Opportunistic Human Fungal Pathogen Research Articles

Phytochemical Analysis and Antimicrobial Activities of Atalantia monophylla (L) Correa and Atalantia racemosa Wight and Arn

Background: Infectious diseases are major leading cause of death in all parts of the world, because of the appearance of new multi drug resistant microbes. Therefore, the discovery of potential drug for effective treatment will help the slaughter of the microbes. The aim of the present study is to evaluate the presence of photochemical and antimicrobial activities of various crude extracts of leaves, fruits and root bark of Atalantia monophylla and Atalantia racemosa against human pathogens by using well diffusion method. Methods: Antimicrobial properties of the various extracts of Atalantia monophylla and Atalantia racemosa were studied against some human pathogenic microbes such as Gram-positive Bacteria, (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus) Gram-negative Bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli) and human opportunistic fungal pathogens (Candida albicans and Aspergillus niger). All the extracts were comparable with standard drugs (Ciprofloxacin, Gentamicin, Nystatin. and Amphotericin B). Minimum inhibitory concentration (MIC), minimum bactericidal /fungicidal concentration (MBC/MFC) values were determined through a microdilution method. The phytochemical analysis of these plant extracts were carried out using standard mthods. Results: Methanolic leaf extract of A. monophylla has showed excellent antimicrobial activity against S. aureus (40mm). As well, the A. racemosa methanolic leaf extract shows notable inhibitory activity against S. aureus (38mm). At the same time, the least inhibition was observed in aqueous extract of A. monophylla against E.coli (9mm). The MIC ranged from 0.78 µg/mL to 50 µg/mL and MBC/MFC 1.56 to 50 µg/mL were recorded. Phytochemical analysis of alkaloids, steroids, saponins, flavonoids, tannins, terpenoids, phenolics and cardiae glycoside were recorded in various extracts of A. monophylla and A. racemosa respectively. Flavonoids, phenolics and cardiac glycoside were present only in methonalic leaf extract of A. monophylla. Conclusion: The result of this study concluded that methanolic leaf extract has possessed novel compounds with significant antimicrobial properties. Hence, we recommend this plant for further studies on the isolation and characterization of that lead antimicrobial potential molecule.

The Ca-loop in thymidylate kinase is critical for growth and contributes to pyrimidine drug sensitivity of Candida albicans

The yeast Candida albicans is the most prevalent opportunistic fungal pathogen in humans. Drug resistance among C. albicans isolates poses a common challenge, and overcoming this resistance represents an unmet need in managing this common pathogen. Here, we investigated CDC8, encoding thymidylate kinase (TMPK), as a potential drug target for the management of C. albicans infections. We found that the region spanning amino acids 106-123, namely the Ca-loop of C. albicans TMPK (CaTMPK), contributes to the hyperactivity of this enzyme compared with the human enzyme (hTMPK) and to the utilization of deoxyuridine monophosphate (dUMP)/deoxy-5-fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, expression of CaTMPK, but not of hTMPK, produced dUTP/5-FdUTP-mediated DNA toxicity in budding yeast (Saccharomyces cerevisiae). CRISPR-mediated deletion of this Ca-loop in C. albicans revealed that the Ca-loop is critical for fungal growth and susceptibility to 5-fluorouridine (5-FUrd). Of note, pathogenic and drug-resistant C. albicans clones were similarly sensitive to 5-FUrd, and we also found that CaTMPK is essential for the growth of C. albicans In conclusion, these findings not only identified a target site for the development of CaTMPK-selective drugs, but also revealed that 5-FUrd may have potential utility as drug for managing C. albicans infections.

Culturing and Mating of Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human fungal pathogen, capable of causing invasive aspergillosis in patients with compromised immune systems. The fungus was long considered a purely asexual organism. However, a sexual cycle was reported in 2009, with methods described to induce mating under laboratory conditions. The presence of a sexual cycle now offers a valuable tool for classical genetic analysis of the fungus, such as allowing determination of whether traits of interest are mono‐ or poly‐genic in nature. For example, the sexual cycle is currently being exploited to determine the genetic basis of traits of medical importance such as resistance to azole antifungals and virulence, and to characterize the genes involved. The sexual cycle can also be used to assess the possibility of gene flow between isolates. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.This unit describes protocols for culturing of A. fumigatus and for inducing sexual reproduction between compatible MAT1‐1 and MAT1‐2 isolates of the species. The unit also provides working methods for harvesting sexual structures, isolating single‐spore progeny and confirming whether sexual recombination has occurred. © The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Characterization of the interaction of the antifungal and cytotoxic cyclic glycolipopeptide hassallidin with sterol-containing lipid membranes

