Calprotectin elicits aberrant iron starvation responses in <i>Pseudomonas aeruginosa</i> under anaerobic conditions

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that uses several mechanisms to survive in the iron-limiting host environment. The innate immune protein calprotectin (CP) sequesters ferrous iron [Fe(II)], among other divalent transition metal ions, to limit its availability to pathogens. CP levels are increased in individuals with cystic fibrosis (CF), a hereditary disease that leads to chronic pulmonary infection by <i>P. aeruginosa</i>. We previously showed that aerobic CP treatment of <i>P. aeruginosa</i> induces a multi-metal starvation response that alters expression of several virulence properties. However, the CF lung is a hypoxic environment due to the growth of <i>P. aeruginosa</i> in dense biofilms. Here, we report that anaerobic CP treatment of <i>P. aeruginosa</i> induces many processes associated with an aerobic iron starvation response, including decreased phenazine production and increased expression of the PrrF small regulatory RNAs (sRNAs). However, the iron starvation response elicited by CP in anaerobic conditions shows characteristics that are distinct from responses observed in aerobic growth, including a lack of siderophore production and increased induction of genes for the FeoAB Fe(II) and Phu heme uptake systems. Also distinct from aerobic conditions, CP treatment induces expression of genes for predicted manganese transporters MntH1 and MntH2 during anaerobic growth while eliciting a less robust zinc starvation response compared to aerobic conditions. Induction of <i>mntH2</i> is dependent on the PrrF sRNAs, suggesting a novel example of metal regulatory cross-talk. Thus, anaerobic CP treatment results in a multi-metal starvation response with key distinctions from aerobic conditions, revealing differences in <i>P. aeruginosa</i> metal homeostasis during anaerobic growth.IMPORTANCEIron is critical for most microbial pathogens, and the innate immune system sequesters this metal to limit microbial growth. Pathogens must overcome iron sequestration to survive during infection. For many pathogens, iron homeostasis has primarily been studied in aerobic conditions. Nevertheless, some host environments are hypoxic, including chronic lung infection sites in individuals with cystic fibrosis (CF). Here, we use the innate immune protein calprotectin, which sequesters divalent metal ions including Fe(II), to study the anaerobic iron starvation response of a common CF lung pathogen, <i>Pseudomonas aeruginosa</i>. We report several distinctions of this response during anaerobiosis, highlighting the importance of carefully considering the host environment when investigating the role of nutritional immunity in host-pathogen interactions.