Silencing of Sporothrix schenckii GP70 Reveals Its Contribution to Fungal Adhesion, Virulence, and the Host-Fungus Interaction.

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 ß-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-1ß 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.

Microevolution of Candida glabrata (Nakaseomyces glabrata) during an infection.

Candida glabrata (Nakaseomyces glabrata) is an emergent and opportunistic fungal pathogen that colonizes and persists in different niches within its human host. In this work, we studied five clinical isolates from one patient (P7), that have a clonal origin, and all of which come from blood cultures except one, P7-3, obtained from a urine culture. We found phenotypic variation such as sensitivity to high temperature, oxidative stress, susceptibility to two classes of antifungal agents, and cell wall porosity. Only isolate P7-3 is highly resistant to the echinocandin caspofungin while the other four isolates from P7 are sensitive. However, this same isolate P7-3, is the only one that displays susceptibility to fluconazole (FLC), while the rest of the isolates are resistant to this antifungal. We sequenced the PDR1 gene which encodes a transcription factor required to induce the expression of several genes involved in the resistance to FLC and found that all the isolates encode for the same Pdr1 amino acid sequence except for the last isolate P7-5, which contains a single amino acid change, G1099C in the putative Pdr1 transactivation domain. Consistent with the resistance to FLC, we found that the CDR1 gene, encoding the main drug efflux pump in C. glabrata, is highly overexpressed in the FLC-resistant isolates, but not in the FLC-sensitive P7-3. In addition, the resistance to FLC observed in these isolates is dependent on the PDR1 gene. Additionally, we found that all P7 isolates have a different proportion of cell wall carbohydrates compared to our standard strains CBS138 and BG14. In P7 isolates, mannan is the most abundant cell wall component, whereas ß-glucan is the most abundant component in our standard strains. Consistently, all P7 isolates have a relatively low cell wall porosity compared to our standard strains. These data show phenotypic and genotypic variability between clonal isolates from different niches within a single host, suggesting microevolution of C. glabrata during an infection.

The Emerging Pathogen Candida metapsilosis: Biological Aspects, Virulence Factors, Diagnosis, and Treatment.

Fungal infections represent a constant and growing menace to public health. This concern is due to the emergence of new fungal species and the increase in antifungal drug resistance. Mycoses caused by Candida species are among the most common nosocomial infections and are associated with high mortality rates when the infection affects deep-seated organs. Candida metapsilosis is part of the Candida parapsilosis complex and has been described as part of the oral microbiota of healthy individuals. Within the complex, this species is considered the least virulent; however, the prevalence has been increasing in recent years, as well as an increment in the resistance to some antifungal drugs. One of the main concerns of candidiasis caused by this species is the wide range of clinical manifestations, ranging from tissue colonization to superficial infections, and in more severe cases it can spread, which makes diagnosis and treatment difficult. The study of virulence factors of this species is limited, however, proteomic comparisons between species indicate that virulence factors in this species could be similar to those already described for C. albicans. However, differences may exist, taking into account changes in the lifestyle of the species. Here, we provide a detailed review of the current literature about this organism, the caused disease, and some sharing aspects with other members of the complex, focusing on its biology, virulence factors, the host-fungus interaction, the identification, diagnosis, and treatment of infection.

Contribution of N-Linked Mannosylation Pathway to Candida parapsilosis and Candida tropicalis Biofilm Formation.

Background: Mycoses are a growing threat to human health, and systemic candidiasis caused by Candida parapsilosis and Candida tropicalis is frequent in immunocompromised patients. Biofilm formation is a virulence factor found in these organisms, as sessile cells adhere to surfaces, the stratification and production of extracellular matrix provides protection and resistance to antifungal drugs. Previous evidence indicated that the N-linked mannosylation pathway is relevant to C. albicans biofilms, but its contribution to other species remains unknown. Methods: C. parapsilosis and C. tropicalis och1∆ mutants, which have a disrupted N-linked mannosylation pathway, were used to form biofilms. In addition, wild-type and mutant cells were also treated to remove N-linked mannans or block this pathway. Biofilms were analyzed by quantifying the included fungal biomass, and extracellular matrix components. Moreover, gene expression and secreted hydrolytic enzymes were also quantified in these biofilms. Results: The och1∆ mutants showed a reduced ability to form biofilms in both fungal species when compared to the wild-type and control strains. This observation was confirmed by trimming N-linked mannans from walls or blocking the pathway with tunicamycin B. According to this observation, mutant, and treated cells showed an altered composition of the extracellular matrix and increased susceptibility to antifungal drugs when compared to control or untreated cells. The gene expression of secreted virulence factors, such as aspartyl proteinases and phospholipases, was normal in all the tested cells but the secreted activity was reduced, suggesting a defect in the secretory pathway, which was later confirmed by treating cells with brefeldin A. Conclusion: Proper N-linked mannosylation is required for biofilm formation in both C. parapsilosis and C. tropicalis. Disruption of this posttranslational modification affected the secretory pathway, offering a link between glycosylation and biofilm formation.

