Phase-specific gene expression underlying morphological adaptations of the dimorphic human pathogenic fungus, Coccidioides posadasii.

Coccidioides posadasii is a dimorphic fungal pathogen that grows as a filamentous saprobe in the soil and as endosporulating spherules within the host. To identify genes specific to the pathogenic phase of Co. posadasii, we carried out a large-scale study of gene expression in two isolates of the species. From the sequenced Co. posadasii genome, we chose 1,000 open reading frames to construct a 70-mer microarray. RNA was recovered from both isolates at three life-cycle phases: hyphae, presegmented spherules, and spherules releasing endospores. Comparative hybridizations were conducted in a circuit design, permitting comparison between both isolates at all three life-cycle phases, and among all life-cycle phases for each isolate. By using this approach, we identified 92 genes that were differentially expressed between pathogenic and saprobic phases in both fungal isolates, and 43 genes with consistent differential expression between the two parasitic developmental phases. Genes with elevated expression in the pathogenic phases of both isolates included a number of genes that were involved in the response to environmental stress as well as in the metabolism of lipids. The latter observation is in agreement with previous studies demonstrating that spherules contain a higher proportion of lipids than saprobic phase tissue. Intriguingly, we discovered statistically significant and divergent levels of gene expression between the two isolates profiled for 64 genes. The results suggest that incorporating more than one isolate in the experimental design offers a means of categorizing the large collection of candidate genes that transcriptional profiling typically identifies into those that are strain-specific and those that characterize the entire species.

A vaccine against coccidioidomycosis is justified and attainable.

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

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

Coccidioides immitis, the causative agent of San Joaquin Valley fever (coccidioidomycosis), produces a urease which has been suggested to contribute to the virulence of this fungal pathogen. Urease catalyzes the hydrolysis of urea and has been proposed to at least partly account for alkalinity of the microenvironment in which C. immitis grows due to the release of ammonia and ammonium ions. The C. immitis urease was purified to homogeneity (1048-fold) from the mycelial cytosol by chromatographic fractionation. The sequence of 12 N-terminal amino-acid residues of the purified, native polypeptide was identical to that predicted by the translated urease gene sequence which has been reported. The isolated enzyme exhibited a specific activity in the presence of urea of 1750 micromol min(-1) mg(-1) protein, has a native molecular mass of 450 kDa, revealed a Km for urea of 4.1 mM, had a pH optimum of 8.0 and is heat stable. Hydroxyurea, acetohydroxamic acid (AHA) and boric acid each inhibited activity of the purified enzyme. Urease activity was enhanced by the presence of 5-10 mM concentrations of Mg2+ or Mn2+, but inhibited by Li+, Ni2+, Cu2+ or Zn2+. The reversible urease inhibitor, AHA, blocked enzyme activity in the crude mycelial cytosolic fraction when added at a concentration of 10 mM. On the other hand, 10 mM AHA added to 4-day-old mycelial cultures only partially decreased the amount of ammonium detected in the culture medium. It is evident, therefore, that C. immitis urease activity does not account for the total amount of ammonia secreted during in vitro growth of the pathogen. Other metabolic sources of ammonia, which may also contribute to the virulence of C. immitis, are under investigation.

Kinetic and structural characterization of urease active site variants.

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.

Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand.

