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Manuela Gómez-Gaviria, Luisa M Contreras-López, Julieta I Aguilera-Domínguez, Héctor M Mora-Montes Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, Gto, MéxicoCorrespondence: Héctor M Mora-Montes, Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, Col. Noria Alta, C.P, Guanajuato, Gto, 36050, México, Tel +52 473-7320006 Ext. 8193, Fax +52 473-7320006 Ext. 8153, Email hmora@ugto.mxAbstract: Fungal infections represent a worldwide concern for public health, due to their prevalence and significant increase in cases each year. Among the most frequent mycoses are those caused by members of the genera Candida, Cryptococcus, Aspergillus, Histoplasma, Pneumocystis, Mucor, and Sporothrix, which have been treated for years with conventional antifungal drugs, such as flucytosine, azoles, polyenes, and echinocandins. However, these microorganisms have acquired the ability to evade the mechanisms of action of these drugs, thus hindering their treatment. Among the most common evasion mechanisms are alterations in sterol biosynthesis, modifications of drug transport through the cell wall and membrane, alterations of drug targets, phenotypic plasticity, horizontal gene transfer, and chromosomal aneuploidies. Taking into account these problems, some research groups have sought new therapeutic alternatives based on drug repositioning. Through repositioning, it is possible to use existing pharmacological compounds for which their mechanism of action is already established for other diseases, and thus exploit their potential antifungal activity. The advantage offered by these drugs is that they may be less prone to resistance. In this article, a comprehensive review was carried out to highlight the most relevant repositioning drugs to treat fungal infections. These include antibiotics, antivirals, anthelmintics, statins, and anti-inflammatory drugs.Keywords: action mechanisms, antifungal resistance, clinical pathogens, repositioning, new treatments

Manuela Gómez-Gaviria, Laura C García-Carnero, Dario A Baruch-Martínez, Héctor M Mora-Montes Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, Gto., MéxicoCorrespondence: Héctor M Mora-Montes, Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, col. Noria Alta, C.P. 36050, Guanajuato, Gto., México, Tel +52 473-7320006 Ext. 8193, Fax +52 473-7320006 Ext. 8153, Email hmora@ugto.mxAbstract: 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.Keywords: antifungal resistance, candidiasis, Candida parapsilosis complex, emerging pathogens, virulence factors

Diana M Clavijo-Giraldo,1 Luis A Pérez-García,1,2 Marco J Hernández-Chávez,1 Iván Martínez-Duncker,3 Héctor M Mora-Montes1 1Departamento de Biología, Universidad de Guanajuato, Guanajuato, Gto., México; 2Facultad de Estudios Profesionales Zona Huasteca, Universidad Autónoma de San Luis Potosí, Ciudad Valles, San Luis Potosí, México; 3Laboratorio de Glicobiología Humana y Diagnóstico Molecular; Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, MéxicoCorrespondence: Héctor M Mora-Montes, Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, Col. Noria Alta, C.P, Guanajuato, Gto., 36050, México, Tel +52 473-7320006 Ext. 8193, Fax +52 473-7320006 Ext. 8153, Email hmora@ugto.mxBackground: 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.Keywords: secretion, virulence factor, phospholipase, aspartyl proteinase, antifungal drug resistance

Manuela Gómez-Gaviria,1 Iván Martínez-Duncker,2 Laura C García-Carnero,1 Héctor M Mora-Montes1 1Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Guanajuato, Guanajuato México; 2Laboratorio de Glicobiología Humana y Diagnóstico Molecular; Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, MéxicoCorrespondence: Héctor M Mora-Montes, Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, Col. Noria Alta, C.P, Guanajuato, Guanajuato, 36050, México, Tel +52 473-7320006 Ext 8193, Fax +52 473-7320006 Ext 8153, Email hmora@ugto.mxBackground: 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.Keywords: cell wall, cytokine production, fungal morphologies, immune response, phagocytosis

