Parsek, Matthew
                            (Ed.)
                        
                    
            
                            ABSTRACT Chronic polymicrobial infections involvingPseudomonas aeruginosaandStaphylococcus aureusare prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated thatP. aeruginosais attracted toS. aureususing type IV pili (TFP)-mediated chemotaxis, but the impact of attraction onS. aureusgrowth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival ofS. aureusduring coculture, we found that interspecies chemotaxis providesP. aeruginosaa competitive advantage by promoting invasion into and disruption ofS. aureusmicrocolonies. This behavior rendersS. aureussusceptible toP. aeruginosaantimicrobials. Conversely, in the absence of TFP motility,P. aeruginosacells exhibit reduced invasion ofS. aureuscolonies. Instead,P. aeruginosabuilds a cellular barrier adjacent toS. aureusand secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into theS. aureuscolonies. Reduced invasion leads to the formation of denser and thickerS. aureuscolonies with increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate treatment strategies. Finally, we show thatP. aeruginosamotility modifications of spatial structure enhance competition againstS. aureus. Overall, these studies expand our understanding of howP. aeruginosaTFP-mediated interspecies chemotaxis facilitates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities. IMPORTANCEThe polymicrobial nature of many chronic infections makes their eradication challenging. Particularly, coisolation ofPseudomonas aeruginosaandStaphylococcus aureusfrom airways of people with cystic fibrosis and chronic wound infections is common and associated with severe clinical outcomes. The complex interplay between these pathogens is not fully understood, highlighting the need for continued research to improve management of chronic infections. Our study unveils thatP. aeruginosais attracted toS. aureus, invades into neighboring colonies, and secretes anti-staphylococcal factors into the interior of the colony. Upon inhibition ofP. aeruginosamotility and thus invasion,S. aureuscolony architecture changes dramatically, wherebyS. aureusis protected fromP. aeruginosaantagonism and responds through physiological alterations that may further hamper treatment. These studies reinforce accumulating evidence that spatial structuring can dictate community resilience and reveal that motility and chemotaxis are critical drivers of interspecies competition. 
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