Fungal Pathogen Coccidioides Immitis Research Articles

Valley Fever on the Rise-Searching for Microbial Antagonists to the Fungal Pathogen Coccidioides immitis.

The incidence of coccidioidomycosis, also known as Valley Fever, is increasing in the Southwestern United States and Mexico. Despite considerable efforts, a vaccine to protect humans from this disease is not forthcoming. The aim of this project was to isolate and phylogenetically compare bacterial species that could serve as biocontrol candidates to suppress the growth of Coccidioides immitis, the causative agent of coccidioidomycosis, in eroded soils or in areas close to human settlements that are being developed. Soil erosion in Coccidioides endemic areas is leading to substantial emissions of fugitive dust that can contain arthroconidia of the pathogen and thus it is becoming a health hazard. Natural microbial antagonists to C. immitis, that are adapted to arid desert soils could be used for biocontrol attempts to suppress the growth of the pathogen in situ to reduce the risk for humans and animals of contracting coccidioidomycosis. Bacteria were isolated from soil samples obtained near Bakersfield, California. Subsequently, pairwise challenge assays with bacterial pure cultures were initially performed against Uncinocarpus reesii, a non-pathogenic relative of C. immitis on media plates. Bacterial isolates that exhibited strongly antifungal properties were then re-challenged against C. immitis. Strongly anti-C. immitis bacterial isolates related to Bacillus subtilis and Streptomyces spp. were isolated, and their antifungal spectrum was investigated using a selection of environmental fungi.

Large-Scale Land Development, Fugitive Dust, and Increased Coccidioidomycosis Incidence in the Antelope Valley of California, 1999-2014.

Ongoing large-scale land development for renewable energy projects in the Antelope Valley, located in the Western Mojave Desert, has been blamed for increased fugitive dust emissions and coccidioidomycosis incidence among the general public in recent years. Soil samples were collected at six sites that were destined for solar farm construction and were analyzed for the presence of the soil-borne fungal pathogen Coccidioides immitis which is endemic to many areas of central and southern California. We used a modified culture-independent nested PCR approach to identify the pathogen in all soil samples and also compared the sampling sites in regard to soil physical and chemical parameters, degree of disturbance, and vegetation. Our results indicated the presence of C. immitis at four of the six sites, predominantly in non-disturbed soils of the Pond-Oban complex, which are characterized by an elevated pH and saltbush communities, but also in grassland characterized by different soil parameters and covered with native and non-native annuals. Overall, we were able to detect the pathogen in 40% of the soil samples (n=42). Incidence of coccidioidomycosis in the Antelope Valley was positively correlated with land use and particulate matter in the air (PM10) (Pearson correlation coefficient>0.5). With the predicted population growth and ongoing large-scale disturbance of soil in the Antelope Valley in coming years, incidence of coccidioidomycosis will likely further increase if policy makers and land developers continue to ignore the risk of grading land without implementing long-term dust mitigation plans in Environmental Impact Reports.

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis.

Coccidioides immitis is a pathogenic fungus populating the southwestern United States and is a causative agent of coccidioidomycosis, sometimes referred to as Valley Fever. Although the genome of this fungus has been sequenced, many operons are not properly annotated. Crystal structures are presented for a putative uncharacterized protein that shares sequence similarity with ζ-class glutathione S-transferases (GSTs) in both apo and glutathione-bound forms. The apo structure reveals a nonsymmetric homodimer with each protomer comprising two subdomains: a C-terminal helical domain and an N-terminal thioredoxin-like domain that is common to all GSTs. Half-site binding is observed in the glutathione-bound form. Considerable movement of some components of the active site relative to the glutathione-free form was observed, indicating an induced-fit mechanism for cofactor binding. The sequence homology, structure and half-site occupancy imply that the protein is a ζ-class glutathione S-transferase, a maleylacetoacetate isomerase (MAAI).

Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

BackgroundRibose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis.ResultsAlthough RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB.ConclusionThe C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether.

Role of signal sequence in vaccine-induced protection against experimental coccidioidomycosis.