Manuela Gómez-Gaviria,* José A Martínez-Álvarez, Joaquín O Chávez-Santiago, Héctor M Mora-Montes* Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Guanajuato, Gto, México*These authors contributed equally to this workCorrespondence: Manuela Gómez-Gaviria; Héctor M Mora-Montes, Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, Col. Noria Alta, Guanajuato, Gto, C. P. 36050, México, Tel +52 473-7320006 Ext. 8193, Fax +52 473-7320006 Ext. 8153, Email m.gomezgaviria@ugto.mx; hmora@ugto.mxAbstract: 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.Keywords: antifungal resistance, candidiasis, cryptic Candida species, emerging pathogens, virulence factors, Candida haemulonii complex, Candida auris

Diana F Mendoza-Reyes, Manuela Gómez-Gaviria,* Héctor M Mora-Montes* Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Guanajuato, Gto, C.P. 36050, México*These authors contributed equally to this workCorrespondence: Manuela Gómez-Gaviria; Héctor M Mora-Montes, Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, col. Noria Alta, Guanajuato, Gto, C.P. 36050, México, Tel +52 473-7320006 Ext. 8193, Fax +52 473-7320006 Ext. 8153, Email m.gomezgaviria@ugto.mx; hmora@ugto.mxAbstract: 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.Keywords: biological aspects, candidiasis, multidrug resistance, opportunistic pathogens, virulence factors

Nallely Nava-Pérez, Lisset G Neri-García, Oscar E Romero-González, Joshua A Terrones-Cruz, Laura C García-Carnero,* Héctor M Mora-Montes* Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Guanajuato, 36050, Gto., México*These authors contributed equally to this workCorrespondence: Laura C García-Carnero; Héctor M Mora-Montes, Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, col. Noria Alta, C.P, Guanajuato, 36050, Gto., México, Tel +52 473-7320006 Ext. 8193, Fax +52 473-7320006 Ext. 8153, Email laura_cgc@hotmail.com; hmora@ugto.mxAbstract: 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.Keywords: antifungal drugs, fungal infection, host–fungus interplay, epidemiology, diagnosis, treatment

Nancy E Lozoya-Pérez,1 Laura C García-Carnero,1 José A Martínez-Álvarez,1 Iván Martínez-Duncker,2 Héctor M Mora-Montes1 1Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Guanajuato, 36050, México; 2Laboratorio de Glicobiología Humana y Diagnóstico Molecular; Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, MéxicoCorrespondence: Héctor M Mora-MontesDepartamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, Col. Noria Alta, C.P, Guanajuato, 36050, MéxicoTel +52 473-7320006 Ext. 8193Fax +52 473-7320006 Ext. 8153Email hmora@ugto.mxBackground: Sporotrichosis is an increasing threat for humans, affecting mainly skin and subcutaneous tissues but that can cause disseminated infection in immunocompromised patients. Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main etiological agents of this mycosis, and each species show different virulence levels. The gold standard to assess fungal virulence is the mouse model that is expensive and time-consuming. Thus, invertebrate models have been reported as an alternative for the evaluation of fungal virulence. Here, we assessed whether Tenebrio molitor larvae could be a new alternative to study Sporothrix spp. virulence.Methods: T. molitor larvae were inoculated with different doses of S. schenckii, S. brasiliensis, and S. globosa, and animal mortality, cytotoxicity, and immunological parameters were analyzed, including the ability to stimulate immunological priming.Results: Mortality curves demonstrated that yeast-like cells were the best fungal morphology to kill larvae and showed a similar ranking in virulence than that reported in other animal models, ie, being S. brasiliensis and S. globosa the species with the highest and lowest virulence, respectively. The usefulness of this model was validated with the analysis of several S. schenckii strains with different virulence degrees, and changes in cytotoxicity, humoral and cellular immunological parameters. Low-virulence strains stimulated low levels of cytotoxicity, phenoloxidase activity, and hemocyte countings, and these immunological cells poorly uptake fungi. Moreover, using recombinant Gp70 from S. schenckii immunological priming was stimulated in larvae and this protected against a lethal dose of fungal cells from any of the three species under study.Conclusion: The study demonstrated that T. molitor larvae are an appropriate alternative invertebrate model to analyze the virulence of S. schenckii, S. brasiliensis, and S. globosa. Additionally, hemocyte levels, phenoloxidase activity, cytotoxicity, uptake by hemocytes, and immunological priming are biological parameters that can be used to study the Sporothrix–T. molitor interaction.Keywords: experimental sporotrichosis, fungal infection, host–fungus interplay, hemocytes, phenoloxidase

