Cooperation and antagonism in polymicrobial infection

PROJECT SUMMARY With the increasing awareness of distinct disease outcomes during polymicrobial infections, there is an urgent need for mechanistic studies to better define microbial interactions. This application seeks to understand community interactions between Pseudomonas aeruginosa and Staphylococcus aureus, which are model organisms to study polymicrobial interactions. They are two of the most common pathogens causing chronic infections in the lungs of people with cystic fibrosis (CF), medical devices, and wounds, suggesting co- existence in vivo. Indeed, chronic co-infections caused by these organisms are more problematic than mono- species infections. However, the relationship between P. aeruginosa and S. aureus is intriguingly complicated. These organisms often compete in vitro but are frequently co-isolated from the same clinical samples. We contribute to the studies of anti-S. aureus mechanisms by proposing a novel role for the P. aeruginosa exopolysaccharide Psl in antagonizing S. aureus growth and interactions with S. aureus protein A (SpA). Studies proposed in aim 1 will utilize several complementary strategies to define mechanisms underlying P. aeruginosa Psl-mediated interactions with S. aureus. In aim 2, we will investigate a unique cooperative behavior associated with P. aeruginosa and S. aureus. This involves production of 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) by P. aeruginosa, and staphyloxanthin (STX), a yellow pigment synthesized by the S. aureus crt operon. STX can promote S. aureus resistance to oxidative stress and neutrophil-mediated killing. We found that STX production in S. aureus, either as surface-grown macrocolonies or planktonic cultures, was elevated when exposed to sub-lethal concentrations of HQNO. When subjected to hydrogen peroxide, human neutrophils, or during in vivo infections, P. aeruginosa survival was significantly higher when mixed with wild- type S. aureus, in comparison to P. aeruginosa cultured alone or with an S. aureus crt mutant deficient in STX production. Therefore, the focus of aim 2 will be to define the pathways and biological relevance of HQNO- mediated induction of STX, resulting in resistance to host ROS during infection. Finally, we discovered that a prior infection of both human- and murine-derived macrophages with S. aureus induces tolerance and prevents responsiveness to subsequent infection. This is due to significant metabolic rewiring of the macrophage innate immune response during primary infection. Thus, aim 3 will use primary macrophages and in vivo animal models to investigate how a S. aureus infection leads to metabolic reprogramming and define how this leads to epigenetic changes and subsequent innate immune gene silencing. P. aeruginosa and S. aureus interactions are clearly multifaceted and likely mediated by bacterial factors, spatial distribution during infection, nutrient availability, and the host microenvironment. These proposed studies will contribute to the understanding of complex polymicrobial interactions and lay the foundation for strategies to modulate these pathways for better control of mixed-species infections.