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PubMed1992

Colony-stimulating factors activate human macrophages to inhibit intracellular growth of Histoplasma capsulatum yeasts.

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M phi) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M phi fungistatic activity when they were added to M phi monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin 3, granulocyte-M phi CSF, or M phi CSF stimulated M phi fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M phi by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M phi fungistatic activity for H. capsulatum. Addition of gamma interferon or tumor necrosis factor alpha to CSF-activated M phi also did not enhance M phi fungistatic activity. These results suggest that interleukin 3, granulocyte-M phi CSF, and M phi CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M phi fungistatic activity.

Cell Differentiation↗
PubMed1992

Intracellular growth inhibition of Histoplasma capsulatum induced in murine macrophages by recombinant gamma interferon is not due to a limitation of the supply of methionine or cysteine to the fungus.

Recombinant murine gamma interferon (rMuIFN-gamma) stimulates mouse peritoneal macrophages to inhibit the intracellular growth of the zoopathogenic fungus Histoplasma capsulatum. In some systems, the inhibition of growth of an intracellular parasite by rIFN-gamma has been related to nutritional constraints induced in the host cells by the lymphokine. Such an explanation might apply to H. capsulatum because the fungus is a functional methionine-cysteine (Met-Cys) auxotroph at 37 degrees C; its sulfite reductase is repressed at that temperature. For this reason, we set about to examine whether or not the antihistoplasma state induced in rMuIFN-gamma is due to a restriction in the availability of Met-Cys. Omission of Met-Cys from the medium in which macrophages were cultivated prevented H. capsulatum from growing within them. Addition of Met or Cys to the macrophage cultures did not antagonize the inhibitory effect induced in the cells by rMuIFN-gamma. Thus, there was no evidence from our work that rMuIFN-gamma evokes the antihistoplasma effect in mouse peritoneal macrophages by limiting the supply of Met-Cys to the fungus.

Animals↗
PubMed1992

An 80-kilodalton antigen from Histoplasma capsulatum that has homology to heat shock protein 70 induces cell-mediated immune responses and protection in mice.

An extract of the cell wall and cell membrane from Histoplasma capsulatum yeast cells was assayed by Western blot (immunoblot) for reactivity with two monoclonal antibodies to heat shock protein 70. Four bands with molecular masses of 80, 66, 54, and 32 kDa bound both antibodies. The 80-kDa protein was isolated, analyzed for homology to heat shock protein 70, and tested for antigenicity and immunogenicity in C57BL/6 mice. The 80-kDa protein reacted with monoclonal antibody to heat shock protein 70. Sera from mice immunized with the antigen recognized H. capsulatum heat shock protein 70. Moreover, the amino-terminal sequence of the 80-kDa protein revealed substantial homology with heat shock protein 70 from several species. The 80-kDa protein induced delayed-type hypersensitivity responses in mice immunized with either viable yeast cells or antigen. Splenocytes from mice immunized with yeast cells or with antigen responded in vitro to the 80-kDa antigen. Immunization of mice with the antigen enhanced host resistance against a sublethal inoculum of H. capsulatum yeast cells, but it did not reduce the mortality of mice given a lethal challenge of yeast cells. Thus, this antigen manifests homology with members of the heat shock protein 70 family. Furthermore, the 80-kDa protein elicits cellular immune responses to H. capsulatum, and it mediates protective immunity.

Amino Acid Sequence↗
PubMed1992

Inhibition of murine macrophage protein kinase C activity by nonviable Histoplasma capsulatum.

Ingestion of Histoplasma capsulatum yeast cells inhibits the oxidative burst response of murine macrophages (M phi's). Since protein kinase C (PKC) is believed to be an integral part of the signal transduction pathway involved in the production of reactive oxygen intermediates, we investigated the relationship between PKC activity and oxidative burst inhibition in H. capsulatum-containing murine peritoneal M phi's. An inhibitory effect on both basal and phorbol myristate acetate-mobilized membrane PKC activities was observed in M phi's which had ingested H. capsulatum but not latex spheres. These results suggest that one way in which H. capsulatum may disrupt the oxidative burst is through a PCK-dependent mechanism.

