The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus is problematic both in hospitals and in the community. Currently, we have limited understanding of mechanisms of innate immune evasion used by S. aureus. To that end, we created an isogenic deletion mutant in strain MW2 (USA400) of the saeR/S 2-component gene regulatory system and studied its role in mouse models of pathogenesis and during human neutrophil interaction. In this study, we demonstrate that saeR/S plays a distinct role in S. aureus pathogenesis and is vital for virulence of MW2 in a mouse model of sepsis. Moreover, deletion of saeR/S significantly impaired survival of MW2 in human blood and after neutrophil phagocytosis. Microarray analysis revealed that SaeR/S of MW2 influences expression of a wide variety of genes with diverse biological functions. These data provide new insight into how virulence is regulated in S. aureus and associates a specific staphylococcal gene-regulatory system with invasive staphylococcal disease.

Figure 1

Generation of isogenic saeR/S deletion mutant from parental S. aureus strain MW2. A. Physical map of the saeR/S genes of S. aureus and construction of the saeR/S deletion mutant. Orfs are depicted by arrows. Details of primers used for the construct are described in Materials and Methods. B. Polymerase chain reaction (PCR) analysis of saeR/S mutant and MW2 using primers for saeS and saeR (saeS forward 5' - TCG AAC GCC ACT TGA GCG TAT T - 3' and saeS reverse 5' - AGC CTA ATC CAG AAC CAC CCG TTT - 3' and saeR forward 5’ – TGACCCACTTACTGATCGTGGATG- 3’ and saeR reverse 5’ – ACGCATAGGGACTTCGTGACCATT- 3’). C. In vitro growth of saeR/S mutant versus parental wild-type strain MW2.

Figure 2

The SaeR/S two-component gene regulatory system is important during invasive S. aureus disease. A. Mice were infected with MW2 and ΔsaeR/S by tail vein inoculation (1 × 10⁸ cfu). Mouse survival results are from 15 mice in each group. * P < .0001 vs. MW2. B, C. The SaeR/S system is not essential for skin abscesses and/or dermonecrosis. Results are from 15 mice infected subcutaneously with 1 × 10⁷ cfu of MW2 and ΔsaeR/S. B. Skin abscess volumes. Results are the average abscess volume in mice infected with MW2 and ΔsaeR/S (P > .05 at all time points except day 5 * P < .05, ANOVA with Bonferroni’s posttest). C. Dermonecrosis caused by MW2 and ΔsaeR/S. Results represent the number of mice with dermonecrosis on each day. D) Number of S. aureus cfus recovered from skin abscesses. Results are from 3 mice per treatment per time point following subcutaneous injection with 1 × 10⁷ cfu of MW2 and ΔsaeR/S. Control mice receiving sterile DPBS were harvested on day 4 and had no bacterial growth (data not shown).

Figure 3

Absence of saeR/S significantly attenuates survival of S. aureus in human blood. A. MW2, compΔsaeR/S, and ΔsaeR/S were incubated in heparinized human blood and percent survival was calculated as described in Materials and Methods. B. Averaged cfu from each time point (average based on counts from two separate plates/time point). Results are from 17 separate blood donors * P < .01 versus wild-type and P < .05 versus compΔsaeR/S, ANOVA with Tukey’s posttest).

Figure 4

SaeR/S is critical for S. aureus survival during PMN phagocytosis. A. Percent of MW2 and saeR/S mutant strains ingested by human PMNs was calculated using the equation: [(number of ingested bacteria per cell/total number of PMN-associated bacteria, bound or ingested) × 100]. Results are from 3 separate donors. B. Killing of MW2 and ΔsaeR/S by PMNs. Percent S. aureus survival during interaction with PMNs was calculated with the equation (CFU_+PMN at t=n/ CFU_+PMN at t=0) × 100. Results are from 7 separate donors. *P < .05 at 0.5 h, P < .01 at 1.0 h, and P < .001 at 1.5 and 3 h vs MW2, ANOVA with Tukey’s posttest. C. Complementation of ΔsaeR/S with saeR/S restores wild-type phenotype. Results are from 10 separate donors *P < .05 vs. MW2 and compΔsaeR/S, ANOVA with Tukey’s posttest. D. PMN lysis after phagocytosis of MW2 and ΔsaeR/S. Percent PMN lysis was determined after 1 h and 5 h of culture with MW2 and ΔsaeR/S strains (PMN to bacteria ratio was ~ 1:10). Percent cytotoxicity was determined with the following equation: (PMNs + CA-MRSA – PMNs in RPMI/H)/(LDH_max-PMNs in RPMI/H) × 100. *P < .001 vs MW2, ANOVA with Tukey’s posttest. Results are from 6 separate donors.

Figure 5

TaqMan real-time RT-PCR confirmation of mutant phenotype. A. Complementation of the saeR/S mutant strain restores saeR/S expression comparable to wild-type MW2. B. Genes (n = 7) identified as differentially transcribed by microarray analysis were selected from several categories for confirmation by TaqMan real-time RT-PCR. Transcript levels were measured either during mid-exponential (ME, OD₆₀₀ = 0.75) or early stationary phases of growth (ES, OD₆₀₀ = 2.0). Samples were analyzed in triplicate and results are from two biological repetitions. There was a positive correlation between TaqMan and microarray results, consistent with previous comparisons.