Manuela Gómez-Gaviria, Héctor M Mora-Montes Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Guanajuato, Gto, MéxicoCorrespondence: Héctor M Mora-MontesDepartamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, Col. Noria Alta, C.P. 36050, Guanajuato, Gto, MéxicoTel +52 473-7320006 Ext. 8193Fax +52 473-7320006 Ext. 8153Email hmora@ugto.mxAbstract: Fungal infections represent a constant and growing menace to human health, because of the emergence of new species as causative agents of diseases and the increment of antifungal drug resistance. Candidiasis is one of the most common fungal infections in humans and is associated with a high mortality rate when the fungi infect deep-seated organs. Candida krusei belongs to the group of candidiasis etiological agents, and although it is not isolated as frequently as other Candida species, the infections caused by this organism are of special relevance in the clinical setting because of its intrinsic resistance to fluconazole. Here, we offer a thorough revision of the current literature dealing with this organism and the caused disease, focusing on its biological aspects, the host-fungus interaction, the diagnosis, and the infection treatment. Of particular relevance, we provide the most recent genomic information, including the gene prediction of some putative virulence factors, like proteases, adhesins, regulators of biofilm formation and dimorphism. Moreover, C. krusei veterinary aspects and the exploration of natural products with anti-C. krusei activity are also included.Keywords: virulence, candidiasis, host-fungus interplay, antifungal drug, immune sensing

María J Navarro-Arias,1 Marco J Hernández-Chávez,1 Laura C García-Carnero,1 Diana G Amezcua-Hernández,1 Nancy E Lozoya-Pérez,1 Eine Estrada-Mata,1 Iván Martínez-Duncker,2 Bernardo Franco,1 Héctor M Mora-Montes1 1Department of Biology, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, 36050, Guanajuato, Gto, México; 2Laboratory of Human Glycobiology and Molecular Diagnostics, Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, 62209, Morelos, México Background: The deep-seated infections caused by the Candida genus are associated with a high mortality rate, and Candida albicans is the most frequent species associated with these diseases. The fungal wall is composed of macromolecules not synthesized by the host, and therefore is a source of ligands recognized by innate immune cells. Methods: We performed a comparative study analyzing the cell wall composition and organization of Candida tropicalis, Candida guilliermondii, Candida krusei, and Candida auris, along with their ability to stimulate cytokine production and phagocytosis by human innate immune cells. Results: We found that the wall of these species had the basic components already described in C. albicans, with most of the chitin and b1,3-glucan located underneath the mannan layer. However, the walls of C. krusei and C. auris were rich in chitin and the former had a lower content of mannans. C. guilliermondii contained changes in the mannan and the b1,3-glucan levels. These species were differentially phagocytosed by human macrophages and stimulated cytokine production in a dectin-1-dependent pathway. C. krusei showed the most significant changes in the tested parameters, whereas C. auris behaved like C. albicans. Conclusion: Our results suggest that the cell wall and innate immune recognition of C. tropicalis, C. guilliermondii, C. krusei, and Candida auris is different from that reported for C. albicans. Keywords: cell wall, protein glycosylation, host–fungus interplay, phagocytosis, cytokine production

