Secondary Bacterial Pneumonia by Staphylococcus aureus Following Influenza A Infection Is SaeR/S Dependent

Abstract

Staphylococcus aureus is a predominant cause of fatal pneumonia following influenza A virus (IAV) infection. Herein we investigate the influence of antecedent IAV infection on S. aureus virulence gene expression. Using a murine model, comparing the USA300 and USA300ΔsaeR/S strains, we demonstrate that S. aureus pathogenesis following IAV infection is SaeR/S dependent. Furthermore, we show that IAV modulates the lung environment to rapidly up-regulate S. aureus virulence factors containing the SaeR-binding domain. Data demonstrate that the pathogen response to IAV infection impacts host outcome and provides evidence that the ability of S. aureus to sense and respond to the lung environment determines severity of pneumonia.

Figure 1.

Staphylococcus aureus bacterial pneumonia following antecedent influenza A virus (IAV) infection is saeR/S dependent. Mice were intranasally mock-infected with phosphate-buffered saline or infected with IAV. Six days postinfection, mice were challenged with S. aureus. A, Survival assay of mice infected with IAV/WSN/1933(H1N1) (WSN) (750 plaque-forming units/50 µL) and challenged with USA300 (5 × 10⁸ colony-forming units [CFU]/50 µL), n = 10 mice per group. *P < .05; **P < .01, log-rank (Mantel-Cox) test. B–D, TaqMan reverse-transcription polymerase chain reaction quantification of select SaeR-target genes. Four hours after S. aureus challenge (5 × 10⁸ CFU/50 µL), RNA was isolated from 3–6 mice per treatment group. Data displayed are the mean fold change of S. aureus expression levels from coinfected lungs compared to lungs infected with S. aureus alone. Gene transcripts were normalized to gyrB expression and calibrated to expression in S. aureus–only infection. B, WSN and USA300. C, PR8 and USA300. D, PR8 and USA400.

Figure 2.

Influenza A virus (IAV) infection promotes rapid up-regulation of Staphylococcus aureus SaeR-regulated genes. Mice were infected intranasally with IAV/WSN/1933(H1N1). Six days post-IAV infection, mice were challenged with USA300 or ∆saeR/S. Four (A), 6 (B), or 8 (C) hours post–S. aureus challenge, mouse lungs were harvested. RNA was purified from harvested lungs and treatment groups were pooled (n = 4–5 mice/treatment group/experiment). Gene transcripts were normalized to gyrB and calibrated to the expression levels of USA300-only infection at corresponding time points. Differential regulation of SaeR target genes was assessed by Quantigene 2.0 assays. “X” indicates transcript levels below the limit of detection. Data are reported as mean fold change of 2 separate experiments.