John N Galgiani

1,3-beta-Glucan synthase is responsible for the synthesis of beta-glucan, an essential cell wall structural component in most fungi. We sought to determine whether Coccidioides posadasii possesses genes homologous to known fungal FKS genes that encode the catalytic subunit of 1,3-beta-glucan synthase. A single gene, designated FKS1, was identified, and examination of its predicted protein product showed a high degree of conservation with Fks proteins from other filamentous fungi. FKS1 is expressed at similar levels in mycelia and early spherulating cultures, and expression decreases as the spherules mature. We used Agrobacterium-mediated transformation to create strains that harbor DeltaFKS1::hygB, a null allele of FKS1, and hypothesize that Fks1p function is essential, due to our inability to purify this allele away from a complementing wild-type FKS1 allele in a heterokaryotic strain. The heterokaryon appears normal with respect to growth rate and arthroconidium production; however, microscopic examination of strains with DeltaFKS1::hygB alleles revealed abnormal swelling of hyphal elements.

Two recombinant antigens which individually protect mice from lethal intranasal infection were studied in combination, either as a mixture of two separately expressed proteins or as a single chimeric expression product. Mice vaccinated with either combination survived longer than mice given single antigens. Immunized mice also exhibited specific IgG immunoglobulins and yielded splenocytes which produced interferon-gamma in response to either antigen. The chimeric antigen has the practical advantage of offering enhanced protection from multiple components without increasing production costs.

Of 150,000 new coccidioidomycosis infections that occur annually in the United States, ≈1% disseminate; one third of those cases are fatal. Immunocompromised hosts have higher rates of dissemination. We identified 8 patients with disseminated coccidioidomycosis who had defects in the interleukin-12/interferon-γ and STAT3 axes, indicating that these are critical host defense pathways.

Valley fever (VF) is difficult to diagnose, partly because the symptoms of VF are confounded with those of other community-acquired pneumonias. Confirmatory diagnostics detect IgM and IgG antibodies against coccidioidal antigens via immunodiffusion (ID). The false-negative rate can be as high as 50% to 70%, with 5% of symptomatic patients never showing detectable antibody levels. In this study, we tested whether the immunosignature diagnostic can resolve VF false negatives. An immunosignature is the pattern of antibody binding to random-sequence peptides on a peptide microarray. A 10,000-peptide microarray was first used to determine whether valley fever patients can be distinguished from 3 other cohorts with similar infections. After determining the VF-specific peptides, a small 96-peptide diagnostic array was created and tested. The performances of the 10,000-peptide array and the 96-peptide diagnostic array were compared to that of the ID diagnostic standard. The 10,000-peptide microarray classified the VF samples from the other 3 infections with 98% accuracy. It also classified VF false-negative patients with 100% sensitivity in a blinded test set versus 28% sensitivity for ID. The immunosignature microarray has potential for simultaneously distinguishing valley fever patients from those with other fungal or bacterial infections. The same 10,000-peptide array can diagnose VF false-negative patients with 100% sensitivity. The smaller 96-peptide diagnostic array was less specific for diagnosing false negatives. We conclude that the performance of the immunosignature diagnostic exceeds that of the existing standard, and the immunosignature can distinguish related infections and might be used in lieu of existing diagnostics.

Previous studies have shown that dendritic cells (DC) pulsed with T27K, an antigenic preparation derived from spherules (of Coccidioides posadasii), activate peripheral blood mononuclear cells (PBMC) from nonimmune subjects as well as from patients with disseminated coccidioidomycosis. In this study, we have assessed the interaction between human DC and C. posadasii spherules in order to better understand the initial response between Coccidioides and the human host. Whole autoclaved spherules induced lymphocyte transformation in PBMC obtained from immune but not from nonimmune donors. Immature DC (iDC) bound fluorescein isothiocyanate-labeled spherules in a time- and temperature-dependent manner. This binding was blocked by the addition of mannan, suggesting mannose receptor involvement in the DC-Coccidioides interaction. Binding was subsequently associated with ingestion and intracellular processing of spherules. Coculturing of spherules with iDC was associated with the development of mature DC that were morphologically, phenotypically, and functionally similar to those induced by tumor necrosis factor alpha and prostaglandin E2. Finally, spherules incubated with iDC induced activation of PBMC from nonimmune donors. These data indicate that human DC are capable of binding, internalizing, and presenting antigens from Coccidioides spherules and suggest that DC may play a critical early role in the formation of a cellular immune response in human coccidioidomycosis.