Hassallidins are cyclic glycolipopeptides produced by cyanobacteria and other prokaryotes. The hassallidin structure consists of a peptide ring of eight amino acids where a fatty acid chain, additional amino acids, and sugar moieties are attached. Hassallidins show antifungal activity against several opportunistic human pathogenic fungi, but does not harbor antibacterial effects. However, they have not been studied on mammalian cells, and the mechanism of action is unknown. We purified hassallidin D from cultured cyanobacterium Anabaena sp. UHCC 0258 and characterized its effect on mammalian and fungal cells. Ultrastructural analysis showed that hassallidin D disrupts cell membranes, causing a lytic/necrotic cell death with rapid presence of disintegrated outer membrane, accompanied by internalization of small molecules such as propidium iodide into the cells. Furthermore, artificial liposomal membrane assay showed that hassallidin D selectively targets sterol-containing membranes. Finally, in silico membrane modeling allowed us to study the interaction between hassallidin D and membranes in detail, and confirm the role of cholesterol for hassallidin-insertion into the membrane. This study demonstrates the mechanism of action of the natural compound hassallidin, and gives further insight into how bioactive lipopeptide metabolites selectively target eukaryotic cell membranes.

Zinc and Iron Homeostasis: Target-Based Drug Screening as New Route for Antifungal Drug Development.

The incidence of fungal diseases is on the rise and the number of fatalities is still unacceptably high. While advances into antifungal drug development have been made there remains an urgent need to develop novel antifungal agents targeting as-yet unexploited pathways, such as metal ion homeostasis. Here we report such an approach by developing a metal sensor screen in the opportunistic human fungal pathogen Candida albicans. Using this reporter strain, we screened a library of 1,200 compounds and discovered several active compounds not previously described as chemical entities with antifungal properties. Two of these, artemisinin and pyrvinium pamoate, have been further characterized and their interference with metal homeostasis and potential as novel antifungal compounds validated. Lastly, we demonstrate that the same strain can be used to report on intracellular conditions within host phagocytes, paving the way toward the development of novel screening platforms that could identify compounds with the potential to perturb ion homeostasis of the pathogen specifically within host cells.

Understand the genomic diversity and evolution of fungal pathogen Candida glabrata by genome-wide analysis of genetic variations

The yeast Candida glabrata, an opportunistic human fungal pathogen, is the second most prevalent cause of candidiasis worldwide, with an infection incidence that has been increasing in the past decades. The completion of the C. glabrata reference genome made fundamental contributions to the understanding of the molecular basis of its pathogenic phenotypes. However, knowledge of genome-wide genetic variations among C. glabrata strains is limited. In this study, we present a population genomic study of C. glabrata based on whole genome re-sequencing of 47 clinical strains to an average coverage of ∼63×. Abundant genetic variations were identified in these strains, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels) and copy number variations (CNVs). The observed patterns of variations revealed clear population structure of these strains. Using population genetic tests, we detected fast evolution of several genes involved in C. glabrata adherence ability, such as EPA9 and EPA10. We also located genome structural variations, including aneuploidies and large fragment CNVs, in regions that are functionally related to virulence. Subtelometric regions were hotspots of CNVs, which may contribute to variation in expression of adhesin genes that are important for virulence. We further conducted a genome-wide association study that identified two SNPs in the 5'UTR region of CST6 that were associated with fluconazole susceptibility. These observations provide convincing evidence for the highly dynamic nature of the C. glabrata genome with potential adaptive evolution to clinical environments, and offer valuable resources for investigating the mechanisms underlying drug resistance and virulence in this fungal pathogen. (249 words).

