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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.

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.

Sporothrix brasiliensis is the most pathogenic species, responsible for the Brazilian cat-transmitted sporotrichosis hyperendemic. In this scenario, an investigation of the pathogen-host interaction can provide relevant information for future treatment strategies. To this end, the invertebrate Galleria mellonella has proven to be a suitable alternative for evaluating the virulence of pathogenic fungi, since the insect immune system is similar to the mammalian innate immune response. The aim of this work was to investigate phenotypic and molecular aspects of the immune response of G. mellonella throughout the S. brasiliensis infection. Hemocyte density and the evolution of the fungal load were evaluated. In parallel, RT-qPCR expression analysis of genes encoding antimicrobial peptides ( Gallerimycin and Galiomycin ) and stress management genes ( C7 Contig 15362 and C8 Contig 19101 ) was conducted. The fungal load and hemocyte densities increased simultaneously and proportionally to the deleterious morphological events and larvae mortality. Gallerimycin , C7 Contig 15362 and C8 Contig 19101 genes were positively regulated (p < 0.05) at distinct moments of S. brasiliensis infection, characterizing a time-dependent and alternately modulated profile. Galiomycin gene expression remained unchanged. Our results contribute to the future proposal of potential alternative pathways for treating and consequently controlling S. brasiliensis zoonosis, a major public health issue in Latin America.

Sporotrichosis is a cutaneous mycosis that affects humans and animals and has a worldwide distribution. This infection is mainly caused by Sporothrix schenckii , Sporothrix brasiliensis , and Sporothrix globosa . Current research about anti- Sporothrix immunity has been mainly focused on S. schenckii and S. brasiliensis , using different types of human or animal immune cells. Granulocytes are a group of cells relevant for cytokine production, with the capacity for phagocytosis and the generation of neutrophil extracellular traps (NETs). Considering their importance, this study aimed to compare the capacity of human granulocytes to stimulate cytokines, uptake, and form NETs when interacting with different Sporothrix species. We found that conidia, germlings, and yeast-like cells from S. schenckii , S. brasiliensis , and S. globosa play an important role in the interaction with these immune cells, establishing morphology- and species-specific cytokine profiles. S. brasil-iensis tended to stimulate an anti-inflammatory cytokine profile, whilst the other two species had a proinflammatory one. S. globosa cells were the most phagocytosed cells, which occurred through a dectin-1-dependent mechanism, while the uptake of S. brasiliensis mainly occurred via TLR4 and CR3. Cell wall N -linked and O -linked glycans, along with β-1,3-glucan, played a significant role in the interaction of these Sporothrix species with human granulocytes. Finally, this study indicates that conidia and yeast-like cells are capable of inducing NETs, with the latter being a better stimulant. To the best of our knowledge, this is the first study that reports the cytokine profiles produced by human granulocytes interacting with Sporothrix cells.

Sporotrichosis is known as a subacute or chronic infection, which is caused by thermodimorphic fungi of the genus Sporothrix . It is a cosmopolitan infection, which is more prevalent in tropical and subtropical regions and can affect both humans and other mammals. The main etiological agents causing this disease are Sporothrix schenckii , Sporothrix brasiliensis , and Sporothrix globosa , which have been recognized as members of the Sporothrix pathogenic clade. Within this clade, S. brasiliensis is considered the most virulent species and represents an important pathogen due to its distribution and prevalence in different regions of South America, such as Brazil, Argentina, Chile, and Paraguay, and Central American countries, such as Panama. In Brazil, S. brasiliensis has been of great concern due to the number of zoonotic cases that have been reported over the years. In this paper, a detailed review of the current literature on this pathogen and its different aspects will be carried out, including its genome, pathogen-host interaction, resistance mechanisms to antifungal drugs, and the caused zoonosis. Furthermore, we provide the prediction of some putative virulence factors encoded by the genome of this fungal species.

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.

Fungal infections caused by Candida species have become a constant threat to public health, especially for immunocompromised patients, who are considered susceptible to this type of opportunistic infections. Candida albicans is known as the most common etiological agent of candidiasis; however, other species, such as Candida tropicalis , Candida parapsilosis , Nakaseomyces glabrata (previously known as Candida glabrata ), Candida auris , Candida guilliermondii , and Pichia kudriavzevii (previously named as Candida krusei ), have also gained great importance in recent years. The increasing frequency of the isolation of this non- albicans Candida species is associated with different factors, such as constant exposure to antifungal drugs, the use of catheters in hospitalized patients, cancer, age, and geographic distribution. The main concerns for the control of these pathogens include their ability to evade the mechanisms of action of different drugs, thus developing resistance to antifungal drugs, and it has also been shown that some of these species also manage to evade the host's immunity. These biological traits make candidiasis treatment a challenging task. In this review manuscript, a detailed update of the recent literature on the six most relevant non- albicans Candida species is provided, focusing on the immune response, evasion mechanisms, and new plant-derived compounds with antifungal properties.

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 Glc 2 Man 9 GlcNAC 2 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.

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.

Sporothrix schenckii 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 Galleria mellonella 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 Candida tropicalis 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 .

The secretory pathway in Candida albicans involves the protein translocation into the lumen of the endoplasmic reticulum and transport to the Golgi complex, where proteins undergo posttranslational modifications, including glycosylation and proteolysis. The Golgi-resident Kex2 protease is involved in such processing and disruption of its encoding gene affected virulence and dimorphism. These previous studies were performed using cells without URA3 or with URA3 ectopically placed into the KEX2 locus. Since these conditions are known to affect the cellular fitness and the host-fungus interaction, here we generated a kex2 Δ null mutant strain with URA3 placed into the neutral locus RPS1 . The characterization of this strain showed defects in the cell wall composition, with a reduction in the N -linked mannan content, and the increment in the levels of O -linked mannans, chitin, and β-glucans. The defects in the mannan content are likely linked to changes in Golgi-resident enzymes, as the α-1,2-mannosyltransferase and α-1,6-mannosyltransferase activities were incremented and reduced, respectively. The mutant cells also showed reduced ability to stimulate cytokine production and phagocytosis by human mononuclear cells and macrophages, respectively. Collectively, these data showed that loss of Kex2 affected the cell wall composition, the protein glycosylation pathways, and interaction with innate immune cells.

The recognition of fungal cells by the host immune system is key during the establishment of a protective anti-fungal response. Even though the immune system has evolved a vast number of processes to control these organisms, they have developed strategies to fight back, avoiding the proper recognition by immune components and thus interfering with the host protective mechanisms. Therefore, the strategies to evade the immune system are as important as the virulence factors and attributes that damage the host tissues and cells. Here, we performed a thorough revision of the main fungal tactics to escape from the host immunosurveillance processes. These include the composition and organization of the cell wall, the fungal capsule, the formation of titan cells, biofilms, and asteroid bodies; the ability to undergo dimorphism; and the escape from nutritional immunity, extracellular traps, phagocytosis, and the action of humoral immune effectors., Competing Interests: The authors declare no conflict of interest.