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

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

Regulation of innate immune responses in macrophages differentiated in the presence of vitamin D and infected with dengue virus 2.

A dysregulated or exacerbated inflammatory response is thought to be the key driver of the pathogenesis of severe disease caused by the mosquito-borne dengue virus (DENV). Compounds that restrict virus replication and modulate the inflammatory response could thus serve as promising therapeutics mitigating the disease pathogenesis. We and others have previously shown that macrophages, which are important cellular targets for DENV replication, differentiated in the presence of bioactive vitamin D (VitD3) are less permissive to viral replication, and produce lower levels of pro-inflammatory cytokines. Therefore, we here evaluated the extent and kinetics of innate immune responses of DENV-2 infected monocytes differentiated into macrophages in the presence (D3-MDMs) or absence of VitD3 (MDMs). We found that D3-MDMs expressed lower levels of RIG I, Toll-like receptor (TLR)3, and TLR7, as well as higher levels of SOCS-1 in response to DENV-2 infection. D3-MDMs produced lower levels of reactive oxygen species, related to a lower expression of TLR9. Moreover, although VitD3 treatment did not modulate either the expression of IFN-α or IFN-ß, higher expression of protein kinase R (PKR) and 2'-5'-oligoadenylate synthetase 1 (OAS1) mRNA were found in D3-MDMs. Importantly, the observed effects were independent of reduced infection, highlighting the intrinsic differences between D3-MDMs and MDMs. Taken together, our results suggest that differentiation of MDMs in the presence of VitD3 modulates innate immunity in responses to DENV-2 infection.

Influences of the Culturing Media in the Virulence and Cell Wall of Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa.

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 ß-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.

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.

Role of Protein Mannosylation in the Candida tropicalis-Host Interaction.

Mannans are components of the fungal wall attached to proteins via N- or O-linkages. In Candida albicans, Och1 is an α1,6-mannosyltransferase that adds the first mannose unit to the N-linked mannan outer chain; whereas Pmr1 is an ion pump that imports Mn2+ into the Golgi lumen. This cation is the cofactor of Golgi-resident mannosyltransferases, and thus Pmr1 is involved in the synthesis of both N- and O-linked mannans. Since we currently have limited information about the genetic network behind the Candida tropicalis protein mannosylation machinery, we disrupted OCH1 and PMR1 in this organism. The C. tropicalis pmr1Δ and och1Δ mutants showed increased doubling times, aberrant colony and cellular morphology, reduction in the wall mannan content, and increased susceptibility to wall perturbing agents. These changes were accompanied by increased exposure of both ß1,3-glucan and chitin at the wall surface of both mutant strains. Our results showed that O-linked mannans are dispensable for cytokine production by human mononuclear cells, but N-linked mannans and ß1,3-glucan are key ligands to trigger cytokine production in a co-stimulatory pathway involving dectin-1 and mannose receptor. Moreover, we found that the N-linked mannan core found on the surface of C. tropicalis och1Δ null mutant was capable of inducing cytokine production; and that a mannan-independent pathway for IL-10 production is present in the C. tropicalis-mononuclear cell interaction. Both mutant strains showed virulence attenuation in the Galleria mellonella and the mouse model of systemic candidiasis. Therefore, mannans are relevant for cell wall composition and organization, and for the C. tropicalis-host interaction.

1,25-Dihydroxyvitamin D3 induces formation of neutrophil extracellular trap-like structures and modulates the transcription of genes whose products are neutrophil extracellular trap-associated proteins: A pilot study.

Neutrophils are components of the innate immune system that participate in controlling infectious diseases through microbicidal mechanisms such as phagocytosis, degranulation and the release of neutrophil extracellular traps (NETs). NETs are DNA structures that are released through the decondensation and spreading of chromatin and the adherence of various proteins, including neutrophil elastase (NE), myeloperoxidase (MPO) and peptidyl arginine deiminase 4 (PDA4). Since NETs recovered after treatment of activated polymorphonuclear neutrophils can enhance IL-1ß and IFN-α production by LPS-activated macrophages, they are thought to be keys to the host's defenses and inflammation. 1,25(OH)2D3 has been shown to play an important role in modulating neutrophils activation and in preventing infections. Therefore, the aim of this study was to assess the effect of 1,25(OH)2D3 in modulating induction of the release of NETs and in regulating the transcription of genes whose products in human neutrophils are NETs-associated proteins, TLRs and interferon. We observed that 1,25(OH)2D3 induced production of NETs-like structures while also upregulating NE/PAD4/COX-3/GAPDH mRNA levels. Additionally, we found an increase in TLR7 and type I interferon (IFN) mRNA levels as a result of neutrophil activation by 1,25(OH)2D3. Since the transcription of genes whose products constitute NETs-associated proteins are differentially-regulated by 1,25(OH)2D3, we proposed that this might restrict the spread of pathogens, such as virus, by inducing NETs, the expression of TLR7 and secretion of IFN-α. Our results suggest the potential importance of this hormone in preventing infections by inducing NETs formation.