Administration, Oral↗
PubMed1994

Recombinant murine gamma interferon stimulates macrophages of the RAW cell line to inhibit intracellular growth of Histoplasma capsulatum.

Macrophages of the RAW 264.7 cell line, activated by pretreatment with recombinant murine gamma interferon, inhibit the intracellular growth of Histoplasma capsulatum. Growth inhibition occurred by a mechanism that was operative only when L-Arg metabolism was allowed to occur. When activated macrophages were cultured in the absence of L-Arg or in the presence of NG-monomethyl-L-Arg, a competitive inhibitor of L-Arg metabolism, activation to the antihistoplasma growth-inhibitory state did not occur. An increase in levels of NO2-, an end product of L-Arg metabolism, was detected only after activation of RAW 264.7 cells to the growth-inhibitory state. In contrast, only baseline levels of NO2- were detected when L-Arg was excluded or when NG-monomethyl-L-Arg was added to the culture medium. Nitric oxide (NO.), a reactive intermediate product of L-Arg metabolism, was implicated as the relevant antihistoplasma effector molecule. When H. capsulatum yeast cells were cultured for 24 to 28 h in a system designed to generate soluble NO., a dose-dependent cytotoxic effect was observed.

Animals↗
PubMed1994

Inhibition of intracellular Histoplasma capsulatum replication by murine macrophages that produce human defensin.

Although purified defensins are effective microbicides in vitro, their operation within intact phagocytes has not been established. To address this question, we inserted cDNA encoding human defensin HNP-1 into a pBabe/neo retroviral vector and transduced it into RAW 264.7 cells, a murine macrophage line that lacks endogenous defensins. We isolated five independent clones of HNP-1-transduced cells, all of which secreted prodefensin and contained small amounts of fully processed HNP-1. The two clones that produced the largest amounts of defensin (clones 5 and 14), together with wild-type RAW cells and pBabe/neo-transduced RAW cells (control), were used for the present study. All cells were grown in Dulbecco's modified Eagle's medium-F12 medium that contained 10% heat-inactivated fetal bovine serum and gentamicin. The medium used for the transduced cells contained aminoglycoside G418 in lieu of gentamicin. Both wild-type and transduced cells were placed in antibiotic-free medium 96 h prior to challenge with a yeast-phase strain of Histoplasma capsulatum. Phagocytosis of yeast cells was allowed to proceed for 90 min and was followed by washing and further incubation for 18.5 h. Whereas the phagocytic index did not differ significantly among the four cell populations under study, the mean level of intracellular growth of H. capsulatum in the defensin-transduced RAW cells was significantly lower than those observed for any other cell types (P < 0.05). These findings constitute the first instance of xenogeneic expression of an antimicrobial peptide by phagocytes and suggest that macrophages can be armed with defensins to enhance their ability to restrict certain intracellular pathogens.

Animals↗
PubMed1995

Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis.

HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice.

Amino Acid Sequence↗
PubMed1995

Immunobiological activity of recombinant H antigen from Histoplasma capsulatum.

The H antigen from Histoplasma capsulatum is one of the known principal antigens of this fungus, but information regarding its identity and its participation in cellular immunity is largely unavailable. Therefore, we sought to determine both the nature of this antigen and the nature of its involvement in the cell-mediated immune response. The antigen was isolated from histoplasmin and digested with selected proteinases, and the cleavage products were subjected to reverse-phase high-performance liquid chromatography. Amino acid sequences of protein fragments were obtained by Edman degradation. A fragment of the gene encoding the H antigen was isolated by using degenerate primers in the PCR. This fragment was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence revealed homology to extracellular beta-glucosidases. A cDNA was generated by reverse transcription PCR and cloned into the expression vector pET 19b. Recombinant H antigen was isolated from inclusion bodies of Escherichia coli and tested for its ability to elicit and induce an in vitro cell-mediated immune response in BALB/c mice. Recombinant antigen stimulated splenocytes from mice immunized with viable yeast cells or with antigen suspended in adjuvant. Mice inoculated with H antigen were not protected against either a sublethal or a lethal inoculum of yeast cells. Thus, H antigen stimulates a cell-mediated immune response but does not induce a protective response to H. capsulatum.

Amino Acid Sequence↗
PubMed1996

Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum.

Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal antibody raised against human hsp7O. Splenocytes from C57BL/6 mice immunized with recombinant hsp7O emulsified in adjuvant, but not yeast cells, reacted in vitro to the antigen. Recombinant hsp7O elicited a cutaneous delayed-type hypersensitivity response in mice immunized with protein or with viable yeast cells. Mice were injected with recombinant hsp7O and challenged intranasally with a sublethal inoculum of yeast cells. Vaccination did not confer protection in this model. Thus, recombinant hsp7O can induce a cell-mediated immune response but does not induce a protective response.

Amino Acid Sequence↗
PubMed1996

Phenotypic variation and persistence of Histoplasma capsulatum yeasts in host cells.

Many Histoplasma capsulatum strains spontaneously give rise to variants during broth culture or subsequent to ingestion by epithelial cells. Unlike their parents, these variants are defective in killing macrophages and lack a major cell wall constituent, alpha-(1,3)-glucan. Inside macrophages, where the variants can persist for several weeks, they adopted an unusual morphology strikingly similar to that reported in the tissues of persistently infected humans or animals. These yeasts were often enlarged or misshapen (allomorphic), but were viable. Decreased cytotoxicity for macrophages was more strongly associated with allomorph formation than was the absence of cell wall alpha-(1,3)-glucan. Allomorphs were also formed in rat and mouse resident macrophages, but not in hamster trachea epithelial cells, indicating that host cell type influences the morphology of these yeasts. We propose that during H. capsulatum infection of mammalian hosts, spontaneous variants arise which can be recognized by their unusual morphologies. In contrast with their virulent parents, such variants "peacefully coexist" within macrophages, potentially contributing to the establishment of latency in vivo.

Animals↗
PubMed1996

Localization of a yeast-phase-specific gene product to the cell wall in Histoplasma capsulatum.

A yeast-phase-specific gene, yps-3, has been identified in the virulent Histoplasma capsulatum strain, G217B. Although DNA sequencing of the genomic yps-3 gene from G217B failed to detect homologies with known proteins, the 5' end of a yps-3 cDNA contained a consensus signal sequence. A 519-bp fragment of the cDNA containing the translational stop codon was linker modified and inserted into the bacterial expression vector, pATH 1. Escherichia coli extracts containing the pATH 1 vector alone expressed a major 34-kDa TrpE polypeptide following induction with indoleacrylic acid, while the pATH 1/yps-3 construct produced a predominant 54-kDa TrpE/yps-3 fusion protein. Polyclonal rabbit sera directed against G217B reacted exclusively with the 54-kDa fusion protein in Western blots (immunoblots); serum samples from three patients with acute pulmonary or disseminated histoplasmosis were also positive. To localize the yps-3 protein within G217B, a monoclonal antibody (MAb 7.1) which recognized the yps-3 portion of the fusion protein was generated. A 17.4-kDa protein was detected with MAb 7.1 in Western blots prepared from cell wall fractions of G217B; cytoplasmic fractions were unreactive. No yps-3 antigen was detected in either fraction of the Downs strain, which fails to express the yps-3 gene. MAb 7.1 also detected a 17.4-kDa antigen in ethanol-precipitated culture supernatants derived from G217B. These findings localize the yps-3 gene product to the cell wall and culture supernatants, where the protein may influence the phase transition or the maintenance of the yeast state.

Animals↗
PubMed1998

Nitric oxide synthase expression in macrophages of Histoplasma capsulatum-infected mice is associated with splenocyte apoptosis and unresponsiveness.

Splenic macrophages from Histoplasma capsulatum-infected mice express inducible nitric oxide synthase (iNOS), and the iNOS expression correlates with severity of the infection. We examined whether production of NO is responsible for apoptosis and the anti-lymphoproliferative response of splenocytes from mice infected with H. capsulatum. In situ terminal deoxynucleotidyl transferase nick end labeling revealed apoptotic nuclei in cryosections of spleen from infected but not normal mice. Splenocytes of infected mice were unresponsive to stimulation by either concanavalin A or heat-killed H. capsulatum yeast cells. Splenocyte responsiveness was restored by addition to the medium of NG-monomethyl-L-arginine, a known inhibitor of NO production. The proliferative response of splenocytes from infected mice was also restored by depletion of macrophages or by replacement with macrophages from normal mice. In addition, expression of iNOS returned to its basal level when the animals had recovered from infection. These results suggest that suppressor cell activity of macrophages is associated with production of NO, which also appears to be an effector molecule for apoptosis of cultured splenocytes from infected mice.

