Laura Cristina García Carnero

Sporotrichosis is a human and animal fungal infection distributed worldwide that is caused by the thermodimorphic species of the Sporothrix pathogenic clade, which includes Sporothrix brasiliensis, Sporothrix schenckii, and Sporothrix globosa. The cell wall composition and the immune response against the Sporothrix species have been studied mainly in S. brasiliensis and S. schenckii, whilst little is known about the S. globosa cell wall and the immune response that its components trigger. Therefore, in this study, we aimed to analyze the cell wall composition of S. globosa in three morphologies (germlings, conidia, and yeast-like cells) and the differences in cytokine production when human peripheral blood mononuclear cells (PBMCs) interact with these morphotypes, using S. schenckii and S. brasiliensis as a comparison. We found that S. globosa conidia and yeast-like cells have a higher cell wall chitin content, while all three morphologies have a higher β-1,3-glucan content, which was found most exposed at the cell surface when compared to S. schenckii and S. brasiliensis. In addition, S. globosa has lower levels of mannose- and rhamnose-based glycoconjugates, as well as of N- and O-linked glycans, indicating that this fungal cell wall has species-specific proportions and organization of its components. When interacting with PBMCs, S. brasiliensis and S. globosa showed a similar cytokine stimulation profile, but with a higher stimulation of IL-10 by S. globosa. Additionally, when the inner cell wall components of S. globosa were exposed at the surface or N- and O-glycans were removed, the cytokine production profile of this species in its three morphotypes did not significantly change, contrasting with the S. schenckii and S. brasiliensis species that showed different cytokine profiles depending on the treatment applied to the walls. In addition, it was found that the anti-inflammatory response stimulated by S. globosa was dependent on the activation of dectin-1, mannose receptor, and TLR2, but not TLR4. All of these results indicate that the cell wall composition and structure of the three Sporothrix species in the three morphologies are different, affecting their interaction with human PBMCs and generating species-specific cytokine profiles.

Moonlighting proteins represent an intriguing area of cell biology, due to their ability to perform two or more unrelated functions in one or many cellular compartments. These proteins have been described in all kingdoms of life and are usually constitutively expressed and conserved proteins with housekeeping functions. Although widely studied in pathogenic bacteria, the information about these proteins in pathogenic fungi is scarce, but there are some reports of their functions in the etiological agents of the main human mycoses, such as Candida spp., Paracoccidioides brasiliensis, Histoplasma capsulatum, Aspergillus fumigatus, Cryptococcus neoformans, and Sporothrix schenckii. In these fungi, most of the described moonlighting proteins are metabolic enzymes, such as enolase and glyceraldehyde-3-phosphate dehydrogenase; chaperones, transcription factors, and redox response proteins, such as peroxiredoxin and catalase, which moonlight at the cell surface and perform virulence-related processes, contributing to immune evasion, adhesions, invasion, and dissemination to host cells and tissues. All moonlighting proteins and their functions described in this review highlight the limited information about this biological aspect in pathogenic fungi, representing this a relevant opportunity area that will contribute to expanding our current knowledge of these organisms’ pathogenesis.

The fungal cell wall is an attractive structure to look for new antifungal drug targets and for understanding the host-fungus interaction. Sporothrix schenckii is one of the main causative agents of both human and animal sporotrichosis and currently is the species most studied of the Sporothrix genus. The cell wall of this organism has been previously analyzed, and rhamnoconjugates are signature molecules found on the surface of both mycelia and yeast-like cells. Similar to other reactions where sugars are covalently linked to other sugars, lipids, or proteins, the rhamnosylation process in this organism is expected to involve glycosyltransferases with the ability to transfer rhamnose from a sugar donor to the acceptor molecule, i.e., rhamnosyltransferases. However, no obvious rhamnosyltransferase has thus far been identified within the S. schenckii proteome or genome. Here, using a Hidden Markov Model profile strategy, we found within the S. schenckii genome five putative genes encoding for rhamnosyltransferases. Expression analyses indicated that only two of them, named RHT1 and RHT2, were significantly expressed in yeast-like cells and during interaction with the host. These two genes were heterologously expressed in Escherichia coli, and the purified recombinant proteins showed rhamnosyltransferase activity, dependent on the presence of UDP-rhamnose as a sugar donor. To the best of our knowledge, this is the first report about rhamnosyltransferases in S. schenckii.

