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ABSTRACT: Genome dynamics that allow pathogens to escape host immune responses are fundamental to our
understanding of host-pathogen interactions. Here we present the first population-based study of the
process of concerted evolution in the repetitive domain of a protein-coding gene. This gene, SOWgp,
encodes the immunodominant protein in the parasitic phase of the human pathogenic fungi Coccidioides
immitis and C. posadasii. We sequenced the entire gene from strains representing the geographic ranges of
the two Coccidioides species. By using phylogenetic and genetic distance analyses we discovered that the
repetitive part of SOWgp evolves by concerted evolution, predominantly by the mechanism of unequal
crossing over. We implemented a mathematical model originally developed for multigene families to
estimate the rate of homogenization and recombination of the repetitive array, and the results indicate
that the pattern of concerted evolution is a result of homogenization of repeat units proceeding at a rate
close to the nucleotide point mutation rate. The release of the SOWgp molecules by the pathogen during
proliferation may mislead the host: we speculate that the pathogen benefits from concerted evolution of
repeated domains in SOWgp by an enhanced ability to misdirect the host’s immune system.
Genetics 06/2013; 171:109-117. · 4.01 Impact Factor
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ABSTRACT: Coccidioides is the causative agent of a potentially life-threatening respiratory disease of humans. A feature of this mycosis is that pH measurements of the microenvironment of pulmonary abscesses are consistently alkaline due to ammonia production during the parasitic cycle. We previously showed that enzymatically active urease is partly responsible for elevated concentrations of extracellular ammonia at sites of lung infection and contributes to both localized host tissue damage and exacerbation of the respiratory disease in BALB/c mice. Disruption of the urease gene (URE) of C. posadasii only partially reduced the amount of ammonia detected during in vitro growth of the parasitic phase, suggesting that other ammonia-producing pathways exist that may also contribute to the virulence of this pathogen. Ureidoglycolate hydrolase (Ugh) expressed by bacteria, fungi and higher plants catalyzes the hydrolysis of ureidoglycolate to yield glyoxylate and the release CO2 and ammonia. This enzymatic pathway is absent in mice or humans. Ureidoglycolate hydrolase gene deletions were conducted in a wild type (WT) isolate of C. posadasii as well as the previously generated Δure knock-out strain. Restorations of UGH in the mutant stains were performed to generate and evaluate the respective revertants. The double mutant revealed a marked decrease in the amount of extracellular ammonia without loss of reproductive competence in vitro compared to both the WT and Δure parental strains. BALB/c mice challenged intranasally with the Δugh/Δure mutant showed 90% survival after 30 days, decreased fungal burden, and well-organized pulmonary granulomas. We conclude that loss of both Ugh and Ure activity significantly reduced the virulence of this fungal pathogen.
Microbial Pathogenesis 04/2013; · 1.94 Impact Factor
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ABSTRACT: Coccidioidomycosis (San Joaquin Valley fever) is a human respiratory disease caused by a soil-borne mold, and is recognized as an intransigent microbial infection by physicians who treat patients with the potentially life-threatening, disseminated form of this mycosis. Epidemiological studies based on surveys of skin-test reactivity of people who reside in the endemic regions of the Southwestern US have shown that at least 150,000 new infections occur annually. The clinical spectrum of coccidioidomycosis ranges from an asymptomatic insult to a severe pulmonary disease in which the pathogen may spread from the lungs to the skin, bones, brain and other body organs. Escalation of symptomatic infections and increased cost of long-term antifungal treatment warrant a concerted effort to develop a vaccine against coccidioidomycosis. This review examines recently reported strategies used to generate such a vaccine and summarizes current understanding of the nature of protective immunity to this formidable disease.
Current Fungal Infection Reports 12/2012; 6(4):235-244.
