An ELISA-based method for Galleria mellonella apolipophorin-III quantification.

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.

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.

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.

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.

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.

Non-albicans Candida Species: Immune Response, Evasion Mechanisms, and New Plant-Derived Alternative Therapies.

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.

Role of Protein Glycosylation in Interactions of Medically Relevant Fungi with the Host.

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.

Early Virulence Predictors during the Candida Species-Galleria mellonella Interaction.

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

Current Aspects in the Biology, Pathogeny, and Treatment of Candida krusei, a Neglected Fungal Pathogen.

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.

Loss of Kex2 Affects the Candida albicans Cell Wall and Interaction with Innate Immune Cells.

The secretory pathway in Candida albicans involves the protein translocation into the lumen of the endoplasmic reticulum and transport to the Golgi complex, where proteins undergo posttranslational modifications, including glycosylation and proteolysis. The Golgi-resident Kex2 protease is involved in such processing and disruption of its encoding gene affected virulence and dimorphism. These previous studies were performed using cells without URA3 or with URA3 ectopically placed into the KEX2 locus. Since these conditions are known to affect the cellular fitness and the host-fungus interaction, here we generated a kex2Δ null mutant strain with URA3 placed into the neutral locus RPS1. The characterization of this strain showed defects in the cell wall composition, with a reduction in the N-linked mannan content, and the increment in the levels of O-linked mannans, chitin, and ß-glucans. The defects in the mannan content are likely linked to changes in Golgi-resident enzymes, as the α-1,2-mannosyltransferase and α-1,6-mannosyltransferase activities were incremented and reduced, respectively. The mutant cells also showed reduced ability to stimulate cytokine production and phagocytosis by human mononuclear cells and macrophages, respectively. Collectively, these data showed that loss of Kex2 affected the cell wall composition, the protein glycosylation pathways, and interaction with innate immune cells.