The vaccine efficacy of the gene sequence encoding the signal peptide of the antigen known as antigen 2 or proline-rich antigen (Ag2/PRA), an immunodominant antigen present in the cell wall of the fungal pathogen Coccidioides immitis, was investigated in a murine model of coccidioidomycosis. Expression plasmids for Ag2/PRA(1-18) DNA (signal sequence), Ag2/PRA(19-194) DNA (lacking the signal sequence), and Ag2/PRA(1-194) DNA (full length) were inserted in the pVR1012 vector, and the constructs were used to vaccinate the highly susceptible BALB/c mouse strain. Immunization with the signal gene sequence significantly reduced the fungal burden in the lungs and spleens of mice 12 days after intraperitoneal challenge with a lethal dose of 2,500 C. immitis arthroconidia, to a level comparable to the protection induced in mice immunized with the full-length Ag2/PRA(1-194) DNA. The Ag2/PRA(19-194) gene protected mice but to a significantly lower level than the signal sequence or the full-length Ag2 gene. The immunizing capacity of Ag2/PRA(1-18) was not attributable to a nonspecific immunostimulatory effect of DNA, as evidenced by the fact that mice immunized with a frameshift mutation of Ag2/PRA(1-18) were not protected against challenge. Furthermore, a synthetic peptide corresponding to the translated sequence of Ag2/PRA(1-18) DNA protected mice, albeit at a lower level than the Ag2/PRA(1-18) DNA vaccine. The protection induced with the signal gene vaccine correlated with the production of gamma interferon when splenocytes from Ag2/PRA(1-18)-immunized mice were stimulated with recombinant full-length Ag2 and was not associated with the production of anti-Coccidioides immunoglobulin G antibody. This is the first study to establish that a signal peptide sequence alone, administered as a gene vaccine or synthetic peptide, can induce protective immunity against a microbial pathogen.

Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis

Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis

Disruption of the gene which encodes a serodiagnostic antigen and chitinase of the human fungal pathogen Coccidioides immitis.

Disruption of genes in medically important fungi has proved to be a powerful tool for evaluation of putative virulence factors and identification of potential protein targets for novel antifungal drugs. Chitinase has been suggested to play a pivotal role in autolysis of the parasitic cell wall of Coccidioides immitis during the asexual reproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (CTS1 and CTS2) of C. immitis have been cloned. Preliminary evidence has suggested that expression of CTS1 is markedly increased during endospore formation. The secreted CTS1 chitinase has also been shown to react with patient anti-Coccidioides complement-fixing (CF) antibody and is a valuable aid in the serodiagnosis of coccidioidomycosis. To examine the role of CTS1 in the morphogenesis of parasitic cells, the CTS1 gene was disrupted by a single, locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construct that contained a 1.1-kb fragment of the chitinase gene into the chromosomal DNA of C. immitis. Results of Southern hybridizations, immunoblot analyses of culture filtrates using both CTS1-specific murine antiserum and serum from a patient with confirmed coccidioidal infection, an immunodiffusion test for CF antigenicity, and substrate gel electrophoresis assays of chitinase activity confirmed that the CTS1 gene was disrupted and nonfunctional. This is the first report of a successful targeted gene disruption in C. immitis. However, loss of CTS1 function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in the parental and Deltacts1 strains suggested that the absence of a functional CTS1 gene can be compensated for by elevated expression of the CTS2 gene. Current investigations are focused on disruption of CTS2 in the Deltacts1 host to further evaluate the significance of chitinase activity in the parasitic cycle of C. immitis.

A test for concordance between the multilocus genealogies of genes and microsatellites in the pathogenic fungus Coccidioides immitis.

