Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis.

We herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis, specifically a more efficient transformation and a gene expression system. We evaluated several parameters that influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions and host cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensis mixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78+/-9 transformants/co-cultivation (5+/-1 transformants/10(6) target cells). P. brasiliensis GFP-expressing isolates were also constructed by insertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopy and flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differences in fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random single gene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use in targeted mutagenesis and linking mutations to phenotypes.

Molecular mycology: a genetic toolbox for Histoplasma capsulatum.

Research in medical mycology has traditionally been a mix of exciting biology and frustrating genetics, although the excitement has been steadily increasing as genetic obstacles have been successfully overcome. Now, a variety of fungal pathogens can be studied using molecular techniques derived from classical bacterial and yeast genetics, but with selective and strategic adaptations. Histoplasma capsulatum is the best-studied of the primary pathogens known as 'dimorphic' fungi, and tailored molecular genetic strategies are beginning to reveal a repertoire of genes and gene products intimately associated with pathogenesis.

Intracellular parasitism by Histoplasma capsulatum: fungal virulence and calcium dependence.

Histoplasma capsulatum is an effective intracellular parasite of macrophages and causes the most prevalent fungal respiratory disease in the United States. A "dimorphic" fungus, H. capsulatum exists as a saprophytic mold in soil and converts to the parasitic yeast form after inhalation. Only the yeasts secrete a calcium-binding protein (CBP) and can grow in calcium-limiting conditions. To probe the relation between calcium limitation and intracellular parasitism, we designed a strategy to disrupt CBP1 in H. capsulatum using a telomeric linear plasmid and a two-step genetic selection. The resultingcbp1 yeasts no longer grew when deprived of calcium, and they were also unable to destroy macrophages in vitro or proliferate in a mouse model of pulmonary infection.

Four genes are required for the system II cytochrome c biogenesis pathway in Bordetella pertussis, a unique bacterial model.

Unlike other cytochromes, c-type cytochromes have two covalent bonds formed between the two vinyl groups of haem and two cysteines of the protein. This haem ligation requires specific assembly proteins in prokaryotes or eukaryotic mitochondria and chloroplasts. Here, it is shown that Bordetella pertussis is an excellent bacterial model for the widespread system II cytochrome c synthesis pathway. Mutations in four different genes (ccsA, ccsB, ccsX and dipZ) result in B. pertussis strains unable to synthesize any of at least seven c-type cytochromes. Using a cytochrome c4:alkaline phosphatase fusion protein as a bifunctional reporter, it was demonstrated that the B. pertussis wild-type and mutant strains secrete an active alkaline phosphatase fusion protein. However, unlike the wild type, all four mutants are unable to attach haem covalently, resulting in a degraded N-terminal apocytochrome c4 component. Thus, apocytochrome c secretion is normal in each of the four mutants, but all are defective in a periplasmic assembly step (or export of haem). CcsX is related to thioredoxins, which possess a conserved CysXxxXxxCys motif. Using phoA gene fusions as reporters, CcsX was proven to be a periplasmic thioredoxin-like protein. Both the B. pertussis dipZ (i. e. dsbD) and ccsX mutants are corrected for their assembly defects by the thiol-reducing compounds, dithiothreitol and 2-mercaptoethanesulphonic acid. These results indicate that DipZ and CcsX are required for the periplasmic reduction of the cysteines of apocytochromes c before ligation. In contrast, the ccsA and ccsB mutants are not corrected by exogenous reducing agents, suggesting that CcsA and CcsB are required for the haem ligation step itself in the periplasm (or export of haem to the periplasm). Related to this suggestion, the topology of CcsB was determined experimentally, demonstrating that CcsB has four transmembrane domains and a large 435-amino-acid periplasmic region.

Phenotypic variation and intracellular parasitism by histoplasma Capsulatum.