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ABSTRACT: Clinical and animal studies of coccidioidomycosis have demonstrated that activated CD4(+) T lymphocytes are essential for protection against this fungal respiratory disease. We previously reported a vaccine against Coccidioides infection which contained three recombinant CD4(+) T cell-reactive proteins and induced a robust, protective immune response in mice. Due to the anticipated high cost of production and clinical assessment of this multivalent vaccine, we generated a single protein which contained immunodominant T cell epitopes of the three polypeptides. Epitopes were initially identified by computational prediction of their ability to bind promiscuously to human major histocompatibility complex class II (MHC II) molecules. Cellular immunoassays confirmed the immunogenicity of the synthesized epitope peptides, while in vitro binding assays revealed a range of peptide affinity for MHC II. A DNA construct was synthesized for bacterial expression of a recombinant protein vaccine which contained five epitopes with the highest affinity for human MHC II, each fused with leader and spacer peptides proposed to optimize epitope processing and presentation to T cell receptors. Recall assays of immune T lymphocytes obtained from human MHC II-expressing HLA-DR4 transgenic mice confirmed that 4 of the 5 epitope peptides were processed. Mice immunized with the epitope-based vaccine admixed with a synthetic oligodeoxynucleotide adjuvant or loaded into yeast glucan particles and then challenged intranasally with Coccidioides showed early lung infiltration of activated T helper-1 (Th1), Th2, and Th17 cells, elevated gamma interferon (IFN-γ) and interleukin (IL)-17 production, significant reduction of fungal burden, and prolongation of survival compared to nonvaccinated mice. This is the first report of an epitope-based vaccine against coccidioidomycosis.
Infection and immunity 09/2012; 80(11):3960-74. · 4.21 Impact Factor
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ABSTRACT: Influenza A virus (flu) is a respiratory tract pathogen causing high morbidity and mortality among the human population. NO is a cellular mediator involved in tissue damage through its apoptosis of target cells and resulting enhancement of local inflammation. Inducible NO synthase (iNOS) is involved in the production of NO following infection. Although NO is a key player in the development of exaggerated lung disease during flu infection, the underlying mechanism, including the role of NO in apoptosis during infection, has not been reported. Similarly, the mechanism of iNOS gene induction during flu infection is not well defined in terms of the host transactivator(s) required for iNOS gene expression. In the current study, we identified Kruppel-like factor 6 (KLF6) as a critical transcription factor essential for iNOS gene expression during flu infection. We also underscored the requirement for iNOS in inducing apoptosis during infection. KLF6 gene silencing in human lung epithelial cells resulted in the drastic loss of NO production, iNOS promoter-specific luciferase activity, and expression of iNOS mRNA following flu infection. Chromatin immunoprecipitation assay revealed a direct interaction of KLF6 with iNOS promoter during in vitro and in vivo flu infection of human lung cells and mouse respiratory tract, respectively. A significant reduction in flu-mediated apoptosis was noted in KLF6-silenced cells, cells treated with iNOS inhibitor, and primary murine macrophages derived from iNOS knockout mice. A similar reduction in apoptosis was noted in the lungs following intratracheal flu infection of iNOS knockout mice.
The Journal of Immunology 06/2012; 189(2):606-15. · 5.79 Impact Factor
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ABSTRACT: Coccidioides is a fungal pathogen and causative agent of a human respiratory disease against which no clinical vaccine exists. In this study we evaluated a novel vaccine adjuvant referred to as EP67, which is a peptide agonist of the biologically active C-terminal region of human complement component C5a. The EP67 peptide was conjugated to live spores of an attenuated vaccine strain (ΔT) of Coccidioides posadasii. The non-conjugated ΔT vaccine provided partial protection to BALB/c mice against coccidioidomycosis. In this report we compared the protective efficacy of the ΔT-EP67 conjugate to the ΔT vaccine in BALB/c mice. Animals immunized subcutaneously with the ΔT-EP67 vaccine showed significant increase in survival and decrease in fungal burden over 75 days postchallenge. Increased pulmonary infiltration of dendritic cells and macrophages was observed on day 7 postchallenge but marked decrease in neutrophil numbers had occurred by 11 days. The reduced influx of neutrophils may have contributed to the observed reduction of inflammatory pathology. Mice immunized with the ΔT-EP67 vaccine also revealed enhanced expression of MHC II molecules on the surface of antigen presenting cells, and in vitro recall assays of immune splenocytes showed elevated Th1- and Th17-type cytokine production. The latter correlated with a marked increase in lung infiltration of IFN-γ- and IL-17-producing CD4(+) T cells. Elevated expression of T-bet and RORc transcription factors in ΔT-EP67-vaccinated mice indicated the promotion of Th1 and Th17 cell differentiation. Higher titers of Coccidioides antigen-specific IgG1 and IgG2a were detected in mice immunized with the EP67-conjugated versus the non-conjugated vaccine. These combined results suggest that the EP67 adjuvant enhances protective efficacy of the live vaccine by augmentation of T-cell immunity, especially through Th1- and Th17-mediated responses to Coccidioides infection.