Nancy E Lozoya-Pérez,1 Sergio Casas-Flores,2 José Roberto Fogaça de Almeida,3 José A Martínez-Álvarez,1 Luz A López-Ramírez,1 Grasielle Pereira Jannuzzi,3 Elías Trujillo-Esquivel,1 Eine Estrada-Mata,1 Sandro R Almeida,3 Bernardo Franco,1 Leila M Lopes-Bezerra,3,4 Héctor M Mora-Montes1 1Department of Biology, Division of Exact and Natural Sciences, Universidad de Guanajuato, Guanajuato, Mexico; 2Division of Molecular Biology, IPICYT, San Luis Potosí, Mexico; 3Laboratory of Clinical Mycology, Faculty of Pharmacy, Universidade de São Paulo, São Paulo, Brazil; 4Laboratory of Cellular Mycology and Proteomics, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil Background: Sporothrix schenckii is a neglected fungal pathogen for the human being and other mammals. In several fungal systems, Och1 is a Golgi α1,6-mannosyltransferase with a key function in the synthesis of N-linked glycans; which are important elements during the host-fungus interplay. The role of OCH1 in fungal virulence seems to be species-specific, being an essential component for Candida albicans virulence and dispensable during the interaction of Aspergillus fumigatus with the host. Methods: Here, we silenced S. schenckii OCH1 and characterized the phenotype of the mutant strains. Results: The mutant strains did not show defects in the cell or colony morphology, the growth rate or the ability to undergo dimorphism; but the cell wall changed in both composition and exposure of inner components at the surface. When interacting with human monocytes, the silenced strains had a reduced ability to stimulate TNFα and IL-6 but stimulated higher levels of IL-10. The interaction with human macrophages was also altered, with reduced numbers of silenced cells phagocytosed. These strains showed virulence attenuation in both Galleria mellonella and in the mouse model of sporotrichosis. Nonetheless, the cytokine levels in infected organs did not vary significantly when compared with the wild-type strain. Conclusion: Our data demonstrate that OCH1 silencing affects different aspects of the S. schenckii-host interaction. Keywords: cell wall, protein glycosylation, sporotrichosis, gene silencing, host–fungus interplay, mannosyltransferase

Laura C García-Carnero,1 Luis A Pérez-García,2 José A Martínez-Álvarez,1 Juana E Reyes-Martínez,1 Héctor M Mora-Montes1 1Department of Biology, Division of Exact and Natural Sciences, Campus Guanajuato, Universidad de Guanajuato, 2Multidisciplinay Academic Unit, Zona Huasteca, Universidad Autónoma de San Luis Potosí, Ciudad Valles, México Abstract: Human fungal infections remain a major challenge in medicine. Only a limited number of antifungal drugs are available, which are often related to severe adverse effects. In addition, there is an increased emergence related to resistant strains, which makes imperative to understand the host-pathogen interactions as well as to develop alternative treatments. Host innate and adaptive immunity play a crucial role controlling fungal infections; therefore, vaccines are a viable tool to prevent and treat fungal pathogens. Innate immunity is triggered by the interaction between the cell surface pattern recognition receptors (PRRs) and the pathogen-associated molecular patterns (PAMPs). Such an initial immunological response is yet little understood in fungal infections, in part due to the complexity and plasticity of the fungal cell walls. Described host cell–fungus interactions and antigenic molecules are addressed in this paper. Furthermore, antigens found in the cell wall and capsule, including peptides, glycoproteins, glycolipids, and glycans, have been used to trigger specific immune responses, and an increased production of antibodies has been observed when attached to immunogenic molecules. The recent biotechnological advances have allowed the development of vaccines against viral and bacterial pathogens with positive results; therefore, this technology has been applied to develop anti-fungal vaccines. Passive immunization has also emerged as an appealing alternative to treat disseminated mycosis, especially in immunocompromised patients. Those approaches have a long way to be seen in clinical cases. However, all studies discussed here open the possibility to have access to new therapies to be applied alone or in combination with current antifungal drugs. Herein, the state of the art of fungal vaccine developments is discussed in this review, highlighting new advances against Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Paracoccidioides brasiliensis, and Sporothrix spp. Keywords: anti-fungal immunity, candidiasis, aspergillosis, cryptococcosis, paracoccidioidomycosis, sporotrichosis

Background: Interleukin (IL)-1beta has an important role in antifungal defense mechanisms. The inflammasome is thought to be required for caspase-1 activation and processing of the inactive precursor pro-IL-1beta. The aim of the present study was to investigate the pathways of IL-1beta production induced by Candida albicans in human monocytes. Methods: Human mononuclear cells were stimulated with C. albicans or mutant strains defective in mannosylation or chitin. Receptors were blocked with specific antagonists, and the IL-1beta concentration was measured. Results: Human primary monocytes produce bioactive IL-1beta when stimulated with C. albicans. The transcription of IL-1beta was induced through mannose receptor (MR), Toll-like receptor (TLR) 2, and dectin-1 but not through TLR4 and TLR9. N-mannan-linked residues, chitin, and beta-glucan from C. albicans are important for IL-1beta stimulation. Surprisingly, processing and secretion of IL-1beta in monocytes did not require pathogen-mediated inflammasome activation, because of the constitutive activation of caspase-1 and the capability of monocytes to release endogenous adenosine-5'-triphosphate. Conclusions: This study is the first dissection of the molecular mechanisms of IL-1beta production by a fungal pathogen. Transcription through mannan/chitin/MR and beta-glucan/dectin-1/TLR2 induces production of IL-1beta by C. albicans in human monocytes, whereas processing of IL-1beta is mediated by constitutively active caspase-1. [ABSTRACT FROM AUTHOR]