Role of protein phosphomannosylation in the Candida tropicalis-macrophage interaction.

Candida tropicalis is an opportunistic fungal pathogen responsible for mucosal and systemic infections. The cell wall is the initial contact point between a fungal cell and the host immune system, and mannoproteins are important components that play key roles when interacting with host cells. In Candida albicans, mannans are modified by mannosyl-phosphate moieties, named phosphomannans, which can work as molecular scaffolds to synthesize ß1,2-mannooligosaccharides, and MNN4 is a positive regulator of the phosphomannosylation pathway. Here, we showed that C. tropicalis also displays phosphomannans on the cell surface, but the amount of this cell wall component varies depending on the fungal strain. We also identified a functional ortholog of CaMNN4 in C. tropicalis. Disruption of this gene caused depletion of phosphomannan content. The C. tropicalis mnn4Δ did not show defects in the ability to stimulate cytokine production by human mononuclear cells but displayed virulence attenuation in an insect model of candidiasis. When the mnn4Δ-macrophage interaction was analyzed, results showed that presence of cell wall phosphomannan was critical for C. tropicalis phagocytosis. Finally, our results strongly suggest a differential role for phosphomannans during phagocytosis of C. albicans and C. tropicalis.

Phosphomannosylation and the Functional Analysis of the Extended Candida albicans MNN4-Like Gene Family.

Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46, or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4, they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.

The Sporothrix schenckii Gene Encoding for the Ribosomal Protein L6 Has Constitutive and Stable Expression and Works as an Endogenous Control in Gene Expression Analysis.

Sporothrix schenckii is one of the causative agents of sporotrichosis, a worldwide-distributed mycosis that affects humans and other mammals. The interest in basic and clinical features of this organism has significantly increased in the last years, yet little progress in molecular aspects has been reported. Gene expression analysis is a set of powerful tools that helps to assess the cell response to changes in the extracellular environment, the genetic networks controlling metabolic pathways, and the adaptation to different growth conditions. Most of the quantitative methodologies used nowadays require data normalization, and this is achieved measuring the expression of endogenous control genes. Reference genes, whose expression is assumed to suffer minimal changes regardless the cell morphology, the stage of the cell cycle or the presence of harsh extracellular conditions are commonly used as controls in Northern blotting assays, microarrays, and semi-quantitative or quantitative RT-PCR. Since the biology of the organisms is usually species specific, it is difficult to find a reliable group of universal genes that can be used as controls for data normalization in experiments addressing the gene expression, regardless the taxonomic classification of the organism under study. Here, we compared the transcriptional stability of the genes encoding for elongation factor 1A, Tfc1, a protein involved in transcription initiation on Pol III promoters, ribosomal protein L6, histone H2A, ß-actin, ß-tubulin, glyceraldehyde 3-phosphate dehydrogenase, UAF30, the upstream activating factor 30, and the transcription initiation factor TFIID subunit 10, during the fungal growth in different culture media and cell morphologies. Our results indicated that only the gene encoding for the ribosomal protein L6 showed a stable and constant expression. Furthermore, it displayed not transcriptional changes when S. schenckii infected larvae of Galleria mellonella or interacted with immune cells. Therefore, this gene could be used as control for data normalization in expression assays. As a proof of concept, this gene was used to assess the expression of genes encoding for glycosidases involved in the protein N-linked glycosylation pathway, a histidine kinase whose expression is regulated during the fungal dimorphism, and a glycosidase that participates in sucrose assimilation.

Understanding the molecular mechanisms of NETs and their role in antiviral innate immunity.

Polymorphonuclear neutrophils (PMNs) are the most abundant cells in the context of innate immunity; they are one of the first cells to arrive at the site of viral infection constituting the first line of defense in response to invading pathogens. Indeed, neutrophils are provided with several defense mechanisms including release of cytokines, cytotoxic granules and the last recently described neutrophil extracellular traps (NETs). The main components of NETs are DNA, granular antimicrobial peptides, and nuclear and cytoplasmic proteins, that together play an important role in the innate immune response. While NETs were first described as a mechanism against bacteria and fungi, recently, several studies are beginning to elucidate how NETs are involved in the host antiviral response and the prominent characteristics of this new mechanism are discussed in the present review.