Sporothrix brasiliensis Infection Modulates Antimicrobial Peptides and Stress Management Gene Expression in the Invertebrate Biomodel Galleria mellonella.

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.

Differential Recognition of Clinically Relevant Sporothrix Species by Human Granulocytes.

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 ß-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.

Differential Recognition of Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa by Human Monocyte-Derived Macrophages and Dendritic Cells.

Background: Sporotrichosis is a mycosis frequently caused by Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa. The cell wall is a species-specific fungal structure with a direct role in activating the host's immune response. The current knowledge about anti-Sporothrix immunity comes from studies using S. schenckii or S. brasiliensis and murine cells. Macrophages and dendritic cells detect and eliminate pathogens, and although the function of these cells links innate with adaptive immunity, little is known about their interaction with Sporothrix spp. Methods: S. schenckii, S. brasiliensis, and S. globosa conidia or yeast-like cells were co-incubated with human monocyte-derived macrophages or dendritic cells, and the phagocytosis and cytokine stimulation were assessed. These interactions were also performed in the presence of specific blocking agents of immune receptors or fungal cells with altered walls to analyze the contribution of these molecules to the immune cell-fungus interaction. Results: Both types of immune cells phagocytosed S. globosa conidia and yeast-like cells to a greater extent, followed by S. brasiliensis and S. schenckii. Furthermore, when the wall internal components were exposed, the phagocytosis level increased for S. schenckii and S. brasiliensis, in contrast to S. globosa. Thus, the cell wall components have different functions during the interaction with macrophages and dendritic cells. S. globosa stimulated an increased proinflammatory response when compared to the other species. In macrophages, this was a dectin-1-, mannose receptor-, and TLR2-dependent response, but dectin-1- and TLR2-dependent stimulation in dendritic cells. For S. schenckii and S. brasiliensis, cytokine production was dependent on the activation of TLR4, CR3, and DC-SIGN. Conclusion: The results of this study indicate that these species are recognized by immune cells differently and that this may depend on both the structure and cell wall organization of the different morphologies.

Current Progress in Sporothrix brasiliensis Basic Aspects.

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.

Differential Recognition of Clinically Relevant Sporothrix Species by Human Mononuclear Cells.

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 ß-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.

Candida haemulonii Complex and Candida auris: Biology, Virulence Factors, Immune Response, and Multidrug Resistance.

There is worldwide concern about the constant increase in infections caused by Candida species that are multiresistant to antifungal drugs. The most common candidiasis is caused by Candida albicans, however, the species of the Candida haemulonii complex and Candida auris are emerging opportunistic pathogens, which isolation from clinical samples has significantly increased in the past years. The special interest in the study of these species lies in their ability to evade the action of antifungal drugs, such as amphotericin B, azoles, and echinocandins. In addition, the phenotypic changes of these species have given them the ability to easily adapt to environmental changes, including the host milieu and immunity. In this paper, a detailed review of the current literature on the C. haemulonii complex and C. auris is shown, analyzing aspects such as biology, immune response, putative virulence factors, infection, treatment, and the current strategies for diagnosis.

Silencing of ROT2, the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii-Host Interaction.

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 ß-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.

Candida lusitaniae: Biology, Pathogenicity, Virulence Factors, Diagnosis, and Treatment.