Vaccine 05/2012; 30(31):4681-90. · 3.77 Impact Factor
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ABSTRACT: The following transformation protocol is based on homologous recombination that occurs between a gene disruption or gene replacement construct and a target gene of Coccidioides. The DNA constructs employed contain either the gene that encodes for hygromycin B or phleomycin resistance, which are present in the pAN7.1 or pAN8.1 plasmid vectors, respectively. Hygromycin B or phleomycin are used to select for transformants at concentrations that inhibit growth of the parental strain. Coccidioides protoplasts generated from germinated arthroconidia are used for the transformation experiments. The plasmid DNA constructs are taken up by the protoplasts in the presence of calcium and polyethylene glycol. Twenty to 100 transformants/μg DNA can be obtained in each transformation experiment. Approximately 5-10% of the transformation events are homologous recombinations. Coccidioides cells in all developmental stages, including arthroconidia, are multinucleate. Since all Coccidioides nuclei are haploid, only one run of transformation is sufficient to create a mutant strain. However, the transformed protoplasts develop into heterokaryotic cells that typically contain both the parental and mutated nuclei. To isolate a homokaryotic strain, we perform multiple subcultures of the single colonies which contain heterokaryotic cells on selection plates with hygromycin B or phleomycin to enrich for the mutated nuclei. Homokaryotic mutants can be obtained after three to four subcultures of isolated colonies. In this protocol, we describe the methodology for preparation of Coccidioides protoplasts, transformation and isolation of homokaryotic mutants.
Methods in molecular biology (Clifton, N.J.) 01/2012; 845:131-47.
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ABSTRACT: Coccidioides immitis and C. posadasii, the causative agents of coccidioidomycosis, are dimorphic fungal pathogens, which grow as hyphae in the saprobic phase in the environment and as spherules in the parasitic phase in the mammalian host. In this study, we use comparative transcriptomics to identify gene expression differences between the saprobic and parasitic growth phases. We prepared Illumina mRNA sequencing libraries for saprobic-phase hyphae and parasitic-phase spherules in vitro for C. immitis isolate RS and C. posadasii isolate C735 in biological triplicate. Of 9,910 total predicted genes in Coccidioides, we observed 1,298 genes up-regulated in the saprobic phase of both C. immitis and C. posadasii and 1,880 genes up-regulated in the parasitic phase of both species. Comparing the saprobic and parasitic growth phases, we observed considerable differential expression of cell surface-associated genes, particularly chitin-related genes. We also observed differential expression of several virulence factors previously identified in Coccidioides and other dimorphic fungal pathogens. These included alpha (1,3) glucan synthase, SOWgp, and several genes in the urease pathway. Furthermore, we observed differential expression in many genes predicted to be under positive selection in two recent Coccidioides comparative genomics studies. These results highlight a number of genes that may be crucial to dimorphic phase-switching and virulence in Coccidioides. These observations will impact priorities for future genetics-based studies in Coccidioides and provide context for studies in other fungal pathogens.
