Candida albicans and Staphylococcus aureus reciprocally promote secretion of virulence factors

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 extracellular virulence factors (ECVFs) contribute to this process, the effects of C. albicans-S. aureus co-culturing on ECVF composition remain unknown. In this study we used mass spectrometry-based proteomics to investigate the effect of co-culturing on the extracellular proteins released by the S. aureus and C. albicans. Co- culturing of C. albicans and S. aureus promoted the secretion of 7 cytolytic, 11 proteolytic, and 3 lipolytic ECVFs. Interestingly, co-culturing of C. albicans Als1p/Als3p mutant alleviated the increase for the majority of the differentially changed C. albicans ECVFs, but not for S. aureus ECVFs, highlighting the importance of Als1p/Als3p in the secretion of C. albicans ECVFs. Of 27 detected S. aureus ECVFs, 17 were significantly increased in co-culturing. Among these, maintenance of pH alone in S. aureus monoculture increased five haemolytic proteins, i.e., alpha haemolysin (Hly/Hla), beta haemolysin (Hlb), and gamma haemolysin (HlgA-C) to a similar extent as the co-culture. In contrast, maintenance of pH diminished the increase of protease-like proteins, (phospho)lipases, delta hemolysin, and leukotoxin, suggesting that both pH-dependent and pH-independent C. albicans factors affect S. aureus ECVFs. A cytotoxicity assay demonstrated that the secretome from co-culture has higher cytotoxicity towards human oral cells (Ca 9-22 and HO1N1) than monoculture. Finally, co-culturing increased the levels of non-extracellular virulence factors from both C. albicans and S. aureus. Taken together, the co-culturing of C. albicans and S. aureus reciprocally promotes their virulence potential, which may provide insights into the synergistic lethality during their co-infection in vivo.