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Sporothrix brasiliensis is one of the leading etiological agents of sporotrichosis, a cutaneous and subcutaneous mycosis worldwide distributed. This organism has been recently associated with epidemic outbreaks in Brazil. Despite the medical relevance of this species, little is known about its virulence factors, and most of the information on this subject is extrapolated from Sporothrix schenckii. Here, we generated S. brasiliensis mutants, where GP70 was silenced. In S. schenckii, this gene encodes a glycoprotein with adhesive properties required for virulence. The S. brasiliensis GP70 silencing led to an abnormal cellular phenotype, with smaller, round yeast-like cells that aggregate. Cell aggregation was disrupted with glucanase, suggesting this phenotype is linked to changes in the cell wall. The cell wall characterization confirmed changes in the structural polysaccharide β-1,3-glucan, which increased in quantity and exposure at the cell surface. This was accompanied by a reduction in protein content and N-linked glycans. Mutant strains with high GP70-silencing levels showed minimal levels of 3-carboxy-cis,cis-muconate cyclase activity, this glycoprotein's predicted enzyme function, and decreased ability to bind laminin and fibronectin. These phenotypical changes coincided with abnormal interaction with human peripheral blood mononuclear cells, where production of IL-1β, IL-17, and IL-22 was reduced and the strong dependence on cytokine stimulation via mannose receptor was lost. Phagocytosis by monocyte-derived macrophages was increased and virulence attenuated in a Galleria mellonella larvae. In conclusion, Gp70 is an abundant cell wall glycoprotein in S. brasiliensis that contributes to virulence and proper interaction with innate immnune cells.

Proteins found within the fungal cell wall usually contain both N- and O-oligosaccharides. N-glycosylation is the process where these oligosaccharides (hereinafter: glycans) are attached to asparagine residues, while in O-glycosylation the glycans are covalently bound to serine or threonine residues. The PMT family is grouped into PMT1, PMT2, and PMT4 subfamilies. Using bioinformatics analysis within the Botrytis cinerea genome database, an ortholog to Saccharomyces cerevisiae Pmt4 and other fungal species was identified. The aim of this study was to assess the relevance of the bcpmt4 gene in B. cinerea glycosylation. For this purpose, the bcpmt4 gene was disrupted by homologous recombination in the B05.10 strain using a hygromycin B resistance cassette. Expression of bcpmt4 in S. cerevisiae ΔScpmt4 or ΔScpmt3 null mutants restored glycan levels like those observed in the parental strain. The phenotypic analysis showed that Δbcpmt4 null mutants exhibited significant changes in hyphal cell wall composition, including reduced mannan levels and increased amounts of chitin and glucan. Furthermore, the loss of bcpmt4 led to decreased glycosylation of glycoproteins in the B. cinerea cell wall. The null mutant lacking PMT4 was hypersensitive to a range of cell wall perturbing agents, antifungal drugs, and high hydrostatic pressure. Thus, in addition to their role in glycosylation, the PMT4 is required to virulence, biofilm formation, and membrane integrity. This study adds to our knowledge of the role of the B. cinerea bcpmt4 gene, which is involved in glycosylation and cell biology, cell wall formation, and antifungal response.

Sporothrix schenckii is one of the etiological agents of sporotrichosis, a cutaneous and subcutaneous infection distributed worldwide. Like other medically relevant fungi, its cell wall is a molecular scaffold to display virulence factors, such as protective pigments, hydrolytic enzymes, and adhesins. Cell wall proteins with adhesive properties have been previously reported, but only a handful of them have been identified and characterized. One of them is Gp70, an abundant cell wall protein mainly found on the surface of yeast-like cells. Since the protein also has a role in the activity of 3-carboxy-cis,cis-muconate cyclase and its abundance is low in highly virulent strains, its role in the Sporothrix–host interaction remains unclear. Here, a set of GP70-silenced strains was generated, and the molecular and phenotypical characterization was performed. The results showed that mutants with high silencing levels showed a significant reduction in the adhesion to laminin and fibrinogen, enzyme activity, and defects in the cell wall composition, which included reduced mannose, rhamnose, and protein content, accompanied by an increment in β-1,3-glucans levels. The cell wall N-linked glycan content was significantly reduced. These strains induced poor TNFα and IL-6 levels when interacting with human peripheral blood mononuclear cells in a dectin-1-, TLR2-, and TLR4-dependent stimulation. The IL-1β and IL-10 levels were significantly higher and were stimulated via dectin-1. Phagocytosis and stimulation of neutrophil extracellular traps by human granulocytes were increased in highly GP70-silenced strains. Furthermore, these mutants showed virulence attenuation in the invertebrate model Galleria mellonella. Our results demonstrate that Gp70 is a versatile protein with adhesin properties, is responsible for the activity of 3-carboxy-cis,cis-muconate cyclase, and is relevant for the S. schenckii–host interaction.