Activation of transposable elements and genetic instability during long-term culture of the human fungal pathogen Candida albicans

It has been repeatedly reported that transposable elements (TE) become active and/or mobile in the genomes of replicatively and stress-induced senescent mammalian cells. However, the biological role of senescence-associated transposon activation and its occurrence and relevance in other eukaryotic cells remain to be elucidated. In the present study, Candida albicans, a prevalent opportunistic fungal pathogen in humans, was used to analyze changes in gene copy number of selected TE, namely Cirt2, Moa and Cmut1 during long-term culture (up to 90 days). The effects of stress stimuli (fluconazole, hydrogen peroxide, hypochlorite) and ploidy state (haploid, diploid, tetraploid cells) were also considered. An increase in copy number of Cirt2 and Moa was the most accented in tetraploid cells after 90 days of culture that was accompanied by changes in karyotype patterns and slightly more limited growth rate compared to haploid and diploid cells. Stress stimuli did not potentiate TE activity. Elevation in chromosomal DNA breaks was also observed during long-term culture of cells of different ploidy, however this was not correlated with increased TE activity. Our results suggest that increased TE activity may promote genomic diversity and plasticity, and cellular heterogeneity during long-term culture of C. albicans cells.

Molecular Characterization of a Debilitation-Associated Partitivirus Infecting the Pathogenic Fungus Aspergillus flavus.

The opportunistic human pathogenic fungus Aspergillus flavus is known to be infected with mycoviruses. In this study, we report a novel mycovirus A. flavus partitivirus 1 (AfPV1) that was originally isolated from the abnormal colonial morphology isolate LD-3-8 of A. flavus. AfPV1 has spherical virus-like particles about 40 nm in diameter, and three double-stranded RNA (dsRNA) segments (dsRNA1, 2, and 3 with lengths of 1.7, 1.4, and 1.1 kbp, respectively) were packaged in the virions. dsRNA1, dsRNA2, and dsRNA3 each contained a single open reading frame and potentially encoded 62, 42, and 32 kDa proteins, respectively. The dsRNA1 encoded protein shows similarity to the RNA-dependent RNA polymerase (RdRp) of partitiviruses, and the dsRNA2 product has no significant similarity to any other capsid protein (CP) in the GenBank databases, beside some homology with the hypothetical “capsid” protein of a few partitiviruses. The dsRNA3 encodes a protein with no similarity to any protein in the GenBank database. SDS-PAGE and polypeptide mass fingerprint-mass spectrum (PMF-MS) analyses indicated that the CP of the AfPV1 was encoded by dsRNA2. Phylogenetic analysis showed that the AfPV1 and relative viruses were found in an unclassified group inside the Partitiviridae family. AfPV1 seems to result in debilitation symptoms, but had no significant effects to murine pathogenicity. These findings provide new insights into the partitiviruses taxonomy and the interactions between viruses and A. flavus.

β-citronellol alters cell surface properties of Candida albicans to influence pathogenicity related traits.

The pathogenicity of Candida albicans, an opportunistic human fungal pathogen, is attributed to several virulence factors. β-citronellol is a monoterpenoid present in several plant essential oils. The present study explores the antifungal potential and mode of action of β-citronellol against C. albicans ATCC 90028 (standard), C. albicans D-27 (FLC-sensitive), and C. albicans S-1 (FLC-resistant). Anti-Candida potential was studied by performing MIC, MFC, growth curves, disc diffusion, spot assay, and WST1 cytotoxic assay. Morphological transition was monitored microscopically in both solid and liquid hyphae inducing media. β-citronellol inhibits yeast to hyphal transition in both liquid and solid hyphae inducing media. It had a significant inhibitory effect on biofilm formation and secretion of extracellular proteinases and phospholipases. We showed that it has an adverse effect on membrane ergosterol levels and modulates expression of related ERG genes. Expression profiles of selected genes associated with C. albicans pathogenicity displayed reduced expression in treated cells. This work suggests that β-citronellol inhibits morphological transition in C. albicans and decreases the secretion of hydrolytic enzymes involved in the early stage of infection as well as modulates the expression of associated genes. Pleiotropic phenotype shown by β-citronellol treated Candida cells suggests various modes of action. Further studies will assess the clinical application of β-citronellol in the treatment of fungal infections.