PLoS ONE 01/2012; 7(7):e41034. · 4.09 Impact Factor
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ABSTRACT: The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS(-/-) mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS(-/-) mice revealed comparable fungal burden in their lungs and spleen at 7 and 11 days postchallenge. However, at 11 days the non-vaccinated, iNOS-deficient mice had significantly higher fungal burden in their spleen compared to WT mice. Additionally, higher numbers of lung-infiltrated neutrophils, macrophages and dendritic cells were observed in WT mice at day 11 postchallenge compared to iNOS(-/-) mice. Moreover, no difference in numbers of T, B, NK or regulatory T cells, or concentrations of selected cytokines and chemokines were detected in lungs of both mouse strains at 7 and 11 days postchallenge. Although iNOS-derived NO production appears to influence the inflammatory response and dissemination of the fungal pathogen, our results suggest that iNOS activity does not play a significant role in the control of coccidioidal infection in mice and that other, still undefined mechanisms of host protection are involved.
Microbial Pathogenesis 09/2011; 51(3):161-8. · 1.94 Impact Factor
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ABSTRACT: Production of reactive oxygen species (ROS) resulting from phagocytic NADPH oxidase (NOX2) activity has been reported to contribute to host defense against numerous microbial pathogens. In this study we explored the role of NOX2 production in experimental coccidioidomycosis, a human respiratory disease caused by a soil-borne fungal pathogen. Activated and non-activated macrophages isolated from either NOX2(-/-) knock-out or wild type (WT) mice showed comparable ROS production and killing efficiency in vitro when infected with parasitic cells of Coccidioides. Both mouse strains also revealed similar fungal burden in their lungs and spleen at 7 and 11 days after intranasal challenge with Coccidioides spores, although the NOX2(-/-) mice died earlier than the WT strain. Immunization of the NOX2(-/-) and WT mice with a live, attenuated vaccine strain of Coccidioides also resulted in comparable reduction of the fungal burden in both lungs and spleen. These combined results initially suggested that NOX2 activity and ROS production are not essential for protection against Coccidioides infection. However, the reduced survival of non-vaccinated NOX2(-/-) mice correlated with high, sustained numbers of lung-infiltrated neutrophils on days 7 and 11 postchallenge, an expansion of the regulatory T cell population in infected lungs in the knock-out mice, and elevated concentrations of pro-inflammatory cytokines and chemokines in lung homogenates compared to infected WT mice. Although NOX2-derived ROS appeared to be dispensable for both innate and acquired immunity to pulmonary Coccidioides infection, evidence is presented that NOX2 production plays a role in limiting pathogenic inflammation in this murine model of coccidioidomycosis.
Microbial Pathogenesis 08/2011; 51(6):432-41. · 1.94 Impact Factor
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ABSTRACT: We have previously reported that C57BL/6 mice vaccinated with a live, attenuated mutant of Coccidioides posadasii, referred to as the ΔT vaccine, are fully protected against pulmonary coccidioidomycosis. This model was used here to explore the nature of vaccine immunity during the initial 2-week period after intranasal challenge. Elevated neutrophil and eosinophil infiltration into the lungs of nonvaccinated mice contrasted with markedly reduced recruitment of these cells in vaccinated animals. The numbers of lung-infiltrated macrophages and dendritic cells showed a progressive increase in vaccinated mice and corresponded with reduction of the lung infection. Concentrations of selected inflammatory cytokines and chemokines were initially higher in lung homogenates of vaccinated mice but then generally decreased at 14 days postchallenge in correlation with containment of the organism and apparent dampening of the inflammation of host tissue. Profiles of cytokines detected in lung homogenates of ΔT-vaccinated mice were indicative of a mixed T helper 1 (Th1)-, Th2-, and Th17-type immune response, a conclusion which was supported by detection of lung infiltration of activated T cells with the respective CD4(+) gamma interferon (IFN-γ)(+), CD4(+) interleukin-5 (IL-5)(+), and CD4(+) IL-17A(+) phenotypes. While Th1 and Th2 immunity was separately dispensed of by genetic manipulation without loss of ΔT vaccine-mediated protection, loss of functional Th17 cells resulted in increased susceptibility to infection in immunized mice. Characterization of the early events of protective immunity to Coccidioides infection in vaccinated mice contributes to the identification of surrogates of immune defense and provides potential insights into the design of immunotherapeutic protocols for treatment of coccidioidomycosis.