The success of Histoplasma capsulatum as an intracellular pathogen depends completely on successful conversion of the saprophytic mycelial (mold) form of this fungus to a parasitic yeast form. It is therefore not surprising that yeast phase-specific genes and gene products are proving to be important for survival and proliferation of H. capsulatum within macrophages. In this study, we have focused on the role and regulation of two yeast-specific characteristics: alpha-(1,3)-glucan, a cell wall polysaccharide modulated by cell-density (quorum) sensing, and a secreted calcium-binding protein (CBP) that is essential for pathogenicity.

Synergistic epithelial responses to endotoxin and a naturally occurring muramyl peptide.

We have investigated the synergistic interactions of a naturally occurring peptidoglycan fragment (muramyl peptide) and bacterial endotoxin in the induction of inflammatory processes within respiratory epithelial cells, at the levels of both signal transduction events and ultimate cellular metabolic effects. The source of the muramyl peptide is Bordetella pertussis, the causative agent of the respiratory disease pertussis. During log-phase growth, B. pertussis releases the muramyl peptide tracheal cytotoxin (TCT), which has the structure N - acetylglucosaminyl - 1,6 - anhydro - N - acetylmuramyl - (L) - alanyl - gamma - (D) - glutamyl - meso - diaminopimelyl - (D) - alanine, equivalent to a monomeric subunit of gram-negative bacterial peptidoglycan. When applied to hamster trachea epithelial (HTE) cells, TCT and endotoxin were found to be highly synergistic in the induction of interleukin-1alpha (IL-1alpha), type II (inducible) nitric oxide synthase (iNOS), nitric oxide production, and inhibition of DNA synthesis. Neither molecule alone significantly triggered these responses. The serine/threonine protein kinase inhibitor H7 blocked induction of both IL-1alpha and iNOS. More selective inhibitors of protein kinase C, cyclic AMP-dependent protein kinase, and cyclic GMP-dependent protein kinase were not capable of blocking the effects of TCT and endotoxin, suggesting that the H7-inhibited component in this pathway is not among the commonly described kinase targets of H7. Treatment of HTE cells with exogenous IL-1 reproduced the induction of iNOS and DNA synthesis inhibition caused by TCT and endotoxin. H7 was not capable of interfering with effects caused by exogenous IL-1, implying that the H7-sensitive step in the pathway is upstream of IL-1 protein production. Similar assays with the phorbol ester phorbol myristate acetate indicate that it could effectively synergize with endotoxin but not with TCT, suggesting that TCT and endotoxin induce different signal transduction events that combine synergistically. The synergy observed with TCT and endotoxin in epithelial cells is significantly different from their interaction with other cell types, revealing a unique inflammatory response by epithelial cells to these natural bacterial products.

Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids.

Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to the saprophytic mycelial phase or the parasitic yeast phase. Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures. In this study, the green fluorescent protein (GFP) was established as a reporter in H. capsulatum to study regulation of CBP1 expression in cultures and in single cells grown under different conditions and inside macrophages. One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells. By monitoring GFP fluorescence during the transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated. Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome. Broth cultures of Histoplasma yeasts carrying a CBP-GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages. Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.

Signalling and cellular specificity of airway nitric oxide production in pertussis.

Bordetella pertussis, the aetiological agent of whooping cough (pertussis), causes selective destruction of ciliated cells of the human airway mucosa. In a hamster tracheal organ culture model, B. pertussis causes identical cytopathology as does tracheal cytotoxin (TCT), a glycopeptide released by the bacterium. The damage caused by B. pertussis or TCT has been shown to be mediated via nitric oxide (NO*). Using immunofluorescence detection of the cytokine-inducible NO synthase (iNOS; NOS type II), we determined that B. pertussis induced epithelial NO* production exclusively within non-ciliated cells. This epithelial iNOS activation could be reproduced by the combination of TCT and endotoxin. However, neither TCT alone nor endotoxin alone was capable of inducing epithelial iNOS. This result mirrors the synergistic activity of TCT and endotoxin exhibited in monolayer cultures of tracheal epithelial cells. Therefore, TCT and endotoxin are both important virulence factors of B. pertussis, combining synergistically to cause the specific epithelial pathology of pertussis.

