The human innate immune protein calprotectin elicits a multi-metal starvation response in Pseudomonas aeruginosa

Abstract

To combat infections, the mammalian host limits availability of essential transition metals such as iron (Fe), zinc (Zn), and manganese (Mn) in a strategy termed "nutritional immunity". The innate immune protein calprotectin (CP) contributes to nutritional immunity by sequestering these metals to exert antimicrobial activity against a broad range of microbial pathogens. One such pathogen is Pseudomonas aeruginosa, which causes opportunistic infections in vulnerable populations including individuals with cystic fibrosis. CP was previously shown to withhold Fe(II) and Zn(II) from P. aeruginosa and induce Fe- and Zn-starvation responses in this pathogen. In this work, we performed quantitative, label-free proteomics to further elucidate how CP impacts metal homeostasis pathways in P. aeruginosa. We report that CP induces an incomplete Fe-starvation response, as many Fe-containing proteins that are repressed by Fe limitation are not affected by CP treatment. The Zn-starvation response elicited by CP seems to be more complete than the Fe-starvation response and includes increases in Zn transporters and Zn-independent proteins. CP also induces the expression of membrane-modifying proteins, and metal-depletion studies indicate this response results from the sequestration of multiple metals. Moreover, the increased expression of membrane-modifying enzymes upon CP treatment correlates with increased resistance to polymyxin B. Thus, response of P. aeruginosa to CP treatment includes both single and multi-metal starvation responses and includes many factors related to virulence potential, broadening our understanding of this pathogens interaction with the host. ImportanceTransition metals are critical for growth and infection by all pathogens, and the innate immune system withholds these metals from pathogens to limit their growth in a strategy termed "nutritional immunity". While multi-metal depletion by the host is appreciated, the majority of metal depletion studies have focused on individual metald. Here we use the innate immune protein calprotectin (CP), which complexes with several metals including iron (Fe), zinc (Zn), and manganese (Mn), and the opportunistic pathogen Pseudomonas aeruginosa to investigate multi-metal starvation. Using an unbiased label-free proteomics response, we demonstrate that multi-metal withholding by CP induces a regulatory response that is not merely additive of individual metal starvation responses, including the induction of Lipid A modification enzymes.