Uncovering the correct phylogeny of closely related species requires analysis of multiple gene genealogies or, alternatively, genealogies inferred from the multiple alleles found at highly polymorphic loci, such as microsatellites. However, a concern in using microsatellites is that constraints on allele sizes may occur, resulting in homoplasious distributions of alleles, leading to incorrect phylogenies. Seven microsatellites from the pathogenic fungus Coccidioides immitis were sequenced for 20 clinical isolates chosen to represent the known genetic diversity of the pathogen. An organismal phylogeny for C. immitis was inferred from microsatellite-flanking sequence polymorphisms and other restriction fragment length polymorphism-containing loci. Two microsatellite genetic distances were then used to determine phylogenies for C. immitis, and the trees found by these three methods were compared. Congruence between the organismal and microsatellite phylogenies occurred when microsatellite distances were based on simple allele frequency data. However, complex mutation events at some loci made distances based on stepwise mutation models unreliable. Estimates of times of divergence for the two species of C. immitis based on microsatellites were significantly lower than those calculated from flanking sequence, most likely due to constraints on microsatellite allele sizes. Flanking-sequence insertions/deletions significantly decreased the accuracy of genealogical information inferred from microsatellite loci and caused interspecific length homoplasies at one of the seven loci. Our analysis shows that microsatellites are useful phylogenetic markers, although care should be taken to choose loci with appropriate flanking sequences when they are intended for use in evolutionary studies.

Soil isolation and molecular identification of Coccidioides immitis

The fungal pathogen Coccidioides immitis, causative agent of coccidioidomycosis or Valley Fever, was first isolated from the environment in 1932. It has been isolated on numerous occasions since then, but always with the use of a mammalian host. The morphology of C. immitis is indistinct from related species, thus, its identification hinges upon its ability to infect and produce spherules in a susceptible animal. In this study, four genetically distinct isolates of C. immitis were isolated from soil samples from the San Joaquin Valley without the use of a host. None of these sites had been definitively associated with human infection. The isolates were identified from over 2400 soil isolates from 720 soil samples using C. immitis specific primers based on the ITS sequence of ribosomal DNA. They were further typed using molecular markers available for clinical isolates of C. immitis.

Soil Isolation and Molecular Identification of Coccidioides immitis

The fungal pathogen Coccidioides immitis, causative agent of coccidioidomycosis or Valley Fever, was first isolated from the environment in 1932. It has been isolated on numerous occasions since th...

The X‐ray structure of a chitinase from the pathogenic fungus Coccidioides immitis

The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 A resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded beta/alpha-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.

Crystallization and preliminary X-ray analysis of a chitinase from the fungal pathogen Coccidioides immitis.

Chitinase is necessary for fungal growth and cell division and, therefore, is an ideal target for the design of inhibitors which may act as antifungal agents. A chitinase from the fungal pathogen Coccidioides immitis has been expressed as a fusion protein with gluathione-S-transferase (GST), which aids in purification. After cleavage from GST, chitinase was crystallized from 30% PEG 4000 in 0. 1 M sodium acetate pH 4.6. The crystals have a tetragonal crystal lattice and belong to space group P41212 or P43212 and diffract to 2. 2 A resolution. The unit-cell parameters are a = b = 91.2, c = 95.4 A; there is only one chitinase molecule in the asymmetric unit.

Isolation and heterologous expression of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from the wheat leaf-spot pathogen, Mycosphaerella graminicola

We describe the isolation and sequence of a gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD (EC 1.13.11.27)) from the wheat leaf-spot fungal pathogen Mycosphaerella graminicola ( Septoria tritici), that directs the synthesis of 2,5-dihydroxyphenylacetate (homogentisic acid, HGA). The sequence of the deduced peptide showed homology to HPPDs from other organisms; the greatest identity was to a T-cell reactive protein, also identified as HPPD, from the human fungal pathogen Coccidioides immitis. As observed for HPPD from other sources, expression of the M. graminicola HPPD gene in Escherichia coli cells could be detected by the gradual development of a brown pigment in cultures as a result of the spontaneous oxidation and polymerisation of HGA. Pigment development in these cultures was prevented by the HPPD inhibitor sulcotrione.

Isolation and heterologous expression of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from the wheat leaf-spot pathogen, Mycosphaerella graminicola.