Infection and immunity 08/2011; 79(11):4511-22. · 4.21 Impact Factor
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ABSTRACT: Dimorphic fungi collectively account for 5-10 million new infections annually worldwide. Ongoing efforts seek to clarify mechanisms of cellular resistance to these agents and develop vaccines. A major limitation in studying the development of protective T cells in this group of organisms is the lack of tools to detect, enumerate, and characterize fungus-specific T cells during vaccination and infection. We generated a TCR transgenic mouse (Bd 1807) whose CD4(+) T cells respond to a native epitope in Blastomyces dermatitidis and also in Histoplasma capsulatum. In this study, we characterize the mouse, reveal its applications, and extend our analysis showing that 1807 cells also respond to the related dimorphic fungi Coccidioides posadasii and Paracoccidioides lutzii. On adoptive transfer into vaccinated wild-type mice, 1807 cells become activated, proliferate, and expand in the draining lymph nodes, and they differentiate into T1 effectors after trafficking to the lung upon lethal experimental challenge. Bd 1807 cells confer vaccine-induced resistance against B. dermatitidis, H. capsulatum, and C. posadasii. Transfer of naive 1807 cells at serial intervals postvaccination uncovered the prolonged duration of fungal Ag presentation. Using 1807 cells, we also found that the administration of vaccine only once induced a maximal pool of effector/memory CD4(+) cells and protective immunity by 4 wk after vaccination. The autologous adoptive transfer system described in this study reveals novel features of antifungal immunity and offers a powerful approach to study the differentiation of Ag-specific T cells responsive to multiple dimorphic fungi and the development of CD4(+) T cell memory needed to protect against fungal infection.
The Journal of Immunology 06/2011; 187(3):1421-31. · 5.79 Impact Factor
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ABSTRACT: We have used the human respiratory pathogen, Coccidioides immitis, as an experimental model to explore possible interrelationships of wall-associated hydrolases, cell growth, and reproduction. Preliminary evidence has been presented that suggests that certain wall hydrolases (glucanase, chitinase) may play key roles in cell development in this systemic pathogen. Initial differentiation of the parasitic cells from cylindrical arthroconidia involves a period of isotropic growth and results in formation of a multinucleate spherule (approximately 60 μm diameter). An endo-1,3-β-glucanase that may participate in this diametric growth phase has been isolated. Two distinct chitinase genes (cts1, cts2) have been isolated from C. immitis and shown to be members of different classes of this wall hydrolase. The class I chitinase (CTS2) demonstrates homology to a reported endochitinase of Saccharomyces cerevisiae that has been shown to be essential for yeast daughter cell release. CTS2 may play a pivotal role in isotropic growth, as well as differentiation and release of endospores from maternal spherules. In the absence of specific gene disruption and transformation experiments, these data are still circumstantial evidence for the functions of wall hydrolases in C. immitis development. However, we suggest our results provide further support for the concept that wall hydrolases represent rational molecular targets for future development of novel antifungal agents. Key words: Coccidioides, cell wall, β-glucanase, chitinase, morphogenesis.
Canadian Journal of Botany 03/2011; 73:1132-1141. · 1.40 Impact Factor
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ABSTRACT: We studied the effect of the presence of Coccidioides on the production of nitric oxide (NO) by primary macrophages previously activated by IFN-γ and LPS. The fungal cells were isolated from cultures of arthroconidia that had been incubated for 24 h in a medium that supported parasitic phase growth and were co-cultured with the macrophages. These live, first-generation parasitic cells of Coccidioides, referred to as spherule initials, suppressed NO production as well as iNOS mRNA expression by activated macrophages. Phagocytosis was not required for suppression of NO. We also showed that the culture supernatant of the spherule initials was capable of suppressing NO production, and that this activity was mediated by an as yet unidentified, secreted fungal factor(s). Heat-, paraformaldehyde- or X-ray-treated spherule initials did not show this inhibitory effect. To our surprise, macrophages obtained from iNOS-deficient mice revealed phagocytic activity and killing efficiency which were comparable to that of macrophages isolated from wild type C57BL/6 mice. Although the cultured fungal pathogen can suppress NO production, this oxidative product is apparently not essential for in vitro killing of Coccidioides by activated macrophages. Our results suggest that other unidentified fungicidal mechanisms exist against Coccidioides which are apparently independent of NO production.
