Mass Spectrometry-Based Proteomic and Immunoproteomic Analyses of the Candida albicans Hyphal Secretome Reveal Diagnostic Biomarker Candidates for Invasive Candidiasis.
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ABSTRACT:
SUBMITTER: Vaz C
PROVIDER: S-EPMC8306665 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Candida albicans is an opportunistic pathogen and a commensal fungus of the mucosa from many healthy patients. Invasive candidiasis (IC) has high levels of morbidity and mortality that are attributed to some extent to the difficulty in its early diagnosis. Diverse and immunogenic proteins with roles such as virulence and immune evasion are secreted by this pathogen. Immunoproteomics is an important technique for the assessment of antibody reactivities to immunogenic proteins and our group previously developed an algorithm to predict the clinical outcome in IC patients. With the ultimate purpose of discovering new biomarkers of IC, immunoproteomics was applied for the analysis of the serological response to hyphal C. albicans extracellular proteins. Gel-free proteomic analysis was performed by LC-MS/MS for the detection of the secreted proteins and 301 proteins were identified. As expected, gene ontology analysis in cellular component showed enrichment in proteins from extracellular region, cell wall, cell surface, cell periphery and intracellular region. Importantly, 46.5% of the intracellular proteins identified were also annotated in extracellular region. Furthermore, from the extracellular proteins identified here, 44 proteins were previously found to be immunogenic against human sera. To carry out immunoproteomic analysis, secreted proteins were separated by two-dimensional gel electrophoresis, electroblotted onto nitrocellulose membranes, and tested by Western blotting with three distinct pools of human sera: P1 (12 IC patients), P2 (11 patients with Candida infection associated with catheters) and P3 (11 patients without IC). A higher reactivity pattern was observed in P1 and P2 in comparison with P3. So far, Glx3 was immunogenic to P1 and P2 but not to P3. Eno1, Pgk1 and Pra1 were found to be more immunoreactive to both P1 and P2 than to the P3. This approach will contribute to a better and faster diagnosis of this life-threatening fungal infection.
2021-07-05 | PXD013933 | Pride
Project description:Candida albicans is the most abundant fungal species in oral cavity. As a smart opportunistic pathogen, it increases the virulence by switching its forms from yeasts to hyphae and becomes the major pathogenic agent for oral candidiasis. However, the overuse of current clinical antifungals and lack of new types of drugs highlight the challenges in the antifungal treatments because of the drug resistance and side effects. Anti-virulence strategy is proved as a practical way to develop new types of anti-infective drugs. Here, seven artemisinins, including artemisinin, dihydroartemisinin, artemisinic acid, dihydroartemisinic acid, artesunate, artemether and arteether, were employed to target at the hyphal development, the most important virulence factor of C. albicans. Artemisinins failed to affect the growth, but significantly inhibited the hyphal development of C. albicans, including the clinical azole resistant isolates, and reduced their damage to oral epithelial cells, while arteether showed the strongest activities. The transcriptome suggested that arteether could affect the energy metabolism of C. albicans. Seven artemisinins were then proved to significantly inhibit the productions of ATP and cAMP, while reduced the hyphal inhibition on RAS1 overexpression strain indicating that artemisinins regulated the Ras1-cAMP-Efg1 pathway to inhibit the hyphal development. Importantly, arteether significantly inhibited the fungal burden and infections with no systemic toxicity in the murine oropharyngeal candidiasis models in vivo caused by both fluconazole sensitive and resistant strains. Our results for the first time indicated that artemisinins can be potential antifungal compounds against C. albicans infections by targeting at its hyphal development.