The ongoing COVID-19 pandemic has quickly become a health threat worldwide, with high mortality and morbidity among patients with comorbidities. This viral infection promotes the perfect setting in patients for the development of opportunistic infections, such as those caused by fungi. Mucormycosis, a rare but deadly fungal infection, has recently increased its incidence, especially in endemic areas, since the onset of the pandemic. COVID-19-associated mucormycosis is an important complication of the pandemic because it is a mycosis hard to diagnose and treat, causing concern among COVID-19-infected patients and even in the already recovered population. The risk factors for the development of mucormycosis in these patients are related to the damage caused by the SARS-CoV-2 itself, the patient’s overstimulated immune response, and the therapy used to treat COVID-19, causing alterations such as hyperglycemia, acidosis, endothelial and lung damage, and immunosuppression. In this review, the molecular aspects of mucormycosis and the main risk factors for the development of COVID-19-associated mucormycosis are explained to understand this virus–fungi–host interaction and highlight the importance of this neglected mycosis.

Sporotrichosis is an important subcutaneous mycosis with high prevalence and threat to human and animal health worldwide. Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main etiological agents of this disease; and even though many efforts have been made recently to understand the Sporothrix-host interaction, little is known about S. globosa, an underestimated species. This organism shows the lowest virulence among the members of the Sporothrix pathogenic clade and represents an important pathogenic agent due to its global distribution. Here, we offer a review with all the known information about S. globosa, including its genome and proteomic information, and compare it with S. schenckii and S. brasiliensis, to explain the differences observed among these species, in terms of virulence, the host immune response, and the antifungal sensitivity. Also, we provide the gene prediction of some S. globosa putative virulence factors.

Sporothrixschenckii is one of the etiological agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. Its cell wall contains a glycoconjugate composed of rhamnose, mannose, glucuronic acid, and proteins, named peptidorhamnomannan, which harbors important Sporothrix-specific immunogenic epitopes. Although the peptidorhamnomannan carbohydrate moiety has been extensively studied, thus far, little is known about the protein core. Here, using LC-MS/MS, we analyzed the S.schenckii peptidorhamnomannan peptide fraction and generated mass signals of 325 proteins, most of them likely to be moonlighting proteins. Among the identified proteins, chaperonin GroEL/Hsp60 and the uncharacterized protein Pap1 were selected for further analysis. Both proteins were heterologously expressed in bacteria, and they showed adhesive properties to the extracellular matrix proteins laminin, elastin, fibrinogen, and fibronectin, although Pap1 also was bound to type-I and type-II collagen. The inoculation of concentrations higher than 40 μg of these proteins, separately, increased immune effectors in the hemolymph of Galleriamellonella larvae and protected animals from an S.schenckii lethal challenge. These observations were confirmed when yeast-like cells, pre-incubated with anti-rHsp60 or anti-rPap1 antibodies were used to inoculate larvae. The animals inoculated with pretreated cells showed increased survival rates when compared to the control groups. In conclusion, we report that Hsp60 and Pap1 are part of the cell wall peptidorhamnomannan, can bind extracellular matrix components, and contribute to the S.schenckii virulence. To our knowledge, this is the first report about moonlighting protein in the S.schenckii cell wall with an important role during the pathogen–host interaction.

Protein glycosylation is a highly conserved post-translational modification among organisms. It plays fundamental roles in many biological processes, ranging from protein trafficking and cell adhesion to host–pathogen interactions. According to the amino acid side chain atoms to which glycans are linked, protein glycosylation can be divided into two major categories: N-glycosylation and O-glycosylation. However, there are other types of modifications such as the addition of GPI to the C-terminal end of the protein. Besides the importance of glycoproteins in biological functions, they are a major component of the fungal cell wall and plasma membrane and contribute to pathogenicity, virulence, and recognition by the host immunity. Given that this structure is absent in host mammalian cells, it stands as an attractive target for developing selective compounds for the treatment of fungal infections. This review focuses on describing the relationship between protein glycosylation and the host–immune interaction in medically relevant fungal species.

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.

Species of the Sporothrix complex are the etiological agents of sporotrichosis, an important subcutaneous mycosis with several clinical forms and an increasing incidence around the world that affects humans and other mammals. The immunological mechanisms involved in the prevention and control of this mycosis are not entirely understood. Many reports have suggested that cell-mediated immunity has an essential role in the development of the disease, being the primary response controlling it, while only recent data supports that the humoral response is essential for the appropriate control. This mycosis is a challenge for diagnosis since the culture and isolation of the organism are time-consuming and complicated; reasons that have led to the study of fungus antigenic molecules capable of generating a detectable humoral response. The treatment for this disease includes the use of several antifungal drugs like itraconazole, amphotericin B, caspofungin, fluconazole, and the combination between them among others such as the extract of Vismia guianensis.