Microbial Pathogenesis 02/2011; 50(2):100-8. · 1.94 Impact Factor
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Daniel E Neafsey,
Bridget M Barker,
Thomas J Sharpton,
Jason E Stajich,
Daniel J Park,
Emily Whiston,
Chiung-Yu Hung,
Cody McMahan,
Jared White,
Sean Sykes, [......],
J Pendexter Macdonald,
Margaret Priest,
Marc J Orbach,
John N Galgiani,
Theo N Kirkland, Garry T Cole,
Bruce W Birren,
Matthew R Henn,
John W Taylor,
Steven D Rounsley
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ABSTRACT: 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.
Genome Research 07/2010; 20(7):938-46. · 13.61 Impact Factor
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Thomas J Sharpton,
Jason E Stajich,
Steven D Rounsley,
Malcolm J Gardner,
Jennifer R Wortman,
Vinita S Jordar,
Rama Maiti,
Chinnappa D Kodira,
Daniel E Neafsey,
Qiandong Zeng, [......],
M Alejandra Mandel,
Ellen M Kellner,
Bridget M Barker,
John N Galgiani,
Marc J Orbach,
Theo N Kirkland, Garry T Cole,
Matthew R Henn,
Bruce W Birren,
John W Taylor
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ABSTRACT: 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.
Genome Research 09/2009; 19(10):1722-31. · 13.61 Impact Factor
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ABSTRACT: This paper describes a method for a single-step, site-specific conjugation of bioactive peptides to proteins that exploits the monitoring advantages provided by the unique UV signature absorbance of a bis-arylhydrazone. The utility of this method is demonstrated by the conjugation of a decapeptide molecular adjuvant, YSFKDMP(MeL)aR (EP67), to two test proteins, ovalbumin (OVA) and bovine serum albumin (BSA), and to proteins expressed on intact influenza virons and fungal arthroconidia (spores) of Coccidioides. Conjugation is accomplished with a version of EP67 in which its N-terminus is modified with succinimidyl-4-benzoylhydrazino-nicotinamide (S4BHyNic) (peptide 7), thus enabling conjugation to these large entities via formation of amide bonds with surface-exposed amino groups. The presence of the strongly absorbing bis-arylhydrazone S4BHyNic (ε(354 nm) = 29 000 L mol(-1) cm(-1)) allows for determination of EP67-to-protein molar substitution ratios (MSR), which are in good agreement with the MSRs determined by amino acid analysis. Conjugation to OVA does not compromise the ability of EP67 to engage C5a receptor bearing antigen presenting cells (APC) as measured by the EP67-mediated release of interleukin-6 (IL-6) from APCs. Mice immunized with the resulting OVA-EP67 vaccine conjugate produce high serum titers of OVA-specific IgG antibodies relative to OVA alone. Also, the conjugation of EP67 does not affect the surface integrity of influenza virons or the biological viability of Coccidioides spores. This method of conjugating bioactive peptides to proteins and other large biological entities may represent a convenient and effective way of generating various bioconjugates for use in mechanistic studies or novel therapeutic entities such as EP67-containing vaccines.
Bioconjugate Chemistry 09/2009; 20(10):1950-7. · 4.93 Impact Factor
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ABSTRACT: Coccidioidomycosis (also known as San Joaquin Valley fever) is an occupational disease. Workers exposed to outdoor dust which contains spores of the soil-inhabiting fungus have a significantly increased risk of respiratory infection. In addition, people with compromised T-cell immunity, the elderly, and certain racial groups, particularly African-Americans and Filipinos, who live in regions of endemicity in the southwestern United States have an elevated incidence of symptomatic infection caused by inhalation of spores of Coccidioides posadasii or Coccidioides immitis. Recurring epidemics and escalation of medical costs have helped to motivate production of a vaccine against valley fever. The major focus has been the development of a defined, T-cell-reactive, recombinant protein vaccine. However, none of the products described to date have provided full protection to coccidioidal disease-susceptible BALB/c mice. Here we describe the first genetically engineered, live, attenuated vaccine that protects both BALB/c and C57BL/6 mice against coccidioidomycosis. Two chitinase genes (CTS2 and CTS3) were disrupted to yield the attenuated strain, which was unable to endosporulate and was no longer infectious. Vaccinated survivors mounted an immune response characterized by production of both T-helper-1- and T-helper-2-type cytokines. Histology revealed well-formed granulomas and markedly diminished inflammation. Significantly fewer organisms were observed in the lungs of survivors than in those of nonvaccinated mice. Additional investigations are required to further define the nature of the live, attenuated vaccine-induced immunity against Coccidioides infection.
