Staphylococcus aureus virulence in the respiratory tract:agr and sar dependent activation of epithelial Ca²⁺ fluxes and IL-8 expression ♦ 849

S. aureus is a common respiratory pathogen in cystic fibrosis particularly in young patients. In this study we used well characterized mutants of S. aureus RN6390 to identify which virulence factors are important in the pathogenesis of pulmonary disease and the induction of inflammation in the lung. A neonatal mouse model of acute pulmonary infection was used to compare the virulence of the parental strain, and genetically constructed mutants lacking either of the global regulatory genes sar (Staphylococcus accessory regulator) agr (accessory gene regulator) or both regulatory loci. Whereas the parental strain caused pneumonia, as defined by recovery of >10³cfu/100 μl of lung tissue, in 100% of the 10-14 day old BALBc/ByJ mice (n=30) when introduced intranasally, 100% bacteremia and 30% mortality; the agr sar mutant infected 75% of the inoculated mice (n=32), caused bacteremia in 38% and 0% mortality (P<0.01). Infection associated with a single mutation in sar gave an intermediate phenotype, with 100% infection, but 0% mortality. To examine the mechanisms through which S. aureus cause pneumonia, we compared the ability of the different strains to activate signal transduction in fura-2 loaded 16HBEo- human respiratory epithelial cells, by monitoring the changes in intracellular Ca²⁺ after exposure to the bacteria. Adherence of the wild type strain promptly stimulated oscillating Ca²⁺ waves, while there was minimal response to the sar mutant, and no response to the sar, agr mutant. However, the sar, agr, and double mutant strains were able to stimulate IL-8 expression from human epithelial cells lines in a dose dependent fashion in the presence of 0.1% fetal bovine serum, to levels comparable to that elicited by an IL-1β positive control. Neither S. aureus culture supernatant, nor dead organisms stimulated an IL-8 response. These results suggest that S. aureus has multiple surface components which trigger epithelial responses, and while the agr and sar regulatory loci are clearly implicated in virulence in vivo, even avirulent mutants can elicit a significant proinflammatory response from respiratory epithelial cells. S. aureus colonization in children with CF, can contribute to ongoing airway inflammation, even in the absence of chronic infection.