Chitin is a polymer of N-acetylglucosamine and an essential component of the fungal cell wall. Chitosan is the deacetylated form of chitin and is also important for maintaining the integrity of this structure. Both polysaccharides are widely distributed in nature and have been shown to have a variety of applications in biomedicine, including their potential in immune sensing and as potential antifungal agents. In addition, chitin has been reported to play an important role in the pathogen-host interaction, involving innate and adaptive immune responses. This paper will explore the role of chitin and chitosan when incorporated into nanobiocomposites to improve their efficacy in detecting fungi of medical interest and inhibiting their growth. Potential applications in diagnostic and therapeutic medicine will be discussed, highlighting their promise in the development of more sensitive and effective tools for the early diagnosis of fungal infections. This review aims to highlight the importance of the convergence of nanotechnology and biology in addressing public health challenges., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall mannans of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H + /Na + pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure-function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences., Competing Interests: Rodolfo García-Contreras, Héctor Manuel Mora-Montes and Bernardo Franco are Academic Editors at PeerJ. The other authors declare that they have no competing interests., (©2024 García-Contreras et al.)

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 β-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking., Results: We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and β-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports., Conclusions: The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development., (© 2024. The Author(s).)

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.

Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns. Galleria mellonella is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The G. mellonella immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the G. mellonella immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with Escherichia coli , Candida albicans , Sporothrix schenckii , Sporothrix globosa , and Sporothrix brasiliensis . The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique., Competing Interests: Hector M. Mora-Mones is an Academic Editor for Peer Journal., (© 2024 Ramírez-Sotelo et al.)

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.

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., Competing Interests: Héctor M. Mora-Montes is an Academic Editor for PeerJ., (© 2022 Arvizu-Rubio et al.)

Organisms need mechanisms to perceive the environment and respond accordingly to environmental changes or the presence of hazards. Transcription factors (TFs) are required for cells to respond to the environment by controlling the expression of genes needed. Escherichia coli has been the model bacterium for many decades, and still, there are features embedded in its genome that remain unstudied. To date, 58 TFs remain poorly characterized, although their binding sites have been experimentally determined. This study showed that these TFs have sequence variation at the third codon position G+C content but maintain the same Codon Adaptation Index (CAI) trend as annotated functional transcription factors. Most of these transcription factors are in areas of the genome where abundant repetitive and mobile elements are present. Sequence divergence points to groups with distinctive sequence signatures but maintaining the same type of DNA binding domain. Finally, the analysis of the promoter sequences of the 58 TFs showed A+T rich regions that agree with the features of horizontally transferred genes. The findings reported here pave the way for future research of these TFs that may uncover their role as spare factors in case of lose-of-function mutations in core TFs and trace back their evolutionary history., Competing Interests: Bernardo Franco is an Academic Editor for PeerJ. Héctor Manuel Mora-Montes is an Academic Editor for PeerJ., (© 2022 Duarte-Velázquez et al.)

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 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Grosjean, Le Jean, Chalot, Mora-Montes, Armengaud, Gross and Blaudez.)

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 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vargas-Macías, Gómez-Gaviria, García-Carnero and Mora-Montes.)

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 the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated with each species but remain largely uncharacterized. The S. schenckii and S. brasiliensis cell wall composition and virulence are influenced by the culturing media, with little or no influence on S. globosa . By keeping constant the culturing media, we compared the cell wall composition of three S. schenckii and two S. brasiliensis strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five Sporothrix spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-β-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-β-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in Sporothrix spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Villalobos-Duno, Barreto, Alvarez-Aular, Mora-Montes, Lozoya-Pérez, Franco, Lopes-Bezerra and Niño-Vega.)