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Project description:Localization of specific mRNAs is an important mechanism through which cells achieve polarity and direct asymmetric growth. Based on a framework established in Saccharomyces cerevisiae, we describe a She3-dependent RNA transport system in Candida albicans, a fungal pathogen of humans that grows as both budding (yeast) and filamentous (hyphal and pseudohyphal) forms. We identify a set of 40 mRNAs that are selectively transported to the buds of yeast-form cells and to the tips of hyphae, and we show that many of the genes encoded by these mRNAs contribute to hyphal development, as does the transport system itself. Although the basic system of mRNA transport is conserved between S. cerevisiae and C. albicans, we find that the cargo mRNAs have diverged considerably, implying that specific mRNAs can easily move in and out of transport control over evolutionary timescales. The differences in mRNA cargos likely reflect the distinct selective pressures acting on the two species.
| S-EPMC2739428 | biostudies-literature
Project description:Invasive candidiasis frequently involves medical device placement. On the surfaces of these devices, Candida can form biofilms and proliferate in adherent layers of fungal cells surrounded by a protective extracellular matrix. Due in part to this extracellular matrix, biofilms resist host defenses and antifungal drugs. Previous work (using neutrophils from healthy donors) found that one mechanism employed to resist host defenses involves the inhibition of neutrophil extracellular traps (NET) formation. NETs contain nuclear DNA, as well as antimicrobial proteins that can ensnare pathogens too large or aggregated to be effectively killed by phagocytosis. Given that these neutrophil structures are anticipated to have activity against the large aggregates of C. albicans biofilms, understanding the role of this inhibition in patients could provide insight into new treatment strategies. However, prior work has not included patients. Here, we examine NET formation by neutrophils collected from patients with invasive candidiasis. When compared to neutrophils from healthy participants, we show that patient neutrophils exhibit a heightened background level of NET release and respond to a positive stimulus by producing 100% more NETs. However, despite these physiologic differences, patient neutrophil responses to C. albicans were similar to healthy neutrophils. For both groups, planktonic cells induce strong NET release and biofilms inhibit NET formation. These results show that a mechanism of immune evasion for fungal biofilms translates to the clinical setting.
| S-EPMC7747767 | biostudies-literature
Project description:The ability to switch between different morphological forms is an important feature of Candida albicans and is relevant to its pathogenesis. Many conserved positive and negative transcription factors are involved in morphogenetic regulation of the two dimorphic fungi Candida albicans and Saccharomyces cerevisiae. In S. cerevisiae, the transcriptional repressor Sfl1 and the activator Flo8 function antagonistically in invasive and filamentous growth. We have previously reported that Candida albicans Flo8 is a transcription factor essential for hyphal development and virulence in C. albicans. To determine whether a similar negative factor exists in C. albicans, we identified Candida albicans Sfl1 as a functional homolog of the S. cerevisiae sfl1 mutant. Sfl1 is a negative regulator of hyphal development in C. albicans. Deletion of C. albicans SFL1 enhanced filamentous growth and hypha-specific gene expression in several media and at several growth temperatures. Overexpression of the SFL1 led to a significant reduction of filament formation. Both deletion and overexpression of the SFL1 attenuated virulence of C. albicans in a mouse model. Deleting FLO8 in an sfl1/sfl1 mutant completely blocked hyphal development in various growth conditions examined, suggesting that C. albicans Sfl1 may act as a negative regulator of filamentous growth by antagonizing Flo8 functions. We suggest that, similar to the case for S. cerevisiae, a combination of dual control by activation and repression of Flo8 and Sfl1 may contribute to the fine regulatory network in C. albicans morphogenesis responding to different environmental cues.