We describe the isolation and sequence of a gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD) (EC 1.13.11.27)) from the wheat leaf-spot fungal pathogen Mycosphaerella graminicola (Septoria tritici), that directs the synthesis of 2,5-dihydroxyphenylacetate (homogentisic acid, HGA). The sequence of the deduced peptide showed homology to HPPDs from other organisms; the greatest identity was to a T-cell reactive protein, also identified as HPPD, from the human fungal pathogen Coccidioides immitis. As observed for HPPD from other sources, expression of the M. graminicola HPPD gene in Escherichia coli cells could be detected by the gradual development of a brown pigment in cultures as a result of the spontaneous oxidation and polymerisation of HGA. Pigment development in these cultures was prevented by the HPPD inhibitor sulcotrione.

Isolation and characterization of the urease gene ( URE) from the pathogenic fungus Coccidioides immitis

The urease (URE)-encoding gene from Coccidioides immitis ( Ci), a respiratory fungal pathogen of humans, was cloned, sequenced, chromosome-mapped and expressed. Both the genomic and cDNA sequences are reported. The transcription start point and poly(A)-addition site were confirmed. The URE gene contains eight introns and a 2517-bp ORF that translates a 839-amino-acid (aa) protein of 91.5 kDa and p I of 5.5, as deduced by computer analysis of the nucleotide sequence. The translated protein revealed eight putative N-glycosylation sites. The deduced URE showed comparable levels of homology to reported URE of the jack bean plant ( Canavalia ensiformis; 71.8%) and URE of several genera of bacteria ( Bp, 71.7%; Hp, 68.3%; Ka, 71.6%; Pm, 71.9%). The URE gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern hybridization. Expression of a 1687-bp fragment of the URE gene in E. coli resulted in the production of a 63-kDa recombinant protein that was recognized in an immunoblot by antiserum raised against the Ka URE homolog. This is the first report of a fungal URE gene.

The hsp60 gene of the human pathogenic fungus Coccidioides immitis encodes a T-cell reactive protein

A heat shock protein-encoding gene ( hsp60) from the human respiratory fungal pathogen, Coccidioides immitis ( Ci), was cloned, sequenced, chromosome-mapped, expressed and immunolocalized in parasitic cells. Both the genomic and cDNA sequences are presented. The transcription start point and poly (A) addition site were confirmed. The hsp60 gene contains two introns and a 1782-bp ORF which translates a 594-amino acid (aa) protein of 62.4 kDa and p I of 5.6. The translated protein revealed two potential N-glycosylation sites. The deduced HSP60 showed 78–83% aa sequence similarity to reported fungal HSP60 proteins. The hsp60 gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern and Northern hybridization. Expression of a 1737-bp cDNA fragment of the hsp60 gene in E. coli resulted in production of a recombinant protein. Amino acid sequence analysis of the recombinant protein confirmed that it was encoded by the Ci hsp60 gene. Antiserum raised in mice against the isolated recombinant protein immunolocalized HSP60 in the cytoplasm and wall of parasitic cells of Ci. The recombinant HSP60 was used to immunize BALB/c mice and was shown to induce proliferation of T cells isolated from lymph nodes of these animals. The hsp60 gene of Ci is the first reported heat-shock protein gene of this human pathogen.

Cloning and expression of a gene encoding a T-cell reactive protein from coccidioides immitis: homology to 4-hydroxyphenylpyruvate dioxygenase and the mammalian F antigen

The gene which encodes a previously described T-cell reactive protein (TCRP) of the human fungal pathogen Coccidioides immitis (Ci) was cloned and sequenced. Both the genomic and cDNA sequences were determined. The transcription start point was confirmed. The tcrP gene has three introns and a 1197-bp ORF which translates to a 399-amino-acid (aa) protein (45.2 kDa). The predicted protein has approx. 50% aa sequence identity and 70% similarity to mammalian 4-hydroxyphenylpyruvate dioxygenase (HPPD) proteins and mammalian F-antigens. Expression of the Ci tcrP in Escherichia coli resulted in production of a deep brown pigment, consistent with E. coli expression of the bacterial HPPD homolog from Shewanella colwelliana. The TCRP is likely the Ci form of HPPD.

