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Item Kinetics and Mechanism of the Oxidation of Human deoxyHemoglobin by Nitrites(Elsevier, 1981-12-10) Doyle, Michael; Pickering, Ruth; DeWeert, Thomas; Hoekstra, James; Pater, DavidThe reactions of human hemoglobin with sodium nitrite and ethyl nitrite in deoxygenated media have been examined in kinetic detail. Nitrous acid has been identified from kinetic pH dependence studies as the principal oxidant of hemoglobin in reactions with sodium nitrite. Nitrosylhemoglobin is produced concurrently with methemoglobin as a result of reductive release of nitric oxide from nitrous acid. However, oxidation of hemoglobin by nitric oxide competes with association, and this process is proposed to arise from the action of the nitric oxide dimer on hemoglobin. Ethyl nitrite, which serves as a model for nitrous acid, reacts with hemoglobin at rates that are at least 10 times slower than those extrapolated for nitrous acid, and hydrolysis of the alkyl nitrite is not competitive with oxidation of hemoglobin. The composite experimental results are interpreted to describe alkyl nitrite, and, presumably, nitrous acid association with hemoglobin followed by rate-limiting electron transfer resulting in nitric oxide and alkoxide (or hydroxide) production. Proton transfer resulting in alcohol (or water) formation occurs subsequent to the rate-limiting step as do reactions of hemoglobin with nitric oxide.Item Oxidation of Hemoglobin and Myoglobin by Alkyl Nitrites. Inhibition by Oxygen(Elsevier, 1981-12-10) Doyle, Michael; LePoire, Diane; Pickering, RuthThe reactions of human hemoglobin and sperm whale myoglobin with ethyl nitrite under aerobic conditions have been examined in kinetic detail. Ethyl nitrite converts two equivalents of oxyhemoglobin or oxymyoglobin to their oxidized counterparts with concurrent production of one equivalent each of molecular oxygen, nitrate ion, and ethyl alcohol. Inverse first order kinetic dependence on the concentration of molecular oxygen has been observed and is interpreted by a mechanism in which oxygen dissociation from the oxyhemoprotein occurs prior to rate-limiting oxidation by ethyl nitrite. The rate constant for ethyl nitrite oxidation of hemoglobin from which the fourth oxygen has dissociated is calculated to be 45 times greater than the corresponding rate constant for oxidation of deoxyhemoglobin. This rate enhancement is proposed to be a reflection of the oxidative susceptibility of the R and T conformational states of hemoglobin. Results obtained for the oxidation of myoglobin confirm this interpretation as do kinetic data for hemoglobin and myoglobin oxidations by iron(III) and copper(II) complexes. The effects of organic phosphates on rates for hemoglobin oxidations are interpreted in terms of oxidation inhibition by molecular oxygen.Item Structural Effects in Alkyl Nitrite Oxidation of Human Hemoglobin(Elsevier, 1983-06-15) Doyle, Michael; Pickering, Ruth; da Conceicao, JoseOxidations of hemoglobin in oxygen-saturated and deoxygenated media by 10 structurally variant alkyl nitrites have been examined in kinetic detail. Pronounced structural influences on rate constants, whose values span a range of 80 in oxidations of both oxy- and deoxyhemoglobin, have been observed. tert-Butyl nitrite provides the slowest oxidation rate that for deoxyhemoglobin terminates after only half of the available iron(II) heme units have been oxidized. Activation parameters have been determined for oxidations of oxyhemoglobin and deoxyhemoglobin by ethyl, isopropyl, and neopentyl nitrites from kinetic evaluation of these reactions as a function of temperature, The differences in free energies of activation (ΔG) between hemoglobin R and T states range from 1.8 to 2.9 kcal/mol for the three alkyl nitrites examined. The composite data portray alkyl nitrite oxidations as inner sphere electron transfer processes whose kinetic characteristics reflect the ligand binding properties of hemoglobin. A sulfhydryl-induced alkyl nitrite oxidation of oxyhemoglobin that is most pronounced in oxygen-saturated media has been observed, and its cause has been traced to nitrosyl exchange of alkyl nitrites with the β-93 cysteine sulfhydryl group of hemoglobin.Item Oxidation of Deoxyhemerythrin to the Semi-Met Level by Nitrite(Elsevier, 1984-10) Doyle, Michael; Kurtz, Donald Jr.; Nocel, Judith; Pickering, RuthIn anaerobic phosphate buffer, pH 6.3-7.5, deoxyhemerythrin is oxidized to semi-methemerythrin (semi-met) by excess sodium nitrite. This oxidation is quantitative as judged by EPR spectroscopy. Further oxidation to methemerythrin is not detected. The absorbance changes of hemerythrin during the oxidation are biphasic. The rate of the faster first phase is linearly dependent