cotH Genes Are Necessary for Normal Spore Formation and Virulence in Mucor lusitanicus.

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of the CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to spore wall formation by affecting chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identify the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

Response of Human Neutrophil Granulocytes to the Hyphae of the Emerging Fungal Pathogen Curvularia lunata.

Curvularia lunata is an ascomycete filamentous fungus causing local and invasive phaeohyphomycoses in both immunocompromised and immunocompetent patients. Neutrophils are crucial participants of the first line host defense against fungal infections. They migrate to the infected site and eliminate the infectious agents by various mechanisms including phagocytoses, oxidative damage, or formation of neutrophil extracellular trap (NET). Neutropenia may be a risk factor for phaeohyphomycoses, and restoration of the neutrophil function can improve the outcome of the infection. In the present study, interaction of primary human neutrophil granulocytes with the hyphae C. lunata was examined and compared to that with the well characterized filamentous fungal pathogen Aspergillus fumigatus. Neutrophils could recognize the serum opsonized hyphae of C. lunata and attach to them. Myeloperoxidase release was also activated by a soluble factor present in the culture supernatant of the fungus. Induction of the oxidative burst was found to depend on serum opsonization of the hyphae. Although extracellular hydrogen peroxide production was induced, the fungus efficiently blocked the oxidative burst by acidifying the reaction environment. This blockage also affected the NET forming ability of the neutrophils.