Muramyl peptide probes derived from tracheal cytotoxin of Bordetella pertussis.

A novel semisynthetic scheme was developed to couple amine-reactive labeling reagents to the muramyl peptide tracheal cytotoxin (TCT) without affecting a critical amine group. Tracheal cytotoxin, N-acetylglucosaminyl-1, 6-anhydro-N-acetylmuramyl-Ala-gamma-Glu-A2pmAla (A2pm, diaminopimelic acid), is released by Bordetella pertussis, the etiologic agent of whooping cough. This glycopeptide reproduces the specific ciliated cell damage observed in the respiratory tract during B. pertussis infection. To examine binding of TCT to target respiratory cells, we have produced labeled TCT analogs. Structure-function studies have shown that the primary amine of the A2pm side chain is essential for TCT toxicity in respiratory tissue. The methodology described here allows coupling of amine-reactive reagents to TCT without affecting this essential amine. The terminal N-acetylglucosamine ring is opened by oxidation with periodic acid, a dihydrazide linker is coupled to the oxidized ring, and pH control is used to selectively derivatize the free hydrazide with an N-hydroxysuccinimide ester, while the A2pm side-chain amine remains free. Using this method, we have coupled the Bolton-Hunter reagent to TCT, producing a biologically active 125I-labeled TCT analog.

Rare homologous gene targeting in Histoplasma capsulatum: disruption of the URA5Hc gene by allelic replacement.

URA5 genes encode orotidine-5'-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the Histoplasma capsulatum URA5 gene (URA5Hc) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized ura5 strains of H. capsulatum. We attempted to target the genomic URA5 locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in H. capsulatum and has been challenging in some other pathogenic fungi. Several strategies commonly used in Saccharomyces cerevisiae and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for URA5 gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (hph, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Deltaura5Hc::hph plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in H. capsulatum, or in any dimorphic systemic fungal pathogen of humans.

Probing the yeast phase-specific expression of the CBP1 gene in Histoplasma capsulatum.

Histoplasma capsulatum is a pathogenic fungus that exists in two distinct forms. The saprophytic mycelial phase inhabits moist soil environments; once inhaled, hyphae and conidia convert to a unicellular yeast phase that is capable of parasitizing macrophage phagolysosomes. Yeasts cultures, but not mycelial cultures, release large quantities of a calcium-binding protein (CBP) which may be important in calcium acquisition during intracellular parasitism. In this study, we show that the gene encoding CBP (CBP1) is transcriptionally regulated. To identify promoter sequences that are important for yeast phase-specific activity, we created a series of fusions between successively truncated CBP1 5' untranslated regulatory sequences and the Escherichia coli lacZ gene. The fusions were constructed on a telomeric shuttle plasmid that can replicate autonomously in the fungus. By assaying for beta-galactosidase activity from H. capsulatum transformants, we identified a 102-bp region that mediates promoter activation and yeast phase promoter activity. Base pair substitution analysis suggests that the sequences between 839 and 877 bp upstream of the start codon are the most important for this positive regulation.

Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum.

We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional beta-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.

Histoplasma acquisition of calcium and expression of CBP1 during intracellular parasitism.

