Human-Fungal Pathogen Interactions from the Perspective of Immunoproteomics Analyses.

Antibody immunity is now known to play a critical role in combating mycotic infections. The identification of molecules that can elicit an antibody response against fungal pathogens is the first step in developing antibody-based therapeutic strategies. Antigenic proteins are molecules recognized by the immune system that can stimulate antibody production and, therefore, can be a direct target for studying human-fungal pathogen interactions. Advances in recent immunoproteomic approaches have substantially aided in determining the key antigenic proteins on a large scale. In this review, we present a collection of antigenic proteins identified in yeast, dimorphic, and filamentous fungal pathogens to date. The general features of antigenic proteins are summarized and reveal that the proteins could commonly function in antistress responses, protein synthesis, and metabolism. The antigenic proteins listed here could serve as starting materials for developing species-specific or broad-spectrum diagnostic tests, therapeutic antibodies, and even vaccines against fungal infections.

The Role of the Glutathione System in Stress Adaptation, Morphogenesis and Virulence of Pathogenic Fungi.

Morphogenesis and stress adaptation are key attributes that allow fungal pathogens to thrive and infect human hosts. During infection, many fungal pathogens undergo morphological changes, and this ability is highly linked to virulence. Furthermore, pathogenic fungi have developed multiple antioxidant defenses to cope with the host-derived oxidative stress produced by phagocytes. Glutathione is a major antioxidant that can prevent cellular damage caused by various oxidative stressors. While the role of glutathione in stress detoxification is known, studies of the glutathione system in fungal morphological switching and virulence are lacking. This review explores the role of glutathione metabolism in fungal adaptation to stress, morphogenesis, and virulence. Our comprehensive analysis of the fungal glutathione metabolism reveals that the role of glutathione extends beyond stressful conditions. Collectively, glutathione and glutathione-related proteins are necessary for vitality, cellular development and pathogenesis.

Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-ß.

Extracellular plaques of amyloid-ß and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-ß fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-ß. Interestingly, many adverse responses to amyloid-ß, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-ß are strongly associated with Alzheimer's disease, are more toxic than amyloid-ß, residues 1-42 (Aß(1-42)) and Aß(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aß may trigger Alzheimer's disease. Aß(3(pE)-42) co-oligomerizes with excess Aß(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aß(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aß(3(pE)-42) plus 95% Aß(1-42) (5% pE-Aß) seed new cytotoxic LNOs through multiple serial dilutions into Aß(1-42) monomers in the absence of additional Aß(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aß(3(pE)-42), and enhanced Aß(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aß(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aß(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aß(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.

The GRF10 homeobox gene regulates filamentous growth in the human fungal pathogen Candida albicans.

Candida albicans is the most common human fungal pathogen and can cause life-threatening infections. Filamentous growth is critical in the pathogenicity of C. albicans, as the transition from yeast to hyphal forms is linked to virulence and is also a pivotal process in fungal biofilm development. Homeodomain-containing transcription factors have been linked to developmental processes in fungi and other eukaryotes. We report here on GRF10, a homeobox transcription factor-encoding gene that plays a role in C. albicans filamentation. Deletion of the GRF10 gene, in both C. albicans SN152 and BWP17 strain backgrounds, results in mutants with strongly decreased hyphal growth. The mutants are defective in chlamydospore and biofilm formation, as well as showing dramatically attenuated virulence in a mouse infection model. Expression of the GRF10 gene is highly induced during stationary phase and filamentation. In summary, our study emphasizes a new role for the homeodomain-containing transcription factor in morphogenesis and pathogenicity of C. albicans.

Antibody screening reveals antigenic proteins involved in Talaromyces marneffei and human interaction.

