Interplay of Cytokines and Chemokines in Aspergillosis.

Aspergillosis is a fungal infection caused by various species of Aspergillus, most notably A. fumigatus. This fungus causes a spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma, chronic pulmonary aspergillosis, and invasive aspergillosis. The clinical manifestations and severity of aspergillosis can vary depending on individual immune status and the specific species of Aspergillus involved. The recognition of Aspergillus involves pathogen-associated molecular patterns (PAMPs) such as glucan, galactomannan, mannose, and conidial surface proteins. These are recognized by the pathogen recognition receptors present on immune cells such as Toll-like receptors (TLR-1,2,3,4, etc.) and C-type lectins (Dectin-1 and Dectin-2). We discuss the roles of cytokines and pathogen recognition in aspergillosis from both the perspective of human and experimental infection. Several cytokines and chemokines have been implicated in the immune response to Aspergillus infection, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), CCR4, CCR17, and other interleukins. For example, allergic bronchopulmonary aspergillosis (ABPA) is characterized by Th2 and Th9 cell-type immunity and involves interleukin (IL)-4, IL-5, IL-13, and IL-10. In contrast, it has been observed that invasive aspergillosis involves Th1 and Th17 cell-type immunity via IFN-γ, IL-1, IL-6, and IL-17. These cytokines activate various immune cells and stimulate the production of other immune molecules, such as antimicrobial peptides and reactive oxygen species, which aid in the clearance of the fungal pathogen. Moreover, they help to initiate and coordinate the immune response, recruit immune cells to the site of infection, and promote clearance of the fungus. Insight into the host response from both human and animal studies may aid in understanding the immune response in aspergillosis, possibly leading to harnessing the power of cytokines or cytokine (receptor) antagonists and transforming them into precise immunotherapeutic strategies. This could advance personalized medicine.

Proteome Profile of Aspergillus terreus Conidia at Germinating Stage: Identification of Probable Virulent Factors and Enzymes from Mycotoxin Pathways.

Aspergillus terreus is an emerging opportunistic fungal pathogen that causes invasive aspergillosis in immunocompromised individuals. The main risk group of individuals for this organism is leukopenic patients, individuals having cancers, bone marrow transplant persons and those who have immunological disorders. The lack of early diagnostic marker for A. terreus and intrinsic resistance to Amphotericin B, further limits the successful therapy of A. terreus-associated infections. The germination of inhaled conidia is the key step to establish successful invasion in host tissues or organs. Thus, profiling of expressed proteins during germination of conidia not only shed light on proteins that are involved in invasion or virulence but may also provide early diagnostic markers. We used nanoLC-Q-TOF to study the proteome of germinating conidia (at 16 h time points) of A. terreus. We observed expression of 373 proteins in germinating conidia of A. terreus. A total of 74 proteins were uncharacterized in the database. The expressed proteins were associated with various processes like cell wall modulation, virulence factors and secondary metabolite biosynthesis. The most abundant proteins were associated with protein biosynthesis, carbohydrate metabolism and unknown functions. Among virulent proteins, mitogen-activated protein kinase (hog1) and mitogen-activated protein kinase (mpkC) are key virulent proteins observed in our study. We observed 7 enzymes from terretonin and 10 enzymes from geodin mycotoxin biosynthesis pathway. Interestingly, we observed expression of terrelysin protein, associated with blood cell lysis. Quantitative RT-PCR analysis showed 26-fold increase in transcripts encoding for dihydrogeodin oxidase and 885-fold for terrelysin gene in germinating conidia in comparison to conidia. Further, we propose that terrelysin protein and secondary metabolite such as geodin could be explored as diagnostic marker for A. terreus-associated infections.

Mycologic Endocrinology.