Sporotrichosis is a fungal disease caused by the members of the Sporothrix pathogenic clade, and one of the etiological agents is Sporothrix schenckii. The cell wall of this organism has been previously analyzed and thus far is known to contain an inner layer composed of chitin and β -glucans, and an outer layer of glycoproteins, which are decorated with mannose and rhamnose-containing oligosaccharides. The L-rhamnose biosynthesis pathway is common in bacteria but rare in members of the Fungi kingdom. Therefore, in this study, we aimed to disrupt this metabolic route to assess the contribution of rhamnose during the S. schenckii-host interaction. We identified and silenced in S. schenckii a functional ortholog of the bacterial rmlD gene, which encodes for an essential reductase for the synthesis of nucleotide-activated L-rhamnose. RmlD silencing did not affect fungal growth or morphology but decreased cell wall rhamnose content. Compensatory, the β-1,3-glucan levels increased and were more exposed at the cell surface. Moreover, when incubated with human peripheral blood mononuclear cells, the RmlD silenced mutants differentially stimulated cytokine production when compared with the wild-type strain, reducing TNFα and IL-6 levels and increasing IL-1 β and IL-10 production. Upon incubation with human monocyte-derived macrophages, the silenced strains were more efficiently phagocytosed than the wild-type strain. In both cases, our data suggest that rhamnose-based oligosaccharides are ligands that interact with TLR4. Finally, our findings showed that cell wall rhamnose is required for the S. schenckii virulence in the G. mellonella model of infection.

Sporothrix schenckii is a member of the Sporothrix pathogenic clade and one of the most common etiological agents of sporotrichosis, a subcutaneous fungal infection that affects both animal and human beings. Like other fungal pathogens, the Sporothrix cell wall is composed of structural polysaccharides and glycoproteins that are covalently modified with both N-linked and O-linked glycans. Thus far, little is known about the N-linked glycosylation pathway in this organism or its contribution to cell wall composition and interaction with the host. Here, we silenced ROT2, which encodes the catalytic subunit of the endoplasmic reticulum α-glucosidase II, a processing enzyme key for the N-linked glycan core processing. Silencing of ROT2 led to the accumulation of the Glc2Man9GlcNAC2 glycan core at the cell wall and a reduction in the total content of N-linked glycans found in the wall. However, the highly silenced mutants showed a compensatory mechanism with increased content of cell wall O-linked glycans. The phenotype of mutants with intermediate levels of ROT2 silencing was more informative, as they showed changes in the cell wall composition and exposure of β-1.3-glucans and chitin at the cell surface. Furthermore, the ability to stimulate cytokine production by human mononuclear cells was affected, along with the phagocytosis by human monocyte-derived macrophages, in a mannose receptor-, complement receptor 3-, and TLR4-dependent stimulation. In an insect model of experimental sporotrichosis, these mutant cells showed virulence attenuation. In conclusion, S. schenckii ROT2 is required for proper N-linked glycosylation, cell wall organization and composition, and interaction with the host.

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are etiological agents of sporotrichosis, a human subcutaneous mycosis. Although the protocols to evaluate Sporothrix virulence in animal models are well described, the cell preparation before inoculation is not standardized, and several culturing media are used to grow yeast-like cells. Here, we found that carbon or nitrogen limitation during fungal cell preparation negatively impacted the ability of S. schenckii and S. brasiliensis to kill Galleria mellonella larvae, but not S. globosa. The fungal growth conditions associated with the short median survival of animals were accompanied by increased hemocyte countings, phenoloxidase activity, and cytotoxicity. The fungal growth under carbon or nitrogen limitation also affected the cell wall composition of both S. schenckii and S. brasiliensis and showed increased exposure of β-1,3-glucan at the cell surface, while those growing conditions had a minimal impact on the S.globosa wall, which had higher levels of this polysaccharide exposed on the wall regardless of the culture condition. This polysaccharide exposure was linked to the increased ability of insect hemocytes to uptake fungal cells, suggesting that this is one of the mechanisms behind the lower virulence of S.globosa or cells from the other species grown in carbon or nitrogen limitation.

