Eric D. Holbrook

Extracellular superoxide dismutase protects Histoplasma yeast cells from host-derived oxidative stress.

In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival.

Definition of the Extracellular Proteome of Pathogenic-Phase Histoplasma capsulatum

The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease. Within the mammalian host, pathogenic Histoplasma yeast infect, replicate within, and ultimately kill host phagocytes. Surprisingly, few factors have been identified that contribute to Histoplasma virulence. To address this deficiency, we have defined the constituents of the extracellular proteome using LC−MS/MS analysis of the proteins in pathogenic-phase culture filtrates of Histoplasma. In addition to secreted Cbp1, the extracellular proteome of pathogenic Histoplasma yeast consists of 33 deduced proteins. The proteins include glycanases, extracellular enzymes related to oxidative stress defense, dehydrogenase enzymes, chaperone-like factors, and five novel culture filtrate proteins (Cfp’s). For independent verification of proteomics-derived identities, we employed RNA interference (RNAi)-based depletion of candidate factors and showed loss of specific proteins from the cell-free culture filtrate. Quantitative RT-PCR revealed the expression of 10 of the extracellular factors was particularly enriched in pathogenic yeast cells as compared to nonpathogenic Histoplasma mycelia, suggesting that these proteins are linked to Histoplasma pathogenesis. In addition, Histoplasma yeast express these factors within macrophages and during infection of murine lungs. As extracellular proteins are positioned at the interface between host and pathogen, the definition of the pathogenic-phase extracellular proteome provides a foundation for the molecular dissection of how Histoplasma alters the host-pathogen interaction to its advantage.

Histoplasma capsulatum pathogenesis : making a lifestyle switch

The dimorphism of Histoplasma reflects a developmental switch in morphology and lifestyle that is necessary for virulence. The dimorphism regulating kinase DRK1 and the Histoplasma WOR1 homolog RYP1 mediate the thermally induced transition to the pathogenic yeast-phase program. The genes expressed as part of this regulon influence the host-pathogen interaction to favor Histoplasma virulence. While surface localized HSP60 supports yeast attachment to host macrophages, yeast alpha-glucan polysaccharides conceal immunostimulatory cell wall beta-glucans from detection by macrophage receptors. Intramacrophage growth of yeast cells is facilitated by CBP a secreted, protease-resistant calcium-binding protein tailored to function within the phagolysosomal environment. In some Histoplasma strains, YPS3 promotes dissemination of yeast from pulmonary infection sites. The Histoplasma yeast-phase program includes additional cell surface and extracellular molecules that potentially function in further aspects of Histoplasma virulence.

Redundant Catalases Detoxify Phagocyte Reactive Oxygen and Facilitate Histoplasma capsulatum Pathogenesis

ABSTRACT Histoplasma capsulatum is a respiratory pathogen that infects phagocytic cells. The mechanisms allowing Histoplasma to overcome toxic reactive oxygen molecules produced by the innate immune system are an integral part of Histoplasmas ability to survive during infection. To probe the contribution of Histoplasma catalases in oxidative stress defense, we created and analyzed the virulence defects of mutants lacking CatB and CatP, which are responsible for extracellular and intracellular catalase activities, respectively. Both CatB and CatP protected Histoplasma from peroxide challenge in vitro and from antimicrobial reactive oxygen produced by human neutrophils and activated macrophages. Optimal protection required both catalases, as the survival of a double mutant lacking both CatB and CatP was lower than that of single-catalase-deficient cells. Although CatB contributed to reactive oxygen species defenses in vitro, CatB was dispensable for lung infection and extrapulmonary dissemination in vivo. Loss of CatB from a strain also lacking superoxide dismutase (Sod3) did not further reduce the survival of Histoplasma yeasts. Nevertheless, some catalase function was required for pathogenesis since simultaneous loss of both CatB and CatP attenuated Histoplasma virulence in vivo. These results demonstrate that Histoplasmas dual catalases comprise a system that enables Histoplasma to efficiently overcome the reactive oxygen produced by the innate immune system.

ENHANCED ANTIBODY DETECTION AND DIAGNOSIS OF COCCIDIOIDOMYCOSIS WITH THE MIRAVISTA IGG AND IGM DETECTION ENZYME IMMUNOASSAY.

ABSTRACT Coccidioidomycosis is a common cause of community-acquired pneumonia in areas of the southwestern United States in which the disease is endemic. Clinical presentations range from self-limited disease to severe disseminated disease. Therefore, early and accurate diagnosis is essential to ensure appropriate treatment and monitoring. Currently available diagnostic tests have variable accuracy, particularly in certain patient populations, and new tests may offer improved accuracy for the diagnosis of coccidioidomycosis. Serum samples from 103 cases of coccidioidomycosis and 373 controls were tested for IgG and IgM antibodies using the MVista anti-Coccidioides antibody enzyme immunoassay. Serum specimens from 170 controls from areas in which the disease is endemic and 44 cases were tested by immunodiffusion at MiraVista Diagnostics. The sensitivity of the MVista antibody assay was 88.3%, and the specificity was 90%. The sensitivity was maintained in the presence of immunocompromising conditions or immunosuppressive therapies. The sensitivity of immunodiffusion was 60.2%, and the specificity was 98.8%. The sensitivity of complement fixation (62 cases) was 66.1%, but the specificity could not be determined. The MVista anti-Coccidioides antibody enzyme immunoassay offers improved sensitivity, compared with immunodiffusion and complement fixation, is not impaired in immunocompromised patients, and permits highly reproducible semiquantification.

Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen

ABSTRACT The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome of Histoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells, suggesting its role in pathogenesis. We have generated three monoclonal antibodies as tools for characterization and detection of Cfp4 and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages or during lung infection in a murine model of histoplasmosis. Coinfection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage to Histoplasma yeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well as a strain from Panama. In addition, human immune sera recognize the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.