Garry T. Cole

Evolution of the mating type locus: Insights gained from the dimorphic primary fungal pathogens Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii

ABSTRACT Sexual reproduction of fungi is governed by the mating type (MAT) locus, a specialized region of the genome encoding key transcriptional regulators that direct regulatory networks to specify cell identity and fate. Knowledge of MAT locus structure and evolution has been considerably advanced in recent years as a result of genomic analyses that enable the definition of MAT locus sequences in many species as well as provide an understanding of the evolutionary plasticity of this unique region of the genome. Here, we extend this analysis to define the mating type locus of three dimorphic primary human fungal pathogens, Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii, using genomic analysis, direct sequencing, and bioinformatics. These studies provide evidence that all three species possess heterothallic bipolar mating type systems, with isolates encoding either a high-mobility-group (HMG) domain or an α-box transcriptional regulator. These genes are intact in all loci examined and have not been subject to loss or decay, providing evidence that the loss of fertility upon passage in H. capsulatum is not attributable to mutations at the MAT locus. These findings also suggest that an extant sexual cycle remains to be defined in both Coccidioides species, in accord with population genetic evidence. Based on these MAT sequences, a facile PCR test was developed that allows the mating type to be rapidly ascertained. Finally, these studies highlight the evolutionary forces shaping the MAT locus, revealing examples in which flanking genes have been inverted or subsumed and incorporated into an expanding MAT locus, allowing us to propose an expanded model for the evolution of the MAT locus in the phylum Ascomycota.

The fungal spore and disease initiation in plants and animals.

I. Spore Attachment and Invasion.- 1. Adhesion of Fungi to the Plant Surface: Prerequisite for Pathogenesis.- 2. Signaling for Infection Structure Formation in Fungi.- 3. The Plant Cell Wall as a Barrier to Fungal Invasion.- 4. Rust Basidiospore Germlings and Disease Initiation.- 5. Attachment of Mycopathogens to Cuticle: The Initial Event of Mycoses in Arthropod Hosts.- 6. The Fate of Fungal Spores in the Insect Gut.- 7. Candida Blastospore Adhesion, Association, and Invasion of the Gastrointestinal Tract of Vertebrates.- 8. Infectious Propagules of Dermatophytes.- II. Fungal Spore Products and Pathogenesis.- 9. Melanin Biosynthesis: Prerequisite for Successful Invasion of the Plant Host by Appressoria of Colletotrichum and Pyricularia.- 10. The Plant Cuticle: A Barrier to Be Overcome by Fungal Plant Pathogens.- 11. Appearance of Pathogen-Related Proteins in Plant Hosts: Relationships between Compatible and Incompatible Interactions.- 12. The Role of Cuticle-Degrading Enzymes in Fungal Pathogenesis in Insects.- 13. Potential for Penetration of Passive Barriers to Fungal Invasion in Humans.- 14. Dihydroxynaphthalene (DHN) Melanin and Its Relationship with Virulence in the Early Stages of Phaeohyphomycosis.- III. Host Response to Early Fungal Invasion.- 15. Invasion of Plants by Powdery Mildew Fungi, and Cellular Mechanisms of Resistance.- 16. Induced Systemic Resistance in Plants.- 17. The Plant Membrane and Its Response to Disease.- 18. The Fungal Spore: Reservoir of Allergens.- 19. Conidia of Coccidioides immitis: Their Significance in Disease Initiation.- 20. Cell-Mediated Host Response to Fungal Aggression.- 21. Suppression of Phagocytic Cell Responses by Conidia and Conidial Products of Aspergillus fumigatus.- IV. Molecular Aspects of Disease Initiation.- 22. Molecular Approaches to the Analysis of Pathogenicity Genes from Fungi Causing Plant Disease.- 23. Current Status of the Molecular Basis of Candida Pathogenicity.- Taxonomic Index.

Urease as a Virulence Factor in Experimental Cryptococcosis

ABSTRACT Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of aure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation withURE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.