HIV-1-derived single-stranded RNA acts as activator of human neutrophils.

Neutrophils are key effector cells of the innate immune system and are involved in the host defense against invading pathogens such as viruses. Recently, it was reported that HIV-1-neutrophil interaction triggers neutrophil activation and promotes expression of Toll-like receptors (TLRs). Here, we assessed the role of single-stranded RNA40 (ssRNA40) derived from HIV-1 in neutrophil activation. We observed functional activation of neutrophils in response to HIV-1-derived ssRNA40 based on the expression of TLR7/8, RIG-I, and MDA5, induction of cytokines (IL-6 and TNF-α), and the production of reactive oxygen species (ROS). Additionally, ssRNA40 promoted the expression of CD62L and TNF-α and the production of ROS in the presence of the TLR2 agonist Pam2CSK4. ssRNA40 together with R848 (a TLR7/8 agonist) increased CD11b expression but decreased CD62L expression. Furthermore, decreased IL-6 expression was observed in the presence of the TLR4 agonist LPS. Finally, we found that ssRNA40 promotes RIG-I and MDA5 expression in the presence of the TLR2, TLR4 and TLR7/8 agonists. This study demonstrates a functional response of TLRs in neutrophils challenged with ssRNA40, suggesting that TLRs could be involved in the innate immune response observed during HIV infection, which might be mediated by its genome.

Analysis of Sporothrix schenckii sensu stricto and Sporothrix brasiliensis virulence in Galleria mellonella.

The study of the host-pathogen interaction is essential to understand the mechanisms underlying adhesion, colonization and tissue damage by pathogens. This is usually achieved by performing in vivo studies using small mammals, such as rats, mice and guinea pigs. Nowadays, the mouse models of systemic or subcutaneous infection are the gold standard assays to analyze the virulence of members of the Sporothrix schenckii complex. There are, however, invertebrates that have been recently used as alternative hosts to assess the virulence of both bacteria and fungi, and among them, larvae of Galleria mellonella are popular because they are easy to breed, and require non-specialized facilities to maintain the colony. Here, we assessed the use of G. mellonella larvae to test the virulence of S. schenckii sensu stricto and Sporothrix brasiliensis strains, and found that infection with yeast-like cells, but not with conidia or germlings, reproduces the virulence data generated in the mouse model of infection. Furthermore, with this insect model we could classify the virulence of some strains as low, intermediate or high, in line with the observations in the mammalian model. Therefore, G. mellonella is suitable, and a new alternative, to test virulence of both S. schenckii sensu stricto and S. brasiliensis.

Involvement of neutrophil hyporesponse and the role of Toll-like receptors in human immunodeficiency virus 1 protection.

OBJECTIVES: Neutrophils contribute to pathogen clearance through pattern recognition receptors (PRRs) activation. However, the role of PRRs in neutrophils in both HIV-1-infected [HIV-1(+)] and HIV-1-exposed seronegative individuals (HESN) is unknown. Here, a study was carried out to evaluate the level of PRR mRNAs and cytokines produced after activation of neutrophils from HIV-1(+), HESN and healthy donors. METHODS: The neutrophils were stimulated with specific agonists for TLR2, TLR4 and TLR9 in the presence of HIV-1 particles. Pro-inflammatory cytokine production, expression of neutrophil activation markers and reactive oxygen species (ROS) production were analyzed in neutrophils from HESN, HIV-1(+) and healthy donors (controls). RESULTS: We found that neutrophils from HESN presented reduced expression of PRR mRNAs (TLR4, TLR9, NOD1, NOD2, NLRC4 and RIG-I) and reduced expression of cytokine mRNAs (IL-1ß, IL-6, IL-18, TNF-α and TGF-ß). Moreover, neutrophils from HESN were less sensitive to stimulation through TLR4. Furthermore, neutrophils from HESN challenged with HIV-1 and stimulated with TLR2 and TLR4 agonists, produced significantly lower levels of reactive oxygen species, versus HIV-1(+). CONCLUSIONS: A differential pattern of PRR expression and release of innate immune factors in neutrophils from HESN is evident. Our results suggest that lower neutrophil activation can be involved in protection against HIV-1 infection.