PROJECT SUMMARY The primary objective of this renewal application is to elucidate how the metal-sequestering host-defense protein calprotectin (CP) modulates the physiology and interactions of Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa) using models that consider environmental cues relevant to infection. Metal ions are essential nutrients for all organisms, and pathogens must acquire these nutrients from the host to replicate and cause infection. In response to pathogen invasion, the human innate immune system enacts a metal- withholding response to limit the bioavailability of transition metal nutrients including manganese (Mn), iron (Fe), and zinc (Zn). This host response includes the deployment of CP and other metal-sequestering proteins at sites of infection. We discovered that CP withholds Fe(II) from and induces Fe-starvation responses by Pa and Sa, two bacterial pathogens that cause chronic polymicrobial infections in diverse patient populations, including lung infections in individuals with cystic fibrosis (CF). This hereditary disease predisposes individuals to life-long pulmonary infections, marked by debilitating exacerbations that reduce lung function. Notably, the CF lung becomes increasingly acidic and hypoxic as disease progresses, and Fe(II) becomes a predominant form of bioavailable Fe in this environment. Nevertheless, few studies have addressed how acidic pH and hypoxia impact microbial Fe homeostasis and the host metal-withholding response. We hypothesize that the Fe(II)-sequestering activity of CP has profound consequences on the physiology and virulence potential of Pa and Sa in diverse environmental milieus relevant to infection. We further propose that interactions between CP and these pathogens depend on environmental cues that vary both temporally and spatially at infection foci. In Aim 1, we will evaluate how mildly-acidic pH affects the metal-sequestration profile of CP as well as the physiology of Pa and Sa and their responses to CP and metal starvation. In Aim 2, we will examine how hypoxia affects the responses of Pa and Sa to metal starvation and CP. In Aim 3, we will extend these investigations to more complex in vitro models that incorporate relevant aspects of infection including co- cultures, biofilms, and additional metal-sequestering host-defense factors such as lactoferrin and S100A12. These investigations will enable future studies that address how CP and Fe drive the progression of CF lung infections and may guide the design and development of novel diagnostic, preventative, and therapeutic approaches to treat bacterial infections.