on [H+] and [NO2-] suggesting that the oxidant is nitrous acid rather than nitrite. During the slower second phase, the characteristic EPR spectrum of semi-methemerythrin appears. The first phase can be interpreted by a scheme in which nitrous acid transforms deoxyhemerythrin (FeIIFeII) to the semi-met nitrosyl adduct (FeIIFeIIINO) and hydroxide. Independent experiments confirm that the combination of semi-met plus NO produces an EPR-silent adduct. The rates of the absorbance changes for the second phase are nearly independent of nitrite concentration and pH in the range 6.3-7.5. This slower phase involves the transformation of the EPR-silent intermediate to the semi-met nitrite adduct (FeIIFeIIINO2-) and is consistent with rate-limiting dissociation of nitric oxide followed by rapid attachment of nitrite. Nitrite appears to be a unique oxidant of deoxyhemerythrin in that when employed in excess, the final, stable product is semi-met- rather than methemerythrin. The lack of reactivity of ethyl nitrite with deoxyhemerythrin suggests that HONO oxidizes deoxyhemerythrin via an "inner-sphere" process in contrast to oxidants such as Fe(CN)6(3-). A proposed generalization is that excesses of "inner-sphere" oxidants convert deoxy to (semi-met)R, which is stabilized with respect to (semi-met)R, which is stabilized with respect to (semi-met)0 and met because the oxidant and/or a product of the oxidant can bind to the iron site.Item Electrophilic Metal Carbenes as Reaction Intermediates in Catalytic Reactions(American Chemical Society, 1986-11-01) Doyle, MichaelItem Highly Efficient Olefin Isomerization Catalyzed by Metal Hydrides Derived from Dirhodium(II) Carboxylates and Catecholborane(De Gruyter Open, 1994-07-01) Doyle, Michael; Devora, GeneDirhodium(ll) tetraacetate in combination with catecholborane catalyzes the isomerization of alkenes and dienes. Effective isomerization occurs at 80-135°C with the use of only 0.1 mol % rhodium acetate. With 2-methyl-1,5-hexadiene the disubstituted double bond is preferentially isomerized. In addition, hydrogen transfer hydrogenation occurs with 1,4-cyclohexadienes. The mechanism of these reactions is proposed to involve organoborane addition across a Rh-0 bond which activates the catalyst for isomerization and hydrogenation.Item New Catalysts and Methods for Highly Enantioselective Metal Carbene Reactions(De Gruyter, 1998-01-01) Doyle, MichaelAsymmetric catalysis of metal carbene transformations with unique chiral dirhodium(II) carboxamidates provides highly enantioselective, diastereoselective, and regioselective syntheses of lactones and lactams via cyclopropanation, cyclopropenation, carbon-hydrogen insertion, and ylide derived reactions of diazo acetates and diazoacetamides. Constructed from a dirhodium(II) core with bridging chiral pyrrolidone, oxazolidinone, azedinone, or imidazolidinone ligands, these catalysts are especially effective for intramolecular transformations. Reactions characteristically occur with high turnover numbers, and products are formed in high yield with enantiomeric excesses that are generally greater than 90%.Item A Major Cell Surface Antigen of Coccidioides immitis Which Elicits Both Humoral and Cellular Immune Responses(American Society for Microbiology, 2000-02-01) Hung, Chiung-Yu; Ampel, Neil M.; Christian, Lara; Seshan, Kalpathi R.; Cole, Garry T.Multinucleate parasitic cells (spherules) of Coccidioides immitis isolates produce a membranous outer wall component (SOW) in vitro which has been reported to be reactive with antibody from patients with coccidioidal infection, elicits a potent proliferative response of murine immune T cells, and has immunoprotective capacity in a murine model of coccidioidomycosis. To identify the antigenic components of SOW, the crude wall material was first subjected to Triton X-114 extraction, and a water-soluble fraction derived from this treatment was examined for protein composition and reactivity in humoral and cellular immunoassays. Protein electrophoresis revealed that the aqueous fraction of three different isolates of C. immitis each contained one or two major glycoproteins (SOWgps), distinguished by their molecular sizes, which ranged from 58 to 82 kDa. The SOWgps, however, showed identical N-terminal amino acid sequences, and each was recognized by sera from patients with C. immitis infection. Antibody raised against the purified 58-kDa glycoprotein (SOWgp58) of the Silveira isolate was used for Western blot and immunolocalization analyses. Expression of SOWgp was shown to be parasitic phase specific, and the antigen was localized to the membranous SOW. The water-soluble fraction of SOW and the purified SOWgp58 were tested for the ability to stimulate proliferation of human peripheral monocytic cells (PBMC). The latter were obtained from healthy volunteers with positive skin test reaction to spherulin, a parasitic-phase antigen of C. immitis, and from volunteers who showed no skin test reaction to the same antigen. The SOW preparations stimulated proliferation of PBMC from skin test-positive but not skin test-negative donors, and the activated cells secreted gamma interferon, which is indicative of a T helper 1 pathway of immune response. Results of this study suggest that SOWgp is a major parasitic cell surface-expressed antigen that elicits both humoral and cellular immune responses in patients with coccidioidal infection.Item Disruption of the Gene Which Encodes a Serodiagnostic Antigen and Chitinase of the Human Fungal Pathogen Coccidioides immitis(American Society for Microbiology, 2000-10-01) Reichard, Utz; Hung, Chiung-Yu; Thomas, Pei W.; Cole, Garry T.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 ofCTS1 function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in the parental and Δcts1 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 Δcts1host to further evaluate the significance of chitinase activity in the parasitic cycle of C. immitis.Item Cloning and Expression of the Gene Which Encodes a Tube Precipitin Antigen and Wall-Associated β-Glucosidase of Coccidioides immitis(American Society for Microbiology, 2001-04-01) Hung, Chiung-Yu; Yu, Jieh-Juen; Lehmann, Paul F.; Cole, Garry T.We report the structure and expression of the Coccidioides immitis BGL2 gene which encodes a previously characterized 120-kDa glycoprotein of this fungal respiratory pathogen. The glycoprotein is recognized by immunoglobulin M tube precipitin (TP) antibody present in sera of patients with coccidioidomycosis, a reaction which has been used for serodiagnosis of early coccidioidal infection. The deduced amino acid sequence of BGL2 shows 12 potential N glycosylation sites and numerous serine-threonine-rich regions which could function as sites for O glycosylation. In addition, the protein sequence includes a domain which is characteristic of family 3 glycosyl hydrolases. Earlier biochemical studies of the purified 120-kDa TP antigen revealed that it functions as a β-glucosidase (EC 3.2.1.21 ). Its amino acid sequence shows high homology to several other reported fungal β-glucosidases which are members of the family 3 glycosyl hydrolases. Results of previous studies have also suggested that the 120-kDa β-glucosidase participates in wall modification during differentiation of the parasitic cells (spherules) of C. immitis. In this study we showed that expression of the BGL2 gene is elevated during isotropic growth of spherules and the peak of wall-associated BGL2 enzyme activity correlates with this same phase of parasitic cell differentiation. These data support our hypothesis that the 120-kDa β-glucosidase plays a morphogenetic role in the parasitic cycle of C. immitis.Item Recombinant Urease and Urease DNA of Coccidioides immitis Elicit an Immunoprotective Response against Coccidioidomycosis in Mice(American Society for Microbiology, 2001-05-01) Li, Kun; Yu, Jieh-Juen; Hung, Chiung-Yu; Lehmann, Paul F.; Cole, Garry T.Coccidioides immitis antigens which stimulate a T helper cell 1 (Th1) pathway of host immune response are considered to be essential components of a vaccine against coccidioidomycosis. Recombinant urease (rURE) and recombinant heat shock protein 60 (rHSP60) of C. immitis were expressed in Escherichia coli and tested as vaccine candidates in BALB/c mice. A synthetic oligodeoxynucleotide which contained unmethylated CpG dinucleotides and was previously shown to enhance a murine Th1 response was used as an immunoadjuvant. T cells isolated from the spleens and lymph nodes of the rURE- and rHSP60-immune mice showed in vitro proliferative responses to the respective recombinant protein, but only those T lymphocytes from rURE-immunized mice revealed markedly elevated levels of expression of selected Th1-type cytokine genes. BALB/c mice immunized subcutaneously with rURE and subsequently challenged by the intraperitoneal (i.p.) route with a lethal inoculum of C. immitis arthroconidia demonstrated a significant reduction in the level of C. immitis infection compared to control animals. rHSP60 was much less effective as a protective antigen. Evaluation of cytokine gene expression in lung tissue and levels of recombinant urease-specific immunoglobulins (immunoglobulin G1 [IgG1] versus IgG2a) in murine sera at 12 days after challenge provided additional evidence that immunization with rURE stimulated a Th1 response to the pathogen. Urease was further evaluated by expression of theURE gene in a mammalian plasmid vector (pSecTag2A.URE) which was used to immunize mice by the intradermal route. In this case, 82% of the vector construct-immunized animals survived more than 40 days after i.p. infection, compared to only 10% of the mice immunized with the vector alone. In addition, 87% of the pSecTag2A.URE-immunized survivors had sterile lungs and spleens. These data support the need for further evaluation of the C. immitis urease as a candidate vaccine against coccidioidomycosis.Item A Parasitic Phase-Specific Adhesin of Coccidioides immitis Contributes to the Virulence of This Respiratory Fungal Pathogen(American Society for Microbiology, 2002-07-01) Hung, Chiung-Yu; Yu, Jieh-Juen; Seshan, Kalpathi R.; Reichard, Utz; Cole, Garry T.