Infection and immunity 07/2009; 77(8):3196-208. · 4.21 Impact Factor
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ABSTRACT: Evaluation of the protective efficacy of recombinant T-cell-reactive proteins of Coccidioides posadasii in a murine model of coccidioidomycosis has led to the discovery of potential vaccines against this respiratory disease. A recombinant proline-rich antigen (rAg2/Pra) has been reported to be a leading vaccine candidate. However, contradictory results exist on the protection afforded by this antigen. Subcutaneous vaccination of either C57BL/6 or BALB/c mice with rAg2/Pra plus adjuvant followed by intraperitoneal challenge with C. posadasii resulted in a significant reduction of the fungal burden at 12 to 14 days postchallenge compared to that in nonvaccinated animals. Use of the same vaccination protocol followed by intranasal (i.n.) challenge of C57BL/6 mice with an equal number of organisms culminated in chronic pulmonary infection or death over a 90-day period. Early studies of Ag2/Pra suggested that it is a component of an immunogenic complex. We reveal in this study that C. posadasii produces a homolog of the reported proline-rich antigen, designated Prp2, which shows 69% protein sequence identity and 86% similarity to Ag2/Pra. Protection against i.n. challenge of C57BL/6 mice was evaluated by vaccination with the single bacterially expressed homolog, rAg2/Pra, or rPrp2 in combination with rAg2/Pra, each in the presence of the same adjuvant. The combined vaccine provided significantly better protection than either of the single recombinant protein vaccines. Results of enzyme-linked immunospot assays of the immunized mice revealed that the two proline-rich homologs contain unique T-cell epitopes. In combination, the recombinant proteins stimulate a more heterogeneous and protective T-cell repertoire than the monovalent vaccines.
Infection and immunity 01/2008; 75(12):5777-87. · 4.21 Impact Factor
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ABSTRACT: Coccidioides is a fungal respiratory pathogen of humans that can cause disease in both immunosuppressed and immunocompetent individuals. We describe here three mechanisms by which the pathogen survives in the hostile host environment: production of a dominant spherule outer wall glycoprotein (SOWgp) that modulates host immune response and results in compromised cell-mediated immunity to coccidioidal infection, depletion of SOWgp presentation on the surface of endospores, which prevents host recognition of the pathogen when the fungal cells are most vulnerable to phagocytic defenses, and induction of elevated production of host arginase I and coccidioidal urease, which contribute to tissue damage at sites of infection. Arginase I competes with inducible nitric oxide synthase (iNOS) in macrophages for the common substrate, L-arginine, and thereby reduces nitric oxide (NO) production and increases the synthesis of host orinithine and urea. Host-derived L-ornithine may promote pathogen growth and proliferation by providing a pool of the monoamine, which could be taken up and used for synthesis of polyamines via metabolic pathways of the parasitic cells. We have shown that high concentrations of Coccidioides- and host-derived urea at infection sites in the presence of urease produced and released by the pathogen, results in secretion of ammonia and contributes to alkalinization of the microenvironment. We propose that ammonia and enzymatically active urease released from spherules during the parasitic cycle of Coccidioides exacerbate the severity of coccidioidal infection by contributing to a compromised immune response to infection and damage of host tissue at foci of infection.
Annals of the New York Academy of Sciences 10/2007; 1111:225-35. · 3.15 Impact Factor