| S-EPMC2168412 | biostudies-literature
Project description:Morphogenesis contributes to the virulence of the opportunistic human fungal pathogen Candida albicans. Ras1-MAPK pathways play a critical role in the virulence of C. albicans by regulating cell growth, morphogenesis, and biofilm formation. Ume6 acts as a transcription factor, and Nrg1 is a transcriptional repressor for the expression of hyphal-specific genes in morphogenesis. Azoles or echinocandin drugs have been extensively prescribed for C. albicans infections, which has led to the development of drug-resistant strains. Therefore, it is necessary to develop new molecules to effectively treat fungal infections. Here, we showed that Molecule B and Molecule C, which contained a carbazole structure, attenuated the pathogenicity of C. albicans through inhibition of the Ras1/MAPK pathway. We found that Molecule B and Molecule C inhibit morphogenesis through repressing protein and RNA levels of Ras/MAPK-related genes, including UME6 and NRG1. Furthermore, we determined the antifungal effects of Molecule B and Molecule C in vivo using a candidiasis murine model. We anticipate our findings are that Molecule B and Molecule C, which inhibits the Ras1/MAPK pathway, are promising compounds for the development of new antifungal agents for the treatment of systemic candidiasis and possibly for other fungal diseases.
| S-EPMC8466151 | biostudies-literature
Project description:Nrg1 is a repressor of hypha formation and hypha-associated gene expression in the fungal pathogen Candida albicans. It has been well studied in the genetic background of the type strain SC5314. Here, we tested Nrg1 function in four other diverse clinical isolates through an analysis of nrg1Δ/Δ mutants, with SC5314 included as a control. In three strains, nrg1Δ/Δ mutants unexpectedly produced aberrant hyphae under inducing conditions, as assayed by microscopic observation and endothelial cell damage. The nrg1Δ/Δ mutant of strain P57055 had the most severe defect. We examined gene expression features under hypha-inducing conditions by RNA-sequencing (RNA-Seq) for the SC5314 and P57055 backgrounds. The SC5314 nrg1Δ/Δ mutant expressed six hypha-associated genes at reduced levels compared with wild-type SC5314. The P57055 nrg1Δ/Δ mutant expressed 17 hypha-associated genes at reduced levels compared with wild-type P57055, including IRF1, RAS2, and ECE1. These findings indicate that Nrg1 has a positive role in hypha-associated gene expression and that this role is magnified in strain P57055. Remarkably, the same hypha-associated genes affected by the nrg1Δ/Δ mutation in strain P57055 were also naturally expressed at lower levels in wild-type P57055 than those in wild-type SC5314. Our results suggest that strain P57055 is defective in a pathway that acts in parallel with Nrg1 to upregulate the expression of several hypha-associated genes. IMPORTANCE Hypha formation is a central virulence trait of the fungal pathogen Candida albicans. Control of hypha formation has been studied in detail in the type strain but not in other diverse C. albicans clinical isolates. Here, we show that the hyphal repressor Nrg1 has an unexpected positive role in hypha formation and hypha-associated gene expression, as revealed by the sensitized P57055 strain background. Our findings indicate that reliance on a single type strain limits understanding of gene function and illustrate that strain diversity is a valuable resource for C. albicans molecular genetic analysis.
| S-EPMC10127614 | biostudies-literature
Project description:Candida albicans, the most common human fungal pathogen, is particularly problematic for immunocompromised individuals. The reversible transition of this fungal pathogen to a filamentous form that invades host tissue is important for its virulence. Although different signaling pathways such as a mitogen-activated protein kinase and a protein kinase A cascade are critical for this morphological transition, the function of polarity establishment proteins in this process has not been determined. We examined the role of four different polarity establishment proteins in C. albicans invasive growth and virulence by using strains in which one copy of each gene was deleted and the other copy expressed behind the regulatable promoter MET3. Strikingly, mutants with ectopic expression of either the Rho G-protein Cdc42 or its exchange factor Cdc24 are unable to form invasive hyphal filaments and germ tubes in response to serum or elevated temperature and yet grow normally as a budding yeast. Furthermore, these mutants are avirulent in a mouse model for systemic infection. This function of the Cdc42 GTPase module is not simply a general feature of polarity establishment proteins. Mutants with ectopic expression of the SH3 domain containing protein Bem1 or the Ras-like G-protein Bud1 can grow in an invasive fashion and are virulent in mice, albeit with reduced efficiency. These results indicate that a specific regulation of Cdc24/Cdc42 activity is required for invasive hyphal growth and suggest that these proteins are required for pathogenicity of C. albicans.