Animals↗
PubMed1998

Evolution of the primary immune response to Histoplasma capsulatum in murine lung.

Histoplasma capsulatum induces a cell-mediated immune response in the lungs and lymphoid organs of mammals. In this study, we analyzed the progression of the cytokine and inflammatory reactions in the lungs of mice infected intranasally with H. capsulatum. We measured cytokine mRNA levels and determined the inflammatory cell populations during the active phase of infection (<3 weeks). Transcription of genes encoding interleukin-2 (IL-2), IL-4, and IL-12 and gamma interferon (IFN-gamma) was detectable as early as day 3 of infection, whereas a signal for IL-10 was never observed. Competitive PCR analysis demonstrated that enhanced expression of IL-12 mRNA was observed by day 3 and that expression of mRNA for IL-2 and IFN-gamma progressively increased from day 5 to day 10. All levels declined by day 14. Analysis of the inflammatory response revealed an initial elevation in myeloid cells (Mac-1+) and natural killer (NK) cells followed by a rise in T cells, predominantly CD4+ cells. Since IFN-gamma is a key factor in host defense, we performed cytoplasmic staining to determine the cell populations that produced this cytokine. The hierarchy of synthesis was CD4+ > CD8+ > NK cells. Thus, H. capsulatum provokes an orderly modulation of the inflammatory and cytokine responses in murine lungs.

Animals↗
PubMed1998

Identification and characterization of a phase-specific, nuclear DNA binding protein from the dimorphic pathogenic fungus Histoplasma capsulatum.

Genes expressed in the parasitic yeast (Y) phase of the dimorphic fungal pathogen Histoplasma capsulatum which are transcriptionally silent in the mycelial (M) phase have recently been cloned and analyzed. To understand the molecular regulation of genes involved in the transition to and maintenance of the Y phase, the presumptive 5' regulatory regions of two Y phase-specific genes (yps-3 and yps 21:E-9) were PCR amplified as labelled probes to identify nuclear DNA binding proteins which may influence phase-specific gene transcription. Protein-DNA interactions were assessed by Southwestern blot analysis in which sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated protein extracts from Y and M phases of the virulent G217B strain of H. capsulatum were visualized by their capability for in situ binding to the labelled 517-bp (G217B yps-3) or the 395-bp (G217B yps 21:E-9) putative 5' regulatory regions. A 30-kDa nuclear protein unique to the M-phase extracts of the highly virulent G217B strain, but absent in the Y phase of the same organism, was identified. In contrast, the low-virulence, thermal-sensitive Downs strain of H. capsulatum lacked detectable p30 binding activity in either yeast- or mycelial phase extracts, regardless of the source of labelled probe (395-bp G217B yps 21:E-9 probe or 512-bp HindIII-EcoRI-labelled Downs yps21:E-9). A decanucleotide motif, TCCTTTTTTT, was identified in the upstream regulatory regions of these yps genes, as well as in the putative alpha-tubulin promoter, and was conserved with 70 to 100% homology. This recognition sequence was sufficient for p30M binding with 32P-labelled ligated oligonucleotides when used in the Southwestern assay. These findings describe the first nuclear DNA binding factor identified in H. capsulatum which binds to target sequences in a phase-specific manner, suggesting that p30M may govern aspects of gene transcription in this pathogenic fungus, in which a temperature-sensitive switch influences morphology and virulence.

Base Sequence↗
PubMed1999

Ferric reduction is a potential iron acquisition mechanism for Histoplasma capsulatum.

