Calprotectin protects <i>Staphylococcus aureus</i> in coculture with <i>Pseudomonas aeruginosa</i> by attenuating quorum sensing and decreasing the production of pseudomonal antimicrobials

<i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i> cause debilitating polymicrobial infections in diverse patient populations. Studies of these bacterial pathogens in coculture have shown that environmental variables, including Fe availability and the host-defense protein calprotectin (CP), impact coculture dynamics. To decipher how CP modulates interactions between <i>P. aeruginosa</i> and <i>S. aureus</i>, we employed dual-species RNA-seq to examine the transcriptional responses of both pathogens in coculture to CP treatment and metal depletion. Analysis of these responses revealed that, for both <i>P. aeruginosa</i> and <i>S. aureus</i>, CP treatment not only induced gene expression changes consistent with single- and multi-metal starvation responses but also induced gene expression changes that were not observed under metal limitation. For <i>P. aeruginosa</i>, CP treatment induced gene expression changes pointing to a shift in chorismate flux away from alkylquinolone and phenazine biosynthesis and toward folate biosynthesis. These observations were consistent with decreased production of alkylquinolones by <i>P. aeruginosa</i>, including the potent anti-staphylococcal alkylquinolone N-oxides. CP treatment altered the levels of two quorum-sensing molecules, 3-oxo-C₁₂-homoserine lactone and C₄-homoserine lactone, produced by <i>P. aeruginosa</i>. In addition, CP treatment enhanced the ability of <i>S. aureus</i> to mount Fe-starvation responses and caused <i>S. aureus</i> to express host virulence genes. This analysis illuminated the physiological consequences of CP treatment that extend beyond metal starvation and impact interspecies interactions. Our findings provide a working model in which CP effectively disarms <i>P. aeruginosa</i> by inhibiting the production of anti-staphylococcal factors and boosts the ability of <i>S. aureus</i> to protect itself from attack.IMPORTANCEThe innate immune protein calprotectin (CP) defends the host against bacterial pathogens by sequestering multiple essential nutrient metal ions at infection sites. In addition to this role in nutritional immunity, CP promotes the survival of <i>Staphylococcus aureus</i> in coculture with <i>Pseudomonas aeruginosa</i>, an effect that is independent of its metal-sequestering function. In this work, we sought to understand how CP modulates this interspecies interaction by evaluating the transcriptional responses of <i>P. aeruginosa</i> and <i>S. aureus</i> to CP and metal limitation in cocultures. Our study revealed that CP attenuates the ability of <i>P. aeruginosa</i> to attack <i>S. aureus</i> with anti-staphylococcal factors and enhances the capacity of <i>S. aureus</i> to withstand this assault, effects that are not recapitulated by metal limitation. This work provides a new understanding of how CP modulates microbial interactions that are relevant to human health.