| S-EPMC141177 | biostudies-literature
Project description:Oropharyngeal candidiasis (OPC) is the most prevalent oral infection in immunocompromised patients, primarily associated with Candida albicans. Increasing evidence points to a significant role of mucosal bacteria on the transition of C. albicans from commensal to pathogenic. In this work, we hypothesized that changes in the abundance or composition of the mucosal bacterial microbiota induced by dietary sucrose during the development of OPC can modulate C. albicans virulence. C. albicans burdens and mucosal lesions were evaluated in a mouse cortisone immunosuppression model amended with sucrose. We also analyzed the mucosal bacterial composition using 16S rRNA gene sequencing and culture methods. In immunocompetent mice, sucrose significantly increased total bacterial burdens and reduced alpha diversity, by increasing the relative abundance of mitis group streptococci. In immunocompromised mice, C. albicans infection was associated with a significantly reduced bacterial alpha diversity due to an increase in the relative abundance of enterococci. When exposed to dietary sucrose, these mice had reduced C. albicans burdens and reduced bacterial alpha diversity, associated with an increase in the relative abundance of Lactobacillus. SparCC correlation networks showed a significant negative correlation between Lactobacillus and Enterococcus in all Candida-infected mice. Depletion of lactobacilli with antibiotic treatment partially restored C. albicans burdens in mice receiving sucrose. In coculture in vitro experiments, mouse oral Lactobacillus johnsonii isolates inhibited growth of Enterococcus faecalis isolates and C. albicans. These results support the hypothesis that the sucrose-induced attenuation of C. albicans virulence was a result of changes in the mucosal bacterial microbiome characterized by a reduction in enterococci and an increase in lactobacilli. IMPORTANCE By comparing Candida albicans virulence and the mucosal bacterial composition in a mouse oral infection model, we were able to dissect the effects of the host environment (immunosuppression), infection with C. albicans, and local modulating factors (availability of sucrose as a carbon source) on the mucosal bacterial microbiome and its role on fungal virulence. We showed that changes in endogenous microbial communities in response to sucrose can lead to attenuation of fungal disease. We also showed that Lactobacillus johnsonii may curtail Candida virulence both by inhibiting its growth and by inhibiting the growth of potentially synergistic bacteria such as enterococci. Our results support the concept that Candida pathogenesis should be viewed in the contexts of both a susceptible host and a mucosal bacterial microbiota conducive to virulence.
| S-EPMC8406182 | biostudies-literature
Project description:Various stimuli, including N-acetylglucosamine (GlcNAc), induce the fungal pathogen Candida albicans to switch from budding to hyphal growth. Previous studies suggested that hyphal morphogenesis is stimulated by transcriptional induction of a set of genes that includes known virulence factors. To better understand hyphal development, we examined the role of GlcNAc metabolism using a triple mutant lacking the genes required to metabolize exogenous GlcNAc (hxk1? nag1? dac1?). Surprisingly, at low ambient pH (?pH 4), GlcNAc stimulated this mutant to form hyphae without obvious induction of hyphal genes. This indicates that GlcNAc can stimulate a separate signal to induce hyphae that is independent of transcriptional responses. Of interest, GlcNAc could induce the triple mutant to express hyphal genes when the medium was buffered to a higher pH (>pH 5), which normally occurs after GlcNAc catabolism. Catabolism of GlcNAc raises the ambient pH rather than acidifying it, as occurs after dextrose catabolism. This synergy between alkalinization and GlcNAc to induce hyphal genes involves the Rim101 pH-sensing pathway; GlcNAc induced rim101? and dfg16? mutants to form hyphae, but hyphal gene expression was partially defective. These results demonstrate that hyphal morphogenesis and gene expression can be regulated independently, which likely contributes to pathogenesis at different host sites.
| S-EPMC4357515 | biostudies-literature