Comparative genomic analyses of the human fungal pathogens Coccidioides and their relatives.

While most Ascomycetes tend to associate principally with plants, the dimorphic fungi Coccidioides immitis and Coccidioides posadasii are primary pathogens of immunocompetent mammals, including humans. Infection results from environmental exposure to Coccidiodies, which is believed to grow as a soil saprophyte in arid deserts. To investigate hypotheses about the life history and evolution of Coccidioides, the genomes of several Onygenales, including C. immitis and C. posadasii; a close, nonpathogenic relative, Uncinocarpus reesii; and a more diverged pathogenic fungus, Histoplasma capsulatum, were sequenced and compared with those of 13 more distantly related Ascomycetes. This analysis identified increases and decreases in gene family size associated with a host/substrate shift from plants to animals in the Onygenales. In addition, comparison among Onygenales genomes revealed evolutionary changes in Coccidioides that may underlie its infectious phenotype, the identification of which may facilitate improved treatment and prevention of coccidioidomycosis. Overall, the results suggest that Coccidioides species are not soil saprophytes, but that they have evolved to remain associated with their dead animal hosts in soil, and that Coccidioides metabolism genes, membrane-related proteins, and putatively antigenic compounds have evolved in response to interaction with an animal host.

Biology of conidial fungi

Biology of conidial fungi , Biology of conidial fungi , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice

Worldwide rates of systemic fungal infections, including three of the major pathogens responsible for such infections in North America (Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis), have soared recently, spurring interest in developing vaccines. The development of Th1 cells is believed to be crucial for protective immunity against pathogenic fungi, whereas the role of Th17 cells is vigorously debated. In models of primary fungal infection, some studies have shown that Th17 cells mediate resistance, while others have shown that they promote disease pathology. Here, we have shown that Th1 immunity is dispensable and that fungus-specific Th17 cells are sufficient for vaccine-induced protection against lethal pulmonary infection with B. dermatitidis in mice. Further, vaccine-induced Th17 cells were necessary and sufficient to protect against the three major systemic mycoses in North America. Mechanistically, Th17 cells engendered protection by recruiting and activating neutrophils and macrophages to the alveolar space, while the induction of Th17 cells and acquisition of vaccine immunity unexpectedly required the adapter molecule Myd88 but not the fungal pathogen recognition receptor Dectin-1. These data suggest that human vaccines against systemic fungal infections should be designed to induce Th17 cells if they are to be effective.

Population genomic sequencing of Coccidioides fungi reveals recent hybridization and transposon control

We have sequenced the genomes of 18 isolates of the closely related human pathogenic fungi Coccidioides immitis and Coccidioides posadasii to more clearly elucidate population genomic structure, bringing the total number of sequenced genomes for each species to 10. Our data confirm earlier microsatellite-based findings that these species are genetically differentiated, but our population genomics approach reveals that hybridization and genetic introgression have recently occurred between the two species. The directionality of introgression is primarily from C. posadasii to C. immitis, and we find more than 800 genes exhibiting strong evidence of introgression in one or more sequenced isolates. We performed PCR-based sequencing of one region exhibiting introgression in 40 C. immitis isolates to confirm and better define the extent of gene flow between the species. We find more coding sequence than expected by chance in the introgressed regions, suggesting that natural selection may play a role in the observed genetic exchange. We find notable heterogeneity in repetitive sequence composition among the sequenced genomes and present the first detailed genome-wide profile of a repeat-induced point mutation (RIP) process distinctly different from what has been observed in Neurospora. We identify promiscuous HLA-I and HLA-II epitopes in both proteomes and discuss the possible implications of introgression and population genomic data for public health and vaccine candidate prioritization. This study highlights the importance of population genomic data for detecting subtle but potentially important phenomena such as introgression.