Talaromycosis is a fungal infection that generally affects immunocompromised hosts and is one of the most frequent systemic mycoses in HIV patients, especially in endemic areas such as Southeast Asia. Talaromyces marneffei, the causative agent of talaromycosis, grows as a mold in the environment but adapts to the human body and host niches by transitioning from conidia to yeast-like cells. Knowledge of the human host and T. marneffei interaction has a direct impact on the diagnosis, yet studies are still lacking. The morbidity and mortality rates are high in taloromycosis patients if the diagnosis and treatments are delayed. Immunogenic proteins are excellent candidates for developing detection tools. Previously, we identified antigenic proteins that were recognized by antibodies from talaromycosis sera. Three of these identified proteins have been previously characterized in detail, while the others have not been explored. To expedite the progress of antigen discovery, the complete list of antigenic proteins and their features was fully reported in this study. Functional annotation and Gene Ontology examination revealed that these proteins showed a high association with membrane trafficking. Further bioinformatics analyses were performed to search for antigenic protein characteristics, including functional domains, critical residues, subcellular localization, secretory signals, and epitope peptide sequences. Expression profiling of these antigenic encoding genes was investigated using quantitative real-time PCR. The results demonstrated that most genes were expressed at low levels in the mold form, but were highly upregulated in the pathogenic yeast phase, consistent with the antigenic role of these genes during the human-host interaction. Most transcripts accumulated in the conidia, suggesting a role during phase transition. The collection of all antigen-encoding DNA sequences described here is freely accessible at GenBank, which could be useful for the research community to develop into biomarkers, diagnostic tests, research detection tools, and even vaccines.

Identification of glutathione metabolic genes from a dimorphic fungus Talaromyces marneffei and their gene expression patterns under different environmental conditions.

Talaromyces marneffei is a human fungal pathogen that causes endemic opportunistic infections, especially in Southeast Asia. The key virulence factors of T. marneffei are the ability to survive host-derived heat and oxidative stress, and the ability to convert morphology from environmental mold to fission yeast forms during infection. Glutathione metabolism plays an essential role in stress response and cellular development in multiple organisms. However, the role of the glutathione system in T. marneffei is elusive. Here, we identified the genes encoding principal enzymes associated with glutathione metabolism in T. marneffei, including glutathione biosynthetic enzymes (Gcs1 and Gcs2), glutathione peroxidase (Gpx1), glutathione reductase (Glr1), and a family of glutathione S-transferase (Gst). Sequence homology search revealed an extended family of the TmGst proteins, consisting of 20 TmGsts that could be divided into several classes. Expression analysis revealed that cells in conidia, mold, and yeast phases exhibited distinct expression profiles of glutathione-related genes. Also, TmGst genes were highly upregulated in response to hydrogen peroxide and xenobiotic exposure. Altogether, our findings suggest that T. marneffei transcriptionally regulates the glutathione genes under stress conditions in a cell-type-specific manner. This study could aid in understanding the role of glutathione in thermal-induced dimorphism and stress response.

Grf10 regulates the response to copper, iron, and phosphate in Candida albicans.

The pathogenic yeast, Candida albicans, and other microbes must be able to handle drastic changes in nutrient availability within the human host. Copper, iron, and phosphate are essential micronutrients for microbes that are sequestered by the human host as nutritional immunity; yet high copper levels are employed by macrophages to induce toxic oxidative stress. Grf10 is a transcription factor important for regulating genes involved in morphogenesis (filamentation, chlamydospore formation) and metabolism (adenylate biosynthesis, 1-carbon metabolism). The grf10Δ mutant exhibited resistance to excess copper in a gene dosage-dependent manner but grew the same as the wild type in response to other metals (calcium, cobalt, iron, manganese, and zinc). Point mutations in the conserved residues D302 and E305, within a protein interaction region, conferred resistance to high copper and induced hyphal formation similar to strains with the null allele. The grf10Δ mutant misregulated genes involved with copper, iron, and phosphate uptake in YPD medium and mounted a normal transcriptional response to high copper. The mutant accumulated lower levels of magnesium and phosphorus, suggesting that copper resistance is linked to phosphate metabolism. Our results highlight new roles for Grf10 in copper and phosphate homeostasis in C. albicans and underscore the fundamental role of Grf10 in connecting these with cell survival.

Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105.

BACKGROUND: Scrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKß-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined. PRINCIPAL FINDINGS: Western blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen's ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm. CONCLUSIONS: O. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.

Functional Mapping of Transcription Factor Grf10 That Regulates Adenine-Responsive and Filamentation Genes in Candida albicans.