Klebsiella aerogenes urease possesses a dinuclear metallocenter in which two nickel atoms are bridged by carbamylated Lys217. To assess whether carbamate-specific chemistry is required for urease activity, site-directed mutagenesis and chemical rescue strategies were combined in efforts to place a carboxylate group at the location of this metal ligand. Urease variants with Lys217 replaced by Glu, Cys, and Ala (K217E, K217C/C319A, and K217A proteins) were purified, shown to be activated by incubation with small organic acids plus Ni(II), and structurally characterized. K217C/C319A urease possessed a second change in which Cys319 was replaced by Ala in order to facilitate efforts to chemically modify Cys217; however, this covalent modification approach did not produce active urease. Chemical rescue of the K217E, K217C/C319A, and K217A variants required 2, 2, and 10 h, respectively, to reach maximal activity levels. The highest activity generated [224 micromol of urea degraded.min-1.(mg of protein)-1, for K217C/C319A urease incubated with 500 mM formic acid and 10 mM Ni at pH 6.5] corresponded to 56% of that measured for in vitro activation of the wild-type apoprotein. While the K217E apoprotein showed minimal structural perturbations, the K217C/C319A apoprotein showed a disordering of some active site residues, and the K217A apoprotein revealed a repositioning of His219 to allow the formation of a hydrogen bond with Thr169, thus replacing the hydrogen bond between the amino group of Lys217 and Thr169 in the native enzyme. Importantly, these structures allow rationalization of the relative rates and yields of chemical rescue experiments. The crystal structures of chemically rescued K217A and K217C/C319A ureases revealed a return of the active site residues to their wild-type positions. In both cases, noncovalently bound formate was structurally equivalent to the Lys-carbamate as the bridging metallocenter ligand. We conclude that carbamate-specific chemistry is not required for urease catalysis.

Mechanism-based inhibitor discrimination in the acyl-CoA dehydrogenases.

The catalytically essential glutamate base in the acyl-CoA dehydrogenase family is found either on the loop between J and K helices (e.g., in short-chain, medium-chain, and glutaryl-CoA dehydrogenases) or on the G helix (long-chain and isovaleryl-CoA dehydrogenases). While active-site bases at either position are functionally equivalent with respect to alpha-proton abstraction, reactions that require removal of a gamma-proton show marked differences between the two enzyme classes. Thus short-chain, medium-chain, and glutaryl-CoA dehydrogenase are rapidly inactivated by 2-pentynoyl-CoA with abstraction of a gamma-proton, whereas isovaleryl-CoA dehydrogenase is not significantly inhibited. This resistance is not due to weak binding: the complex between isovaleryl-CoA dehydrogenase and 2-pentynoyl-CoA shows a Kd of 1.8 microM at pH 7.6. Migration of the catalytic base to the loop between J and K helices (using the Glu254Gly/Ala375Glu double mutant) makes isovaleryl-CoA dehydrogenase sensitive to irreversible inhibition by 2-pentynoyl-CoA. Molecular modeling suggests that this mutation brings the catalytic base close enough to abstract a gamma-proton from the bound inhibitor. Experiments with two mechanism-based inactivators that target the FAD of the medium- and short-chain acyl-CoA dehydrogenases support this conclusion. 3-Methyl-3-butenoyl-CoA requires activation by alpha-proton abstraction and rapidly yields a reduced flavin adduct with wild-type isovaleryl-CoA dehydrogenase. In contrast, the isomeric 3-methyl-2-butenoyl-CoA is inert toward this enzyme because it cannot be activated by gamma-proton abstraction. Molecular modeling supports these observations. This unusual selectivity toward mechanism-based inactivators provides additional discrimination between members of the acyl-CoA dehydrogenase family.

Oxidative inactivation of a charge transfer complex in the medium-chain acyl-CoA dehydrogenase.

The intense charge transfer complex between the enolate of 3-thia-octanoyl-CoA and the oxidized flavin of the medium-chain acyl-CoA dehydrogenase is discharged by the ferricenium ion with irreversible inactivation of the enzyme. Charge transfer complex formation is a necessary, but insufficient, condition for oxidative inactivation: the 3-oxa-octanoyl-CoA complex is also inactivated, whereas the comparable trans-3-octenoyl-CoA species is not. Complete inactivation of the dehydrogenase with 3-thia-octanoyl-CoA requires 1 molecule of thioester and apparently 3 molecules of ferricenium hexafluorophosphate. Experiments with 8-Cl-FAD substituted enzyme and the crystal structure of enzyme.ligand complexes argue that ferricenium ion-mediated oxidation proceeds through the flavin prosthetic group. Synthesis of [2-14C]-3-thia-octanoyl-CoA, followed by isolation of radiolabeled peptide from the modified medium-chain dehydrogenase, showed that inactivation results in labeling the catalytic base, GLU376. Oxidative modification is accompanied by the release of CoASH. A mechanism for inactivation is proposed involving generation of a sulfonium salt which efficiently captures the carboxylate nucleophile.