The interactions of fungi and chemical messenger molecules, hormones or pheromones, are addressed in this chapter. These interactions include mammalian fungal pathogens, also plant pathogens, or non-pathogenic fungi, which can result in functional responses in receptor- or non-receptor-mediated fashions. Endogenous ligands in the fungi have been demonstrated to be important for mating in a number of systems. Mammalian hormones have been demonstrated to have stimulatory or inhibitory effects on growth for organisms such as Candida albicans, Paracoccidioides brasiliensis, Saccharomyces cerevisiae, Rhizopus nigricans, Aspergillus fumigatus, Coccidioides, and dermatophytic fungi. A number of fungi have been shown to have specific binding proteins for corticosteroid, estrogen and progesterone that are stereo-specific and high affinity. In some instances, the interactions of a mammalian hormone with the organism, in vivo, affects pathogenesis. Genome expression profiles of C. albicans in the presence of estradiol or progesterone, and S. cerevisiae with progesterone, indicate major up-regulation of various drug resistance pumps, like CDR1, and CDR2, can affect antifungal susceptibility. Azole antifungal interactions occur with fungal hormone binding proteins. Azoles also can block mammalian steroidogenesis. The finding of interactions of mammalian hormones with fungi and subsequent functional responses by the fungi, suggest that hormonal interactions with fungal systems has been conserved throughout evolution and have an important role in fungal pathogenesis, as well as in the overall biology of the organisms.

Influence of 17ß-estradiol on gene expression of Paracoccidioides during mycelia-to-yeast transition.

BACKGROUND: Paracoccidioides is the causative agent of paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Infection is initiated by inhalation of conidia (C) or mycelial (M) fragments, which subsequently differentiate into yeast (Y). Epidemiological studies show a striking predominance of paracoccidioidomycosis in adult men compared to premenopausal women. In vitro and in vivo studies suggest that the female hormone (17ß-estradiol, E(2)) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E(2) inhibits M-to-Y transition. METHODOLOGY: We assessed temporal gene expression in strain Pb01 in the presence or absence of E(2) at various time points through 9 days of the M-to-Y transition using an 11,000 element random-shear genomic DNA microarray and verified the results using quantitative real time-PCR. E(2)-regulated clones were sequenced to identify genes and biological function. PRINCIPAL FINDINGS: E(2)-treatment affected gene expression of 550 array elements, with 331 showing up-regulation and 219 showing down-regulation at one or more time points (p≤0.001). Genes with low expression after 4 or 12 h exposure to E(2) belonged to pathways involved in heat shock response (hsp90 and hsp70), energy metabolism, and several retrotransposable elements. Y-related genes, α-1,3-glucan synthase, mannosyltransferase and Y20, demonstrated low or delayed expression in E(2)-treated cultures. Genes potentially involved in signaling, such as palmitoyltransferase (erf2), small GTPase RhoA, phosphatidylinositol-4-kinase, and protein kinase (serine/threonine) showed low expression in the presence of E(2), whereas a gene encoding for an arrestin domain-containing protein showed high expression. Genes related to ubiquitin-mediated protein degradation, and oxidative stress response genes were up-regulated by E(2). CONCLUSION: This study characterizes the effect of E(2) at the molecular level on the inhibition of the M-to-Y transition and is indicative that the inhibitory actions of E(2) may be working through signaling genes that regulate dimorphism.

Hormones and the resistance of women to paracoccidioidomycosis.

Paracoccidioidomycosis, one of the most important endemic and systemic mycoses in Latin America, presents several clinical pictures. Epidemiological studies indicate a striking rarity of disease (but not infection) in females, but only during the reproductive years. This suggested a hormonal interaction between female hormones and the etiologic dimorphic fungus Paracoccidioides brasiliensis. Many fungi have been shown to use hormonal (pheromonal) fungal molecules for intercellular communication, and there are increasing numbers of examples of interactions between mammalian hormones and fungi, including the specific binding of mammalian hormones by fungal proteins, and suggestions of mammalian hormonal modulation of fungal behavior. This suggests an evolutionary conservation of hormonal receptor systems. We recount studies showing the specific hormonal binding of mammalian estrogen to proteins in P. brasiliensis and an action of estrogen to specifically block the transition from the saprophytic form to the invasive form of the fungus in vitro. This block has been demonstrated to occur in vivo in animal studies. These unique observations are consistent with an estrogen-fungus receptor-mediated effect on pathogenesis. The fungal genes responsive to estrogen action are under study.

Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro.

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25 % of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12,000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.