Grf10, a homeodomain-containing transcription factor, regulates adenylate and one-carbon metabolism and morphogenesis in the human fungal pathogen Candida albicans Here, we identified functional domains and key residues involved in transcription factor activity using one-hybrid and mutational analyses. We localized activation domains to the C-terminal half of the Grf10 protein by one-hybrid analysis and identified motifs using bioinformatic analyses; one of the characterized activation domains (AD1) responded to temperature. The LexA-Grf10 fusion protein activated the lexAop-HIS1 reporter in an adenine-dependent fashion, and this activation was independent of Bas1, showing that the adenine limitation signal is transmitted directly to Grf10. Overexpression of LexA-Grf10 led to filamentation, and this required a functioning homeodomain, consistent with Grf10 controlling the expression of key filamentation genes; filamentation induced by LexA-Grf10 overexpression was independent of adenine levels and Bas1. Alanine substitutions were made within the conserved interaction regions (IR) of LexA-Grf10 and Grf10 to investigate roles in transcription. In LexA-Grf10, the D302A mutation activated transcription constitutively, and the E305A mutation was regulated by adenine. When these mutations were introduced into the native gene locus, the D302A mutation was unable to complement the ADE phenotype and did not promote filamentation under hypha-inducing conditions; the E305A mutant behaved as the native gene with respect to the ADE phenotype and was partially defective in inducing hyphae. These results demonstrate allele-specific responses with respect to the different phenotypes, consistent with perturbations in the ability of Grf10 to interact with multiple partner proteins.IMPORTANCE Metabolic adaptation and morphogenesis are essential for Candida albicans, a major human fungal pathogen, to survive and infect diverse body sites in the mammalian host. C. albicans utilizes transcription factors to tightly control the transcription of metabolic genes and morphogenesis genes. Grf10, a critical homeodomain transcription factor, controls purine and one-carbon metabolism in response to adenine limitation, and Grf10 is necessary for the yeast-to-hypha morphological switching, a known virulence factor. Here, we carried out one-hybrid and mutational analyses to identify functional domains of Grf10. Our results show that Grf10 separately regulates metabolic and morphogenesis genes, and it contains a conserved protein domain for protein partner interaction, allowing Grf10 to control the transcription of multiple distinct pathways. Our findings contribute significantly to understanding the role and mechanism of transcription factors that control multiple pathogenic traits in C. albicans.

Grf10 and Bas1 Regulate Transcription of Adenylate and One-Carbon Biosynthesis Genes and Affect Virulence in the Human Fungal Pathogen Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that causes superficial fungal infections and lethal systemic infections. To colonize and establish infections, C. albicans coordinates the expression of virulence and metabolic genes. Previous work showed that the homeodomain transcription factor Grf10 is required for formation of hyphae, a virulence factor. Here we report global gene expression analysis of a grf10Δ strain using a DNA microarray and identify genes for de novo adenylate biosynthesis (ADE genes), one-carbon metabolism, and a nucleoside permease (NUP). Upregulation of these genes in response to adenine limitation required both Grf10 and the myb protein Bas1, as shown by quantitative real-time PCR (qRT-PCR). Phenotypic analysis showed that both mutants exhibited growth defects when grown in the absence of adenine, and the doubling time was slower for the bas1Δ mutant. Bas1 is required for basal expression of these genes, whereas NUP expression is more dependent upon Grf10. Disruption of BAS1 led to only modest defects in hypha formation and weak attenuation of virulence in a systemic mouse model of infection, as opposed to the previously reported strong effects found in the grf10Δ mutant. Our data are consistent with a model in which Grf10 coordinates metabolic effects on nucleotide metabolism by interaction with Bas1 and indicate that AMP biosynthesis and its regulation are important for C. albicans growth and virulence. IMPORTANCECandida albicans is a commensal and a common constituent of the human microbiota; however, it can become pathogenic and cause infections in both immunocompetent and immunocompromised people. C. albicans exhibits remarkable metabolic versatility as it can colonize multiple body sites as a commensal or pathogen. Understanding how C. albicans adapts metabolically to each ecological niche is essential for developing novel therapeutic approaches. Purine metabolism has been targeted pharmaceutically in several diseases; however, the regulation of this pathway has not been fully elucidated in C. albicans. Here, we report how C. albicans controls the AMP de novo biosynthesis pathway in response to purine availability. We show that the lack of the transcription factors Grf10 and Bas1 leads to purine metabolic dysfunction, and this dysfunction affects the ability of C. albicans to establish infections.