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.Item Attempted Synthesis of Casbene by Intramolecular Cyclopropanation(ARKAT USA, 2002-11-14) Doyle, Michael; Yan, MingThe antifungal diterpene casbene has been prepared in low yield by intramolecular cyclopropanation of the vinyldiazomethane reactant formed from geranylgeraniol. Careful analysis of the reaction mixture revealed relatively unselective addition catalyzed by dirhodium(II) and copper(I) catalysts.Item Enantioselectivity for Catalytic Cyclopropanation with Diazomalonates(ARKAT USA, 2003-03-24) Doyle, Michael; Hu, WenhaoThe use of chiral azetidinone-ligated dirhodium(II) catalysts activates dinitrogen extrusion from diazomalonates and provides access to cyclopropanation products with selectivities as high as 40-50% ee.Item A Recombinant β-1,3-Glucanosyltransferase Homolog of Coccidioides posadasii Protects Mice against Coccidioidomycosis(American Society for Microbiology, 2003-06-01) Delgado, Nelson; Xue, Jianmin; Yu, Jieh-Juen; Hung, Chiung-Yu; Cole, Garry T.Coccidioides posadasii is a fungal respiratory pathogen which is responsible for recurrent epidemics of San Joaquin Valley fever (coccidioidomycosis) in desert regions of the southwestern United States. Numerous studies have revealed that the cell wall of the parasitic phase of the fungus is a reservoir of immunoreactive macromolecules and a potential source of a vaccine against this mycosis. A 495-bp fragment of a C. posadasii gene which encodes a putative wall-associated, glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase was identified by computational analysis of the partially sequenced genome of this pathogen. The translated, full-length gene (GEL1) showed high sequence homology to a reported β-1,3-glucanosyltransferase of Aspergillus fumigatus (70% identity, 90% similarity) and was selected for further study. The GEL1 mRNA of C. posadasii was detected at the highest level during the endosporulation stage of the parasitic cycle, and the mature protein was immunolocalized to the surface of endospores. BALB/c or C57BL/6 mice were immunized subcutaneously with the bacterium-expressed recombinant protein (rGel1p) to evaluate its protective efficacy against a lethal challenge of C. posadasii by either the intraperitoneal or intranasal route. In both cases, rGel1p-immune mice infected with the pathogen showed a significant reduction in fungal burden and increased survival compared to nonimmune mice. The recombinant β-1,3-glucanosyltransferase is a valuable addition to an arsenal of immunoreactive proteins which could be incorporated into a human vaccine against coccidioidomycosis.Item Production of Cellulose and Curli Fimbriae by Members of the Family Enterobacteriaceae Isolated from the Human Gastrointestinal Tract(American Society for Microbiology, 2003-07-01) Zogaj, Xhavit; Bokranz, Werner; Nimtz, Manfred; Römling, UteCitrobacter spp., Enterobacter spp., and Klebsiella spp. isolated from the human gut were investigated for the biosynthesis of cellulose and curli fimbriae (csg). While Citrobacter spp. produced curli fimbriae and cellulose and Enterobacter spp. produced cellulose with various temperature-regulatory programs, Klebsiella spp. did not show pronounced expression of those extracellular matrix components. Investigation of multicellular behavior in two Citrobacter species and Enterobacter sakazakii showed an extracellular matrix, cell clumping, pellicle formation, and biofilm formation associated with the expression of cellulose and curli fimbriae. In those three strains, the csgD-csgBA region and the cellulose synthase gene bcsA were conserved. PCR screening for the presence of csgD, csgA and bcsA revealed that besides Klebsiella pneumoniae and Klebsiella oxytoca, all species investigated harbored the genetic information for expression of curli fimbriae and cellulose. Since Citrobacter spp., Enterobacter spp., and Klebsiella spp. are frequently found to cause biofilm-related infections such as catheter-associated urinary tract infections, the human gut could serve as a reservoir for dissemination of biofilm-forming isolates.Item A Metalloproteinase of Coccidioides posadasii Contributes to Evasion of Host Detection(American Society for Microbiology, 2005-10-01) Hung, Chiung-Yu; Seshan, Kalpathi R.; Yu, Jieh-Juen; Schaller, Ruth; Xue, Jianmin; Basrur, Venkatesha; Gardner, Malcolm J.; Cole, Garry T.