This study reports on a Mexican mestizo patient with a multi-systemic syndrome including neurological involvement and a type I serum transferrin profile. Clinical exome sequencing revealed complex alleles in ALG1 , the encoding gene for the chitobiosyldiphosphodolichol beta-mannosyltransferase that participates in the formation of the dolichol-pyrophosphate-GlcNAc2Man5, a lipid-linked glycan intermediate during N -glycan synthesis. The identified complex alleles were NM&#95;019109.5(ALG1): c.[208 + 16&#95;208 + 19dup; 208 + 25G > T] and NM&#95;019109.5(ALG1): c.[208 + 16&#95;208 + 19dup; 1312C > T]. Although both alleles carried the benign variant c.208 + 16&#95;208 + 19dup, one allele carried a known ALG1 pathogenic variant (c.1312C > T), while the other carried a new uncharacterized variant (c.208 + 25G > T) causing non-functional alternative splicing that, in conjunction with the benign variant, defines the pathogenic protein effect (p.N70S&#95;S71ins9). The presence in the patient's serum of the pathognomonic N-linked mannose-deprived tetrasaccharide marker for ALG1-CDG (Neu5Acα2,6Galβ1,4-GlcNAcβ1,4GlcNAc) further supported this diagnosis. This is the first report of an ALG1-CDG patient from Latin America., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 González-Domínguez, Fiesco-Roa, Gómez-Carmona, Kleinert-Altamirano, He, Daniel, Raymond, Abreu-González, Manrique-Hernández, González-Jaimes, Salinas-Marín, Molina-Garay, Carrillo-Sánchez, Flores-Lagunes, Jiménez-Olivares, Muñoz-Rivas, Cruz-Muñoz, Ruíz-García, Freeze, Mora-Montes, Alaez-Verson and Martínez-Duncker.)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Sporothrix schenckii is a dimorphic fungus existing as mould in the environment and as yeast in the host. The morphological shift between mycelial/yeast phases is crucial for its virulence, but the transcriptional networks implicated in dimorphic transition are still not fully understood. Here, we report the global transcriptomic differences occurring between mould and yeast phases of S. schenckii , including changes in gene expression profiles associated with these distinct cellular phenotypes. Moreover, we also propose a new genome annotation, which reveals a more complex transcriptional architecture than previously assumed. Using RNA-seq, we identified a total of 17 307 genes, of which 11 217 were classified as protein-encoding genes, whereas 6090 were designated as non-coding RNAs (ncRNAs). Approximately ~71 % of all annotated genes were found to overlap and the different-strand overlapping type was the most common. Gene expression analysis revealed that 8795 genes were differentially regulated among yeast and mould forms. Differential gene expression was also observed for antisense ncRNAs overlapping neighbouring protein-encoding genes. The release of transcriptome-wide data and the establishment of the Sporothrix Genome DataBase (http://sporothrixgenomedatabase.unime.it) represent an important milestone for Sporothrix research, because they provide a strong basis for future studies on the molecular pathways involved in numerous biological processes.

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 .

We designed experiments to assess whether fungal cell wall mannans function as an immune shield or an immune agonist. Fungal cell wall β-(1,3)-glucan normally plays a major and dominant role in immune activation. The outer mannan layer has been variously described as an immune shield, because it has the potential to mask the underlying β-(1,3)-glucan, or an immune activator, as it also has the potential to engage with a wide range of mannose detecting PRRs. To resolve this conundrum we examined species-specific differences in host immune recognition in the och 1Δ N- mannosylation-deficient mutant background in four species of yeast-like fungi. Irrespective of the fungal species, the cytokine response (TNFα and IL-6) induced by the och 1Δ mutants in human monocytes was reduced compared to that of the wild type. In contrast, TNFα production induced by och 1Δ was increased, relative to wild type, due to increased β-glucan exposure, when mouse or human macrophages were used. These observations suggest that N -mannan is not a major PAMP for macrophages and that in these cells mannan does shield the fungus from recognition of the inner cell wall β-glucan. However, N -mannan is a significant inducer of cytokine for monocytes. Therefore the metaphor of the fungal "mannan shield" can only be applied to some, but not all, myeloid cells used in immune profiling experiments of fungal species., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)