Fungal infections are a serious and increasing threat for human health, and one of the most frequent etiological agents for systemic mycoses is Candida spp. The gold standard to assess Candida virulence is the mouse model of systemic candidiasis, a restrictive, expensive, and time-consuming approach; therefore, invertebrate models have been proposed as alternatives. Galleria mellonella larvae have several traits that make them good candidates to study the fungal virulence. Here, we showed that a reduction in circulating hemocytes, increased melanin production, phenoloxidase, and lactate dehydrogenase activities were observed at 12 and 24 h postinoculation of highly virulent Candidatropicalis strains, while minimal changes in these parameters were observed in low-virulent strains. Similarly, the most virulent species Candida albicans, Candida tropicalis, Candida auris, Candida parapsilosis, and Candida orthopsilosis have led to significant changes in those parameters; while the low virulent species Candida guilliermondii, Candida krusei, and Candida metapsilosis induced modest variations in these immunological and cytotoxicity parameters. Since changes in circulating hemocytes, melanin production, phenoloxidase and lactate dehydrogenase activities showed a correlation with the larval median survival rates at 12 and 24 h postinoculation, we proposed them as candidates for early virulence predictors in G. mellonella.

Candida parapsilosis is an important, emerging opportunistic fungal pathogen. Highly mannosylated fungal cell wall proteins are initial contact points with host immune systems. In Candida albicans, Och1 is a Golgi 1,6-mannosyltransferase that plays a key role in the elaboration of the N-linked mannan outer chain. Here, we disrupted C. parapsilosis OCH1 to gain insights into the contribution of N-linked mannosylation to cell fitness and to interactions with immune cells. Loss of Och1 in C. parapsilosis resulted in cellular aggregation, failure of morphogenesis, enhanced susceptibility to cell wall perturbing agents and defects in wall composition. We removed the cell wall O-linked mannans by -elimination, and assessed the relevance of mannans during interaction with human monocytes. Results indicated that O-linked mannans are important for IL-1 stimulation in a dectin-1 and TLR4-dependent pathway; whereas both, N- and O-linked mannans are equally important ligands for TNF and IL-6 stimulation, but neither is involved in IL-10 production. Furthermore, mice infected with C. parapsilosis och1 null mutant cells had significantly lower fungal burdens compared to wild-type (WT)-challenged counterparts. Therefore, our data are the first to demonstrate that C. parapsilosis N- and O-linked mannans have different roles in host interactions than those reported for C. albicans.

Candidemia is an opportunistic mycosis with high morbidity and mortality rates. Even though Candida albicans is the main causative agent, other Candida species, such as Candida tropicalis, are relevant etiological agents of candidiasis and candidemia. Compared with C. albicans, there is currently limited information about C. tropicalis’ biological aspects, including those related to the cell wall and the interaction with the host. Currently, it is known that its cell wall contains O-linked mannans, and the contribution of these structures to cell fitness has previously been addressed using cells subjected to chemical treatments or in mutants where O-linked mannans and other wall components are affected. Here, we generated a C. tropicalis pmt2∆ null mutant, which was affected in the first step of the O-linked mannosylation pathway. The null mutant was viable, contrasting with C. albicans where this gene is essential. The phenotypical characterization showed that O-linked mannans were required for filamentation; proper cell wall integrity and organization; biofilm formation; protein secretion; and adhesion to extracellular matrix components, in particular to fibronectin; and type I and type II collagen. When interacting with human innate immune cells, it was found that this cell wall structure is dispensable for cytokine production, but mutant cells were more phagocytosed by monocyte-derived macrophages. Furthermore, the null mutant cells showed virulence attenuation in Galleria mellonella larvae. Thus, O-linked mannans are minor components of the cell wall that are involved in different aspects of C. tropicalis’ biology.