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.Item Urease Produced by Coccidioides posadasii Contributes to the Virulence of This Respiratory Pathogen(American Society for Microbiology, 2006-01-01) Mirbod-Donovan, Fariba; Schaller, Ruth; Hung, Chiung-Yu; Xue, Jianmin; Reichard, Utz; Cole, Garry T.Urease activity during in vitro growth in the saprobic and parasitic phases of Coccidioides spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of Coccidioides posadasii lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of C. posadasii is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of C. posadasii resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a well-organized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of C. posadasii contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.Item A Recombinant Aspartyl Protease of Coccidioides posadasii Induces Protection against Pulmonary Coccidioidomycosis in Mice(American Society for Microbiology, 2006-01-01) Tarcha, Eric J.; Basrur, Venkatesha; Hung, Chiung-Yu; Gardner, Malcolm J.; Cole, Garry T.Coccidioidomycosis is a respiratory disease of humans caused by the desert soil-borne fungal pathogens Coccidioides spp. Recurrent epidemics of this mycosis in the southwestern United States have contributed significantly to escalated health care costs. Clinical and experimental studies indicate that prior symptomatic coccidioidomycosis induces immunity against subsequent infection, and activation of T cells is essential for containment of the pathogen and its clearance from host tissue. Development of a human vaccine against coccidioidomycosis has focused on recombinant T-cell-reactive antigens which elicit a durable protective immune response against pulmonary infection in mice. In this study we fractionated a protective multicomponent parasitic cell wall extract in an attempt to identify T-cell antigens. Immunoblots of electrophoretic separations of this extract identified patient seroreactive proteins which were subsequently excised from two-dimensional polyacrylamide gel electrophoresis gels, trypsin digested, and sequenced by tandem mass spectrometry. The full-length gene which encodes a dominant protein in the immunoblot was identified using established methods of bioinformatics. The gene was cloned and expressed, and the recombinant protein was shown to stimulate immune T cells in vitro. The deduced protein was predicted to contain epitopes that bind to human major histocompatibility complex class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes induced gamma interferon production by immune T lymphocytes. The T-cell-reactive antigen, which is homologous to secreted fungal aspartyl proteases, protected mice against pulmonary infection with Coccidioides posadasii. We argue that this immunoproteomic/bioinformatic approach to the identification of candidate vaccines against coccidioidomycosis is both efficient and productive.Item Multivalent Recombinant Protein Vaccine against Coccidioidomycosis(American Society for Microbiology, 2006-10-01) Tarcha, Eric J.; Basrur, Venkatesha; Hung, Chiung-Yu; Gardner, Malcolm J.; Cole, Garry T.Coccidioidomycosis is a human respiratory disease that is endemic to the southwestern United States and is caused by inhalation of the spores of a desert soilborne fungus. Efforts to develop a vaccine against this disease have focused on identification of T-cell-reactive antigens derived from the parasitic cell wall which can stimulate protective immunity against Coccidioides posadasii infection in mice. We previously described a productive immunoproteomic/bioinformatic approach to the discovery of vaccine candidates which makes use of the translated genome of C. posadasii and a computer-based method of scanning deduced sequences of seroreactive proteins for epitopes that are predicted to bind to human major histocompatibility (MHC) class II-restricted molecules. In this study we identified a set of putative cell wall proteins predicted to contain multiple, promiscuous MHC II binding epitopes. Three of these were expressed by Escherichia coli, combined in a vaccine, and tested for protective efficacy in C57BL/6 mice. Approximately 90% of the mice survived beyond 90 days after intranasal challenge, and the majority cleared the pathogen. We suggest that the multicomponent vaccine stimulates a broader range of T-cell clones than the single recombinant protein vaccines and thereby may be capable of inducing protection in an immunologically heterogeneous human population.