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

The goal of this study was to decipher how calprotectin (CP) treatment and metal depletion impact interspecies interactions and culture dynamics for Pseudomonas aeruginosa and Staphylococcus aureus in monoculture and in coculture. The RNA-seq study was designed to profile transcriptional responses to CP and metal depletion. To achieve this goal, the study design employed a chemically defined medium (CDM) prepared from trace metals basis reagents, which is supplemented with defined metal concentrations representative of physiologically relevant metal levels in sputum samples from patients with cystic fibrosis. Metal-replete CDM is supplemented with 0.3 micromolar Mn, 5 micromolar Fe, 0.1 micromolar Cu, 0.1 micromolar Ni, and 6 micromolar Zn. To model single-metal depletion, Mn, Fe or Zn was omitted from the supplementation of metal-replete CDM (Mn-depleted, Fe-depleted and Zn-depleted CDM). To model multi-metal depletion, Mn, Fe and Zn were omitted from the supplementation of metal-replete CDM (metal-depleted CDM). For CP treatment, 20 micromolar of CP in working buffer (20 mM HEPES, 100 mM NaCl, pH 7.5) was added to metal-replete CDM. In all other cases, the equivalent volume of working buffer was added as a control. Overall, 30 samples were analyzed in total in this experiment, and metal-replete CDM served as the untreated control. For P. aeruginosa and S. aureus monocultures, three biological replicates of cultures grown in metal-replete CDM supplemented with or without CP were analyzed for each species, for a total of 12 samples. For P. aeruginosa and S. aureus cocultures, three biological replicates of cultures grown in metal-replete CDM supplemented with or without CP, Mn-depleted CDM, Fe-depleted CDM, Zn-depleted CDM and metal-depleted CDM were analyzed, for a total of 18 samples. All samples were grown for 6 hours at 37 degrees Celsius in aerobic conditions with shaking at 250 rpm.