therapeutic approach.

Keywords: Staphylococcus aureus; cytotoxins; immune evasion; iron homeostasis; virulence.

FIG 1

Inactivation of the mspA gene results in a loss of toxicity (cytolytic activity) for S. aureus. (a and b) In both the JE2 (a) and SH1000 (b) backgrounds, the inactivation of mspA resulted in a loss of cytolytic activity, represented by a significant decrease in cell death. The loss of cytolytic activity was complemented in both backgrounds by expressing the mspA gene from a plasmid (pmspA). (c and d) Furthermore, the inactivation of mspA resulted in a loss of toxicity to a lung epithelial cell line (A549) (c) and human red blood corpuscles (d) in both the JE2 and SH1000 backgrounds. Inactivation of mspA also resulted in a reduction in the abundance of alpha toxin in the bacterial supernatants in both backgrounds, determined by Western blotting. (e) Densitometry values for triplicate blots are shown. (f) The effect of the inactivation of mspA on secretion of PSMs was quantified by HPLC-MS, where the secretion of delta toxin was the most affected in both backgrounds (P < 0.001). AU, arbitrary units. (g) The activity of the Agr system is repressed by the inactivation of mspA, as illustrated by qRT-PCR quantification of rnaIII transcription. (h) Protter (21) predicts that MspA is a membrane-bound protein with four membrane-spanning domains. The dots represent biological replicates, the bars represent the mean of the replicates, and the error bars are the standard errors of the means. Statistics were performed using a one-way ANOVA, and significance was determined as the following P values: *, <0.05; **, 0.01; ***, 0.001; ****, 0.0001.

FIG 2

MspA confers protection against aspects of innate immunity. (a) The carotenoid pigment staphyloxanthin is less abundant in the membranes of both the crtM and mspA mutants than in those of both the wild-type strain and floA mutant in the JE2 background. (b and c) Survival of S. aureus upon exposure to oleic acid was reduced in the mspA mutants in both S. aureus backgrounds. The survival phenotype was complemented in both backgrounds by expressing the mspA gene from a plasmid (pmspA). (d and e) Survival of S. aureus upon exposure to the human neutrophil defensin-1 (hNP-1) was reduced in the mspA mutants in both S. aureus backgrounds, and this phenotype was complemented in both backgrounds by expressing the mspA gene from a plasmid (pmspA). (f) Survival of S. aureus following phagocytosis is reduced in the mspA mutants in both S. aureus backgrounds. (g) Survival in human blood is reduced in the absence of mspA in both backgrounds. The dots represent biological replicates, the bars represent the means from the replicates, and the error bars are the standard errors of the means. Statistics were performed using a one-way ANOVA, and significance was determined as the following P values: *, <0.05; **, 0.01; ***, 0.001; ****, 0.0001; ns, not significant.

FIG 3

Inactivation of MspA affects membrane stability. (a) Membrane stability was determined by quantifying the sensitivity of the bacteria to the detergent SDS, where the mspA mutant was significantly impaired. The sensitivity of mutants with transposons in crtM, floA, and agrA was unaffected and is provided for comparison. Means from three independent biological experiments are shown, and the error bars represent the standard errors of the means. Statistics were performed using a one-way ANOVA, and significance was determined as the following P values: *, <0.05; **, 0.01; ***, 0.001. (b and c) The MspA protein inhibits the penetration of the bacterial membrane by propidium iodide. Wild-type and mspA mutant cells in the JE2 (b) and SH1000 (c) backgrounds were incubated with PI, and the fluorescence of the cells was analyzed by flow cytometry. The shift of the peak to the right for the mspA mutants demonstrates an increased PI signal, indicating that PI was better able to penetrate these cells and stain the DNA. Representative images from three independent experiments are shown.