A Parasitic Phase-Specific Adhesin of Coccidioides immitis Contributes to the Virulence of This Respiratory Fungal Pathogen

ABSTRACT We report the isolation of a Coccidioides immitis gene (SOWgp) which encodes an immunodominant, spherule outer wall glycoprotein that is presented as a component of a parasitic phase-specific, membranous layer at the cell surface. The open reading frame of the gene from C. immitis isolate C735 translates a 422-amino-acid (aa) polypeptide that contains 6 copies of a 41- to 47-residue tandem repeat enriched in proline (20.4 mol%) and aspartate (19.7%). Two additional isolates of C. immitis produce SOWgps of different molecular sizes (328 and 375 aa) and show a corresponding difference in the number of tandem repeats (four and five, respectively). The accurate molecular sizes of these proline-rich antigens, as determined by surface-enhanced laser desorption/ionization mass spectrometry, are comparable to the predicted sizes from the translated protein sequences rather than the estimated sizes based on gel-electrophoretic separation. The results of Northern hybridization confirmed that SOWgp expression is parasitic phase specific, and immunoblot studies showed that elevated levels of production of this antigen occurred during early spherule development. The recombinant polypeptide (rSOWp) was shown to bind to mammalian extracellular matrix (ECM) proteins in an in vitro assay (laminin > fibronectin > collagen type IV), suggesting that the parasitic cell surface antigen may function as an adhesin. Deletion of the SOWgp gene by using a targeted gene replacement strategy resulted in partial loss of the ability of intact spherules to bind to ECM proteins and a significant reduction in virulence of the mutant strain. The wild-type gene was restored in the mutant by homologous recombination, and the revertant strain was shown to be as virulent as the parental isolate in our murine model of coccidioidomycosis. The parasitic cell surface glycoprotein encoded by the SOWgp gene appears to function as an adhesin and contributes to the virulence of C. immitis.

Prospects of vaccines for medically important fungi: A vaccine against coccidioidomycosis is justified and attainable

Coccidioides is a fungal pathogen of humans which can cause a life-threatening respiratory disease in immunocompetent individuals. Recurrent epidemics of coccidioidal infections in Southwestern United States has raised the specter of awareness of this soil-borne microbe, particularly among residents of Arizona and Southern California, and has galvanized research efforts to develop a human vaccine against coccidioidomycosis. In this review, we discuss the rationale for such a vaccine, examine the features of host innate and acquired immune response to Coccidioides infection, describe strategies used to identify and evaluate vaccine candidates, and provide an update on progress toward development of a vaccine against this endemic pathogen.

A Metalloproteinase of Coccidioides posadasii Contributes to Evasion of Host Detection

ABSTRACT Coccidioides posadasii is a fungal respiratory pathogen of humans that can cause disease in immunocompetent individuals. Coccidioidomycosis ranges from a mild to a severe infection. It is frequently characterized either as a persistent disease that requires months to resolve or as an essentially asymptomatic infection that can reactivate several years after the original insult. In this report we describe a mechanism by which the pathogen evades host detection during the pivotal reproductive (endosporulation) phase of the parasitic cycle. A metalloproteinase (Mep1) secreted during endospore differentiation digests an immunodominant cell surface antigen (SOWgp) and prevents host recognition of endospores during the phase of development when these fungal cells are most vulnerable to phagocytic cell defenses. C57BL/6 mice were immunized with recombinant SOWgp and then challenged with a mutant strain of C. posadasii in which the MEP1 gene was disrupted. The animals showed a significant increase in percent survival compared to SOWgp-immune mice challenged with the parental strain. To explain these results, we proposed that retention of SOWgp on the surfaces of endospores of the mutant strain in the presence of high titers of antibody to the immunodominant antigen contributes to opsonization, increased phagocytosis, and killing of the fungal cells. In vitro studies of the interaction between a murine alveolar macrophage cell line and parasitic cells coated with SOWgp showed that the addition of anti-SOWgp antibody could enhance phagocytosis and killing of Coccidioides. We suggest that Mep1 plays a pivotal role as a pathogenicity determinant during coccidioidal infections and contributes to the ability of the pathogen to persist within the mammalian host.