Surface proteins and exotoxins are required for the pathogenesis of Staphylococcus aureus pneumonia

Abstract

A model of Staphylococcus aureus-induced pneumonia in adult, immunocompetent C57BL/6J mice is described. This model closely mimics the clinical and pathological features of pneumonia in human patients. Using this system, we defined a role for S. aureus strain Newman surface proteins and secreted exotoxins in pneumonia-related mortality.

FIG. 1.

Inoculum-based mortality and proliferation of S. aureus Newman in murine lung tissue. (A) C57BL/6J animals were inoculated with either PBS or various doses of live S. aureus Newman via the intranasal route. The levels of survival were recorded at 24, 48, and 72 h postinfection. Animals that appeared to be moribund were killed and counted as dead animals at the appropriate time. The results were analyzed to determine statistical significance using Fisher's exact test (P ≤ 0.02) (indicated by asterisks). (B) Animals were inoculated with 3 × 10⁸ to 4 × 10⁸ CFU of S. aureus Newman, and the bacterial CFU in both lungs (5 min) or the right lung (6, 24, 48, and 72 h) were enumerated at different times postinfection. Data were analyzed to determine significance using Student's t test.

FIG. 2.

Gross pathology of animals infected with S. aureus via the intranasal route. Representative infected animals were compared to uninfected animals in order to obtain gross pathological findings for lungs in situ (A) or following dissection of the left lung (B).

FIG. 3.

Histopathologic findings following intranasal inoculation of S. aureus. Lung tissue harvested from animals infected with S. aureus Newman was prepared and visualized by hematoxylin and eosin staining. Representative histology for an uninfected control having normal lung parenchyma is shown along with a series of images obtained for animals examined at the times indicated. Aggregates of purple-stained immune cells were seen as early as 6 h postinfection (arrowhead), and dense accumulation of bacteria was evident in tissues at 24 h postinfection (arrowhead).

FIG. 4.

S. aureus mutants lacking protein A (spa) or all surface proteins (srtA) or exoproteins (agrA and hla) are defective in the ability to cause pneumonia-related mortality. Animals infected with 3 × 10⁸ to 4 × 10⁸ CFU of wild-type S. aureus Newman or isogenic mutant strains were scored for acute lethal disease, which demonstrated that there was a significant reduction in mortality for animals infected with both the srtA and spa strains (A). Analysis of mutants with bursa aurealis insertions in agrA and hla (alpha-toxin) likewise demonstrated that there was a marked reduction in the ability to cause acute lethal disease (B). Statistical significance was evaluated by Fisher's exact test (one asterisk, P = 0.001; two asterisks, P < 0.002).