For the fungus Histoplasma capsulatum, and for other microbial pathogens, iron is an essential nutrient. Iron sequestration in response to infection is a demonstrated host defense mechanism; thus, iron acquisition may be considered an important pathogenic determinant. H. capsulatum is known to secrete Fe(III)-binding hydroxamate siderophores, which is one common microbial process for acquiring iron. Here, we report H. capsulatum ferric reduction activities in whole yeast cells and in both high- and low-molecular-weight fractions of culture supernatants. Each of these activities was induced or derepressed by growth under iron-limiting conditions, a phenomenon often associated with specific iron acquisition mechanisms. The high-molecular-weight culture supernatant activity was enhanced by the addition of reduced glutathione, was proteinase K sensitive and heat labile, and could utilize ferric chloride, ferric citrate, and human holotransferrin as substrates. The low-molecular-weight culture supernatant activity was resistant to proteinase K digestion. These results are consistent with the expression by H. capsulatum of both enzymatic ferric reductase and nonproteinaceous ferric reductant, both of which are regulated by iron availability. Such components could be involved in fungal acquisition of iron from inorganic or organic ferric salts, from H. capsulatum hydroxamate siderophores, or from host Fe(III)-binding proteins, such as transferrin.

Culture Media↗
PubMed1999

The URA5 gene is necessary for histoplasma capsulatum growth during infection of mouse and human cells.

The Histoplasma capsulatum URA5 gene, which has recently been cloned and disrupted by allelic replacement, encodes orotidine-5'-monophosphate pyrophosphorylase. Inactivation of URA5 by either targeted or UV mutagenesis results in disruption of the pyrimidine biosynthetic pathway and uracil auxotrophy. We examined the effect of uracil auxotrophy due to a ura5 mutation on H. capsulatum virulence in both cell culture and whole-animal models. Uracil auxotrophs of two H. capsulatum restriction fragment length polymorphism classes were found to be avirulent in cultured murine and human cells, as well as in mice. Moreover, virulence could be restored either by supplying a functional URA5 gene in trans or by supplying exogenous uracil during infection in vitro. These experiments demonstrate that the pyrimidine biosynthetic pathway is essential for H. capsulatum growth and virulence.

Animals↗
PubMed1999

Molecular cloning, characterization, and expression of the M antigen of Histoplasma capsulatum.

The major diagnostic antigens of Histoplasma capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to H. capsulatum are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic H. capsulatum DNA. This fragment was used to screen an H. capsulatum genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2, 187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from Aspergillus fumigatus, Aspergillus niger, and Eimericella nidulans. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of H. capsulatum is a presumptive catalase, and the recombinant protein retains serodiagnostic activity.

Amino Acid Sequence↗
PubMed1999

Histoplasma capsulatum strain variation in both H antigen production and beta-glucosidase activity and overexpression of HAG1 from a telomeric linear plasmid.

The H antigen of the dimorphic fungal pathogen Histoplasma capsulatum was first described over 40 years ago. It is a secreted glycoprotein that is immunogenic during infection. Recent cloning of the H antigen gene (HAG1) indicated sequence homology with genes for fungal beta-glucosidases. To understand the biological role of this immunodominant antigen in H. capsulatum, enzymatic assays were performed to determine whether H. capsulatum contained a beta-glucosidase enzyme activity and whether this activity was encoded by the HAG1 gene. Substrate gels with H. capsulatum culture supernatants revealed beta-glucosidase activity near the predicted mobility of the H antigen. Quantitative microtiter plate assays revealed marked differences in secreted beta-glucosidase activities from three H. capsulatum restriction fragment length polymorphism (RFLP) classes, with RFLP class II strains displaying high levels of enzyme activity, in contrast to the low levels of activity exhibited by class I and III strains. Immunoblotting of culture supernatants with an H antigen-specific antiserum demonstrated differences in H protein expression levels between the H. capsulatum classes, with a correlation between secreted enzyme activity and H protein levels. We took advantage of these class differences to demonstrate multicopy plasmid H gene overexpression by transformation of an HAG1 plasmid into H. capsulatum. Both a class II strain (G217Bura5-23) and a class III strain (G184ASura5-11) transformed with the telomeric overexpression plasmid pMAD401 displayed increased levels of beta-glucosidase enzyme activity and H protein expression compared to the levels in control transformants containing only the single genomic copy of HAG1. This is the first demonstration of telomeric plasmid-mediated protein overexpression in this pathogenic fungus, and the findings support the identification of the H antigen as a beta-glucosidase.

Animals↗