A highly adapted parasite of macrophages, the yeast phase of Histoplasma capsulatum, survives and proliferates within phagolysosomes, while the mycelial phase exists only as a saprophyte in the soil. We have shown previously that these two phases of Histoplasma differ in their calcium requirements for growth and in the production of a released calcium-binding protein (CBP). Cloning and sequencing the CBP1 gene revealed two introns, a putative signal peptide and potential calcium-binding sites. We also evaluated CBP1 expression by reverse transcription-polymerase chain reaction (RT-PCR) of yeasts grown in broth culture and within two host cell types, a macrophage-like cell line and respiratory epithelial cells. H. capsulatum yeasts expressed CBP1 in all of these settings. Splenocytes from mice immunized with H. capsulatum yeasts responded to purified CBP in proliferation assays, providing evidence for the production of CBP during the infection of mammalian hosts. In addition, after H. capsulatum yeasts were subjected to a calcium-free shock, exogenously added CBP allowed yeasts to incorporate more calcium than yeasts incubated without added CBP. These results suggest that CBP may function to provide yeasts with calcium when they are in a low-calcium environment, such as the phagolysosomal compartment within macrophages.

Fate of transforming DNA in pathogenic fungi.

Genetic engineering is an important tool in helping us to define the molecular basis of pathogenicity and is also useful in helping us to identify new therapeutic targets in pathogenic fungi. Molecular genetic manipulation of micro-organisms requires the development of plasmid-mediated transformation systems that include: (i) infusion of exogenous DNA into recipient cells, (ii) expression of genes present on the incoming DNA, and (iii) stable maintenance and replication of the inserted DNA leading to expression of the desired phenotypic trait. Transformation systems have been developed for only a handful of fungi that are pathogenic to humans including several species of Candida, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces derrmatitidis, Aspergillus fumigatus, Wangiella dermatitidis (Exophiala dermatitidis) and Coccidioides immitis. Except for Candida species and A. fumigatus, where passage of exogenous DNA into recipient cells has been achieved readily using methods developed for transformation of Saccharomyces cerevisiae and Aspergillus nidulans, respectively, development of transformation systems in other pathogenic fungi has been delayed considerably and has only been possible recently with the introduction of electroporation and biolistic methods. Conventional spheroplasting methods or cell wall permeabilization methods using lithium acetate have not been successful for transformation of C. neoformans and work with only low efficiency in H. capsulatum. The fate of incoming DNA varies greatly in these pathogenic species regardless of their phylogenetic relationships. Understanding the fate of incoming DNA is critical for the construction of transforming vectors and the molecular manipulation of the organisms. In this symposium, recent advances in molecular genetic systems including transformation systems, the fate of incoming DNA and strategies for targeted integration are discussed in relation to four pathogenic fungi.

Calcium dependence and binding in cultures of Histoplasma capsulatum.

Histoplasma capsulatum is a pathogenic fungus with two distinct morphologies and lifestyles. The saprophytic form of this organism, a mold, thrives in soil and is especially abundant in the Ohio and Mississippi River valleys. Its parasitic counterpart, a yeast, colonizes phagolysosomes of mammalian macrophages. We have observed a major difference in the calcium requirements of the two forms of Histoplasma, potentially implicating the phagolysosome as a calcium-limiting compartment. Deprivation of calcium by the addition of EGTA to culture media inhibited the growth of mycelial H. capsulatum but had no effect on yeast growth in vitro. In addition, yeasts released a calcium-binding protein (CBP) detectable by a 45CaCl2 blotting technique. CBP was a major component of yeast culture supernatant and was also detectable by ruthenium red staining, another assay for calcium-binding activity. Conversely, mycelial H. capsulatum did not produce CBP, a finding that correlates with the dependence of mycelia on calcium for growth. We also describe here the purification of CBP from yeast culture supernatant by reversed-phase high-pressure liquid chromatography.

Alterations to the cell wall of Histoplasma capsulatum yeasts during infection of macrophages or epithelial cells.

Many Histoplasma capsulatum strains have alpha-(1,3)-glucan in their cell walls and spontaneously produce variants that lack this polymer. The variants, in contrast to the parents, exist in aberrant shapes within macrophages. Here, the ultrastructure of the parental and variant cell walls was examined. All yeasts had identical electron-lucent, thick walls when grown in broth culture. However, ingestion by either macrophages or hamster trachea epithelial (HTE) cells caused the walls of variants to become electron-dense, thin, and sinuous. Parental strains remained unchanged