FIG 4

mspA mutants contain higher levels of intracellular iron. (a to d) Resistance to streptonigin was used to compare the level of intracellular iron in the mspA mutants. Streptonigrin (1.5 μl at a concentration of 2.5 mg/ml) was spotted onto a lawn of JE2 (a), JE2 mspA::tn (b), SH1000 (c), and SH1000 mspA::tn (d) cells, and the plates were incubated to allow the bacteria to grow. The zone of clearance as a result of the diffusion of the antibiotic was larger for both mspA mutants, suggesting they contain higher levels of intracellular iron. Representative images from three independent experiments are shown. (e to g) The effect of streptonigrin was also determined in broth, where bacterial growth was determined after 8 h in increasing concentrations of streptonigrin. A mutant with a transposon in the ferric uptake regulator (fur) in the JE2 background was included as a control. Streptonigrin was consistently more effective at inhibiting the growth of the mspA mutant strains. Means from six independent biological experiments are shown, and error bars represent the standard errors of the means. Statistics were performed using Student’s paired t test, and significance was determined as the following P values: *, <0.05; **, 0.01; ***, 0.001; ****, 0.0001.

FIG 5

Ability of S. aureus to adapt to hemin is affected by the loss of MspA. (a) Illustration of our hemin adaptation assay. Bacteria were cultured either in normal TSB or in TSB supplemented with 10 μM hemin. Bacteria then were subcultured into a range of hemin concentrations (40, 20, 10, 2.5, and 0 μM). Schematic was prepared on BioRender. (b and c) Preculture in hemin conferred an advantage for subsequent growth in high hemin concentrations in both JE2 (b) and SH1000 (c) backgrounds. O/N, overnight. The loss of mspA was disadvantageous for adaption to high hemin environments compared to that of the wild-type strains. Means from six independent biological experiments are shown, and error bars represent the standard errors of the means.

FIG 6

Increased intracellular hemin phenocopies the effects of the loss of MspA. When cultured in increasing concentrations of hemin, the ability of JE2 (a) and SH1000 (b) to cause toxicity in THP-1 cells is diminished in a concentration-dependent manner. (c and d) Transcript levels of RNAIII are, compared to levels of mspA-deficient JE2 and SH1000, further reduced in both backgrounds when exposed to hemin. (e and f) Carotenoid biosynthesis is also reduced during culture with hemin, and this effect was observed in a concentration-dependent manner in both backgrounds. (g and h) A shift in the PI staining for JE2 and SH1000 when cultured with hemin suggests that this also affects membrane integrity. Representative images from three independent flow cytometry analyses are shown. The dots represent biological replicates, the bars represent the means from the replicates, and the error bars are the standard errors of the means. Statistics were performed using a one-way ANOVA, and significance was determined as the following P values: *, <0.05; **, 0.01; ***, 0.001; ****, 0.0001.

FIG 7

Inactivation of mspA affects the ability of S. aureus to cause disease in both a superficial and systemic infection model. BALB/c mice were infected subcutaneously with 2 × 10⁷ CFU wild-type (JE2) and isogenic strains in which the mspA and agrB genes were inactivated. (a) Abscess lesion area was assessed daily, with representative lesions from the dorsal area of mice from each group shown. (b) The bacterial burden in the skin was assessed by viable counting (CFU/mg) at 3 and 6 days postinfection. (c) Abscess lesion area from panel a expressed as total lesion size (cm²) ± SEM. (d to g) Mice were inoculated via tail vein injection of 2 × 10⁷ CFU the wild-type JE2 S. aureus strain and the mspA and agrB mutants. The tail vein of C57BL/6J mice was inoculated with a sublethal dose of the wild-type JE2 S. aureus. Blood (d), spleens (e), livers (f), and kidneys (g) were harvested at 6 and 24 h postinfection, and the burden of bacteria in each sample was quantified. For the superficial infection, n = 10, representative of 2 independent pooled experiments. For the systemic infection, n = 5 from one independent experiment. Statistics were performed using a one-way ANOVA with a Tukey posttest (superficial) and Sidak’s multiple-comparison test (systemic), and significance was determined as the following P values: *, <0.05; **, 0.01; ***, 0.001; ****, 0.0001.

FIG 8

Summary of the effect inactivation of the mspA gene has on the bacterial cell. In the absence of MspA, the bacteria are unable to withstand membrane attack and phagocytosis. The Agr system does not become activated, which subsequently affects the production of cytolytic toxins. The abundance and stoichiometry of the Hrt proteins are also affected in the mspA mutant, which affects heme-iron homeostasis, and this may contribute to the toxicity and immune susceptibility phenotypes reported here.