The incidence of fungal infections is increasing at an alarming rate and has posed a great challenge for science in recent years. The rise in these infections has been related to the increase in immunocompromised patients and the resistance of different species to antifungal drugs. Infections caused by the different Candida species, especially Candida albicans, are one of the most common mycoses in humans, and the etiological agents are considered opportunistic pathogens associated with high mortality rates when disseminated infections occur. Candida lusitaniae is considered an emerging opportunistic pathogen that most frequently affects immunocompromised patients with some comorbidity. Although it is a low-frequency pathogen, and the mortality rate of C. lusitaniae-caused candidemia does not exceed 5%, some isolates are known to be resistant to antifungals such as amphotericin B, 5-fluorocytosine, and fluconazole. In this paper, a detailed review of the current literature on this organism and its different aspects, such as its biology, possible virulence factors, pathogen-host interaction, diagnosis, and treatment of infection, is provided. Of particular interest, through Blastp analysis we predicted possible virulence factors in this species.

Special Issue "Sporothrix and Sporotrichosis 2.0".

Sporotrichosis is a chronic fungal disease of humans and other mammals that often affects the skin and subcutaneous tissues and, rarely, deep-seated organs (most frequently in immunocompetent hosts) [...].

Genome-Wide Mutant Screening in Yeast Reveals that the Cell Wall is a First Shield to Discriminate Light From Heavy Lanthanides.

The rapidly expanding utilization of lanthanides (Ln) for the development of new technologies, green energies, and agriculture has raised concerns regarding their impacts on the environment and human health. The absence of characterization of the underlying cellular and molecular mechanisms regarding their toxicity is a caveat in the apprehension of their environmental impacts. We performed genomic phenotyping and molecular physiology analyses of Saccharomyces cerevisiae mutants exposed to La and Yb to uncover genes and pathways affecting Ln resistance and toxicity. Ln responses strongly differed from well-known transition metal and from common responses mediated by oxidative compounds. Shared response pathways to La and Yb exposure were associated to lipid metabolism, ion homeostasis, vesicular trafficking, and endocytosis, which represents a putative way of entry for Ln. Cell wall organization and related signaling pathways allowed for the discrimination of light and heavy Ln. Mutants in cell wall integrity-related proteins (e.g., Kre1p, Kre6p) or in the activation of secretory pathway and cell wall proteins (e.g., Kex2p, Kex1p) were resistant to Yb but sensitive to La. Exposure of WT yeast to the serine protease inhibitor tosyl phenylalanyl chloromethyl ketone mimicked the phenotype of kex2∆ under Ln, strengthening these results. Our data also suggest that the relative proportions of chitin and phosphomannan could modulate the proportion of functional groups (phosphates and carboxylates) to which La and Yb could differentially bind. Moreover, we showed that kex2∆, kex1∆, kre1∆, and kre6∆ strains were all sensitive to light Ln (La to Eu), while being increasingly resistant to heavier Ln. Finally, shotgun proteomic analyses identified modulated proteins in kex2∆ exposed to Ln, among which several plasmalemma ion transporters that were less abundant and that could play a role in Yb uptake. By combining these different approaches, we unraveled that cell wall components not only act in Ln adsorption but are also active signal effectors allowing cells to differentiate light and heavy Ln. This work paves the way for future investigations to the better understanding of Ln toxicity in higher eukaryotes.

Biological and Clinical Attributes of Sporothrix globosa, a Causative Agent of Sporotrichosis.

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.

Mucormycosis and COVID-19-Associated Mucormycosis: Insights of a Deadly but Neglected Mycosis.

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.

The Search for Cryptic L-Rhamnosyltransferases on the Sporothrix schenckii Genome.

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.

Current Models to Study the Sporothrix-Host Interaction.

Sporotrichosis is a worldwide distributed subcutaneous mycosis that affects mammals, including human beings. The infection is caused by members of the Sporothrix pathogenic clade, which includes Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa. The fungus can be acquired through traumatic inoculation of conidia growing in vegetal debris or by zoonotic transmission from sick animals. Although is not considered a life-threatening disease, it is an emergent health problem that affects mostly immunocompromised patients. The sporotrichosis causative agents differ in their virulence, host range, and sensitivity to antifungal drugs; therefore, it is relevant to understand the molecular bases of their pathogenesis, interaction with immune effectors, and mechanisms to acquired resistance to antifungal compounds. Murine models are considered the gold standard to address these questions; however, some alternative hosts offer numerous advantages over mammalian models, such as invertebrates like Galleria mellonella and Tenebrio molitor, or ex vivo models, which are useful tools to approach questions beyond virulence, without the ethical or budgetary features associated with the use of animal models. In this review, we analyze the different models currently used to study the host-Sporothrix interaction.