Francisco J. Gomez

Monoclonal Antibodies to Heat Shock Protein 60 Alter the Pathogenesis of Histoplasma capsulatum

ABSTRACT Heat shock proteins with molecular masses of ∼60 kDa (Hsp60) are widely distributed in nature and are highly conserved immunogenic molecules that can function as molecular chaperones and enhance cellular survival under physiological stress conditions. The fungus Histoplasma capsulatum displays an Hsp60 on its cell surface that is a key target of the cellular immune response during histoplasmosis, and immunization with this protein is protective. However, the role of humoral responses to Hsp60 has not been fully elucidated. We generated immunoglobulin G (IgG) isotype monoclonal antibodies (MAbs) to H. capsulatum Hsp60. IgG1 and IgG2a MAbs significantly prolonged the survival of mice infected with H. capsulatum. An IgG2b MAb was not protective. The protective MAbs reduced intracellular fungal survival and increased phagolysosomal fusion of macrophages in vitro. Histological examination of infected mice showed that protective MAbs reduced the fungal burden and organ damage. Organs of infected animals treated with protective MAbs had significantly increased levels of interleukin-2 (IL-2), IL-12, and tumor necrosis factor alpha and decreased levels of IL-4 and IL-10. Hence, IgG1 and IgG2a MAbs to Hsp60 can modify H. capsulatum pathogenesis in part by altering the intracellular fate of the fungus and inducing the production of Th1-associated cytokines.

Vaccination with Heat Shock Protein 60 Induces a Protective Immune Response against Experimental Paracoccidioides brasiliensis Pulmonary Infection

ABSTRACT Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in Latin America. The successful resolution of infection with this fungus is dependent on the activation of cellular immunity. We previously identified heat shock protein 60 (HSP60) as a target of the humoral response in paracoccidioidomycosis. Herein we expressed the gene encoding HSP60 in Escherichia coli and analyzed the immunological activity of this recombinant antigen. The immunization of BALB/c mice with recombinant protein emulsified in adjuvant stimulated a cellular immune response. Splenocytes from immunized mice proliferated in response to antigen and released interleukin-12 and gamma interferon (IFN-γ). Vaccination with HSP60 reduced the fungal burden in mice given 106 or 107 yeasts and protected mice from a lethal challenge. The efficacy of the vaccination was blunted by the neutralization of IFN-γ. CD4+ cells were necessary for the efficacy of the vaccination in both the afferent and efferent phases. Thus, we have demonstrated that this immunodominant antigen is a candidate for the development of a vaccine against this fungus.

Heat shock protein 60: Identification of specific epitopes for binding to primary macrophages

In the present study, we characterized regions of human heat shock protein (HSP) 60 responsible for binding to primary macrophages. Studies using 20‐mer peptides of the HSP60 sequence to compete with HSP60‐binding to macrophages from C57BL/6J mice showed that regions aa241–260, aa391–410 and aa461–480 are involved in surface‐binding. HSP60 mutants, lacking the N‐terminal 137, 243 or 359 amino acids, inhibited HSP60‐binding to primary macrophages to different degrees, demonstrating that all three regions are required for optimal binding. Analysis of different pro‐ and eukaryotic HSP60 species indicated that phylogenetically separate HSP60 species use different binding sites on primary macrophages.

Pathogen-Specific Induction of CD154 Is Impaired in CD4+ T Cells from Human Immunodeficiency Virus–Infected Patients

The pathogenesis of immunodeficiency associated with human immunodeficiency virus (HIV) infection remains incompletely understood. CD154, a molecule that is expressed primarily on activated CD4(+) T cells, is pivotal for regulation of cell-mediated and humoral immunity and is crucial for control of many opportunistic infections. We investigated whether CD4(+) T cells from HIV-infected patients exhibit defective induction of CD154 in response to opportunistic pathogens. Incubation of purified human CD4(+) T cells with monocytes plus antigenic preparations of either Candida albicans, cytomegalovirus, or Toxoplasma gondii resulted in induction of CD154. Expression of CD154 in response to these pathogens was impaired in CD4(+) T cells from HIV-infected patients. This defect correlated with decreased production of interleukin (IL)-12 and interferon (IFN)-gamma in response to T. gondii. Recombinant CD154 partially restored secretion of IL-12 and IFN-gamma in response to T. gondii in cells from HIV-infected patients. Together, defective induction of CD154 is likely to contribute to impaired cell-mediated immunity against opportunistic pathogens in HIV-infected patients.

TNF Receptor-Associated Factor 6-Dependent CD40 Signaling Primes Macrophages to Acquire Antimicrobial Activity in Response to TNF-α

IFN-γ is considered an essential stimulus that allows macrophages to acquire activity against intracellular pathogens in response to a second signal such as TNF-α. However, protection against important pathogens can take place in the absence of IFN-γ through mechanisms that are still dependent on TNF-α. Engagement of CD40 modulates antimicrobial activity in macrophages. However, it is not known whether CD40 can replace IFN-γ as priming signal for induction of this response. We show that CD40 primes mouse macrophages to acquire antimicrobial activity in response to TNF-α. The effect of CD40 was not caused by modulation of IL-10 and TGF-β production or TNFR expression and did not require IFN-αβ signaling. Induction of antimicrobial activity required cooperation between TNFR-associated factor 6-dependent CD40 signaling and TNFR2. These results support a paradigm where TNFR-associated factor 6 signaling downstream of CD40 alters the pattern of response of macrophages to TNF-α leading to induction of antimicrobial activity.

Vbeta4(+) T cells promote clearance of infection in murine pulmonary histoplasmosis.

T cells are essential for controlling infection with Histoplasma capsulatum. Because the T cell receptor is vital for transducing the biological activities of these cells, we sought to determine if exposure to this fungus induced an alteration in the Vbeta repertoire in lungs of C57BL/6 mice infected intranasally. Vbeta2(+) cells were elevated on day 3 after infection; Vbeta4(+) cells were higher than controls on days 7, 10, and 14 after infection. Vbeta10(+) cells were increased on days 14 and 21, and Vbeta11(+) exceeded controls only on day 14. We investigated the clonality and function of Vbeta4(+) cells because their expansion transpired during the critical time of infection, that is, when cellular immunity is activated. Sequence analysis demonstrated preferential use of a restricted set of sequences in the complementarity-determining region 3. Elimination of Vbeta4(+) cells from mice impaired their ability to resolve infection. In contrast, depletion of Vbeta7(+) cells, the abundance of which was similar to that of Vbeta4(+), did not alter elimination of the fungus. The identification of clonotypes of Vbeta4(+) cells suggests that a few antigenic determinants may drive proliferation of this subset, which is necessary for optimal clearance.

Characterization of a gene which encodes a mannosyltransferase homolog of Paracoccidioides brasiliensis.

We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM). A sequence (PbYmnt) was obtained and characterized. A genomic clone was obtained by PCR of P. brasiliensis total DNA. The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa. The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources. A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases. Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein. The PbYmnt was expressed preferentially in the yeast parasitic phase. The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353. A recombinant protein was generated in Escherichia coli. Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.

Histoplasma capsulatum proteome response to decreased iron availability

BackgroundA fundamental pathogenic feature of the fungus Histoplasma capsulatum is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. H. capsulatum can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis.ResultsTo investigate the proteomic response by H. capsulatum to decreasing iron availability we have created H. capsulatum protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the H. capsulatum G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 H. capsulatum proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown.ConclusionWe have created a bioinformatics platform for H. capsulatum and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in H. capsulatum at the protein level. We also identified H. capsulatum proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by H. capsulatum to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for H. capsulatum proteomic analysis.