Production of a murine monoclonal antibody that recognizes an epitope specific to Coccidioides immitis antigen 2

Antigen 2 (Ag2) has been implicated as a T-cell-reactive component of the pathogenic fungus Coccidioides immitis. We report the production of a murine monoclonal antibody (MAb) of the immunoglobulin G2a isotype that recognizes an epitope specific to C. immitis Ag2. This specificity was evidenced by the finding that the MAb did not recognize other antigens present in coccidioidin or spherulin and did not show reactivity with antigenic extracts from Histoplasma capsulatum or Blastomyces dermatitidis. The epitope was labile to enzymatic digestion with pronase but resistant to treatment with glycolytic enzymes and to periodate oxidation. This peptide epitope appears to require conformational structure on the basis that it was not recognized by the MAb in immunoblots of antigen that had been electrophoresed in polyacrylamide gels under denaturing, reducing conditions. Immunoaffinity chromatography of spherulin on columns containing the MAb established that the MAb was effective as a ligand for isolating Ag2 from heterogeneous extracts. The production of a MAb which recognizes an Ag2-specific epitope and its utility as a ligand for isolating Ag2 will provide a valuable reagent for studies of this immunologically important antigen.

Isolation of antigens with proteolytic activity from Coccidioides immitis

Three antigens with proteolytic activity have been isolated from crude, water-soluble fractions of the saprobic phase of the fungal pathogen Coccidioides immitis. Two proteinases, identified in our immunoelectrophoresis reference system as Ag11 and AgCS, were isolated from the soluble conidial wall fraction (SCWF). Ag11 was previously shown to be a serine proteinase and was characterized in this study as a 60-kilodalton (kDa) fraction by gel filtration (GF). The purified proteinase demonstrated little or no reactivity with 21 serum samples from coccidioidomycosis patients in the enzyme-linked immunosorbent assay; this may be due to limited presentation of this antigen to the host during the course of coccidioidomycosis. AgCS was separated by GF chromatography into two fractions identified by molecular masses of 39 and 19 kDa. Most proteolytic activity was shown by substrate gel electrophoresis to be associated with the lower-molecular-mass fraction. AgCS was reactive with 18 of the 21 serum samples and shown to be the major component of a heat-stable antigen previously reported to be immunospecific for C. immitis. The third antigen with proteolytic activity was isolated from the 5-day mycelial culture filtrate and identified by GF as a 56-kDa fraction. Uniformly high levels of immunoreactivity between 18 of the 21 patient sera and the 56-kDa antigen were demonstrated. Antigens with proteolytic activity may play important roles in fungus-host interactions as well as morphogenesis of the pathogen.

Proteinase production by the parasitic cycle of the pathogenic fungus Coccidioides immitis.

Coccidioides immitis is the causative agent of coccidioidomycosis (valley fever), a potentially disseminated fungal disease. We hypothesized that proteinases are expressed by the parasitic life cycle of C. immitis and that they might play an important role in the pathogenesis of coccidioidomycosis by facilitating spherule rupture, endospore dissemination, and tissue invasion and destruction. Filtrate from cultures of the parasitic life cycle of C. immitis was therefore assayed for proteolytic activity at neutral pH. The filtrate degraded 68% of a radiolabeled model of an elastin-rich extracellular matrix. The principal activity was against elastin and glycoprotein in the matrix. Degradation of purified elastin by filtrate was 222 micrograms/h per mg of filtrate protein at 37 degrees C. Denatured type I collagen (Azocoll) degradation was 13.5 mg/h per mg of filtrate protein at 37 degrees C. Proteinase activity peaked at 60 h of culture, correlating with release of endospores from mature spherules in the in vitro culture system. Elastase activity was attributed to a serine proteinase which exhibited an active-site preference for phenylalanine at the P1 site. The subunit molecular mass of the elastase determined by [3H]diisopropylfluorophosphate labeling was approximately 25 kilodaltons. Inhibition of the azocollytic activity of crude filtrate by 2 mM 1,10-phenanthroline and 10 mM EDTA, and stimulation by 2 mM CaCl2, suggested that a metalloproteinase was also present. Gelatin substrate gel electrophoresis with and without inhibitors confirmed that two proteinases were expressed, and they were separated by fast protein liquid chromatography.