Candida albicans and Staphylococcus aureus reciprocally promote their virulence factor secretion and pro-inflammatory effects

Co-infections of Candida albicans and Staphylococcus aureus can significantly increase morbidity and mortality. This synergism is linked to the interactions between C. albicans and S. aureus that allow for staphylococcal co-invasion and dissemination. While it is known that virulence factors contribute to this process, the effect of C. albicans-S. aureus coculturing on virulence factors composition and macrophage-mediated inflammation remain unknown. In this study, we used mass spectrometry-proteomics to investigate the effect of coculturing on the composition of the >3 kDa protein fraction of the S. aureus and C. albicans secretome, as well as the secretomal cytotoxicity and proinflammatory effects on human oral cells and immune cells. Results showed that coculturing of C. albicans and S. aureus promoted the secretion of 7 cytolytic, 11 proteolytic, and 3 lipolytic extracellular virulence factors (ECVFs). While the majority of differentially released C. albicans ECVFs were attributable to Als1/Als3-mediated interactions, changes in S. aureus secretomal ECVF composition were mainly attributable to the presence of C. albicans or its pH maintenance. By analysing both secreted and non-secreted virulence factors, it appears that coculturing promotes C. albicans hypha formation together with β-glucan masking, suggesting that coculturing enhances both C. albicans invasion and immune evasion. In addition, coculturing increased the cytotoxic potential of the secretome towards human oral epithelial cells and macrophages compared to the summed cytotoxic effect of both monoculture secretomes. Under non-cytotoxic conditions, the secretome of C. albicans-S. aureus coculture promoted macrophage activation and increased gene transcription of pro-inflammatory pathways compared to monoculture secretomes. Surprisingly, ALS1/ALS3 knock out has marginal effects on the secretome-increased proinflammation in macrophages. Our findings support that C. albicans and S. aureus reciprocally promotes their virulence potential, cytotoxicity, and proinflammatory effects, which may provide insights into the synergistic lethality during their coinfection in vivo.