Characterising the human intestinal mycobiome during healthy ageing

Although fungi are fundamental to the human microbiome, the diversity and dynamics of the mycobiome is poorly understood, particularly in considering their association with infectious disease, autoimmune disorders and atopy that affect immunocompromised individuals and infants. Characterising the human mycobiome faces several challenges relating to their low abundance and lack of standardized procedures for sample collection and isolation of viable cells and/or quality genetic material for culture-dependent and independent taxonomic and functional characterisation. To address these issues, we have developed a mycobiome analysis pipeline employing both culture-dependent and independent methods to identify as well as isolate, where possible, the fungal taxa populating the human intestinal tract. In a proof-of-concept study this pipeline has been used to identify fungal populations in faecal samples obtained from a small cohort of young infants, aged 2 years or younger. All were born prematurely, and severely immunocompromised and at risk from invasive and potentially lethal microbial infections, including those caused by fungal overgrowth. We have used this combined approach successfully to identify the fungi present in each individual infant, and to recover viable isolates. To date, Candida albicans and C. parapsilosis are the most frequently isolated fungi. While both are major opportunistic human fungal pathogens, C. parapsilosis is particularly problematic to preterm babies, due to its innate ability to form biofilms. Detailed characterisation of these isolates is currently underway. Two large-scale longitudinal microbiome studies have started at the Quadram Institute, and our validated analysis pipeline will be incorporated to define the fungal component of each study participant.

Monitoring Phenotypic Switching in Candida albicans and the Use of Next-Gen Fluorescence Reporters.

Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.

Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans.

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence.

Overproduction of Phospholipids by the Kennedy Pathway Leads to Hypervirulence in Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) pathway causes avirulence in a mouse model of systemic infection. The synthesis of PE by this pathway plays a crucial role in virulence, but it was unknown if downstream conversion of PE to phosphatidylcholine (PC) is required for pathogenicity. Therefore, the enzymes responsible for methylating PE to PC, Pem1 and Pem2, were disrupted. The resulting pem1Δ/Δ pem2Δ/Δ mutant was not less virulent in mice, but rather hypervirulent. Since the pem1Δ/Δ pem2Δ/Δ mutant accumulated PE, this led to the hypothesis that increased PE synthesis increases virulence. To test this, the alternative Kennedy pathway for PE/PC synthesis was exploited. This pathway makes PE and PC from exogenous ethanolamine and choline, respectively, using three enzymatic steps. In contrast to Saccharomyces cerevisiae, C. albicans was found to use one enzyme, Ept1, for the final enzymatic step (ethanolamine/cholinephosphotransferase) that generates both PE and PC. EPT1 was overexpressed, which resulted in increases in both PE and PC synthesis. Moreover, the EPT1 overexpression strain is hypervirulent in mice and causes them to succumb to system infection more rapidly than wild-type. In contrast, disruption of EPT1 causes loss of PE and PC synthesis by the Kennedy pathway, and decreased kidney fungal burden during the mouse systemic infection model, indicating a mild loss of virulence. In addition, the ept1Δ/Δ mutant exhibits decreased cytotoxicity against oral epithelial cells in vitro, whereas the EPT1 overexpression strain exhibits increased cytotoxicity. Taken altogether, our data indicate that mutations that result in increased PE synthesis cause greater virulence and mutations that decrease PE synthesis attenuate virulence.

Candida glabrata: A Lot More Than Meets the Eye

Candida glabrata is an opportunistic human fungal pathogen that causes superficial mucosal and life-threatening bloodstream infections in individuals with a compromised immune system. Evolutionarily, it is closer to the non-pathogenic yeast Saccharomyces cerevisiae than to the most prevalent Candida bloodstream pathogen, C. albicans. C. glabrata is a haploid budding yeast that predominantly reproduces clonally. In this review, we summarize interactions of C. glabrata with the host immune, epithelial and endothelial cells, and the ingenious strategies it deploys to acquire iron and phosphate from the external environment. We outline various attributes including cell surface-associated adhesins and aspartyl proteases, biofilm formation and stress response mechanisms, that contribute to the virulence of C. glabrata. We further discuss how, C. glabrata, despite lacking morphological switching and secreted proteolytic activity, is able to disarm macrophage, dampen the host inflammatory immune response and replicate intracellularly.

Cryptococcus neoformans Mating and Genetic Crosses.

The Cryptococcus pathogenic species complex is a group of opportunistic human fungal pathogens that cause cryptococcal meningoencephalitis, an infection associated with unacceptably high mortality rates. The public health relevance of these pathogens has galvanized extensive research over the past several decades and led to characterization of their sexual cycles. This research has allowed several Cryptococcus species to develop into model fungal organisms for both pathogenesis and basic science studies. Many of these studies require observation of the meiotic process and its associated mating structures as well as generation of meiotic progeny with novel phenotypes and genotypes. Herein, we describe how to set up genetic crosses between Cryptococcus strains and observe their mating phenotypes as well as how to recover progeny from these crosses for further analysis. © 2019 by John Wiley & Sons, Inc.

The Significance of Lipids to Biofilm Formation in Candida albicans: An Emerging Perspective.

Candida albicans, the dimorphic opportunistic human fungal pathogen, is capable of forming highly drug-resistant biofilms in the human host. Formation of biofilm is a multistep and multiregulatory process involving various adaptive mechanisms. The ability of cells in a biofilm to alter membrane lipid composition is one such adaptation crucial for biofilm development in C. albicans. Lipids modulate mixed species biofilm formation in vivo and inherent antifungal resistance associated with these organized communities. Cells in C. albicans biofilms display phase-dependent changes in phospholipid classes and in levels of lipid raft formation. Systematic studies with genetically modified strains in which the membrane phospholipid composition can be manipulated are limited in C. albicans. In this review, we summarize the knowledge accumulated on the impact that alterations in phospholipids may have on the biofilm forming ability of C. albicans in the human host. This review may provide the requisite impetus to analyze lipids from a therapeutic standpoint in managing C. albicans biofilms.

The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes

Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.

Aspergillus fumigatus conidial metalloprotease Mep1p cleaves host complement proteins

Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against Aspergillus fumigatus, one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from A. fumigatus mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores (i.e. conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that A. fumigatus conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from A. fumigatus conidia likely facilitates early immune evasion by disarming the complement defense in the human host.

Keratinocytes contribute to the recruitment and M1 polarisation of macrophages during C. albicans colonisation

Candida albicans (C. albicans) is an opportunistic human fungal pathogen that colonises the skin. Both keratinocytes and macrophages play crucial roles in host defence against C. albicans. However, the interaction of keratinocytes with macrophages during C. albicans colonisation has not been well studied. In this study, macrophages were cultured in conditioned medium from keratinocytes treated with heat-inactivated C. albicans (CM-C. albicans), macrophage migration and polarised activation and were then assessed by a Transwell assay, flow cytometry, quantitative real-time PCR (qPCR), Western blot and an enzyme-linked immunosorbent assay (ELISA). The results showed that CM-C. albicans-stimulated macrophages display significantly increased migration and phagocytosis, and they display an upregulation of proinflammatory cytokines (tumour necrosis factor alpha (TNF-a), interleukin (IL)-12 and nitric oxide (NO)). Markers characteristic of M1macrophages,such as human leukocyte antigen (HLA)-DR, CD86 and inducible nitric oxide synthase (iNOS), are upregulated, whereas markers of M2macrophages,such as mannose receptor (MR) and Arginase1(Arg1), are not affected. Additionally, the levels of TNF-a, IL-12 and monocyte chemotactic protein 1 (MCP-1) in CM-C. albicans are markedly upregulated, whereas the levels of IL-4 and IL-10 are not affected. And the CM-C. albicans-induced M1macrophage polarisation, proinflammatory cytokine production and phagocytosis could be blocked by an anti-TNF-a neutralising antibody. This study showed that keratinocytes may promote macrophage recruitment and M1polarisation during C. albicans colonisation at least in part by secreting TNF-a.

Identification of cell-associated and secreted serine-type peptidases in multidrug-resistant emergent pathogens belonging to the Candida haemulonii complex.

The Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera, and Candida duobushaemulonii) comprises emerging opportunistic human fungal pathogens with recognized multidrug-resistance profiles. Little is known about the virulence markers produced by this fungal complex. However, it is recognized that Candida spp. express a large array of peptidases, which play multiple roles in different aspects of fungal-host interactions. In the present study, we have identified proteolytic enzymes in clinical isolates of the C. haemulonii complex using zymographic assays. Peptidases able to hydrolyze gelatin, casein, albumin, hemoglobin, and immunoglobulin G were detected in cell-free supernatants and cellular extracts taken from the three species forming the C. haemulonii complex. Overall, peptidases were preferentially evidenced at physiological pH and temperatures of 37-42°C, with molar masses between 35 and 85kDa. Peptidase profiles of C. haemulonii and C. haemulonii var. vulnera isolates were quite similar, contrasting to the peptidases produced by C. duobushaemulonii. Almost all peptidases were inhibited by phenylmethanesulfonyl fluoride (PMSF), thus classifying them as serine-type peptidases. Additionally, proteolytic cleavage of soluble azoalbumin was blocked by PMSF (65-95% inhibition depending on the fungal isolate). These unprecedented results have demonstrated the capability of the C. haemulonii complex to produce serine-type peptidases with an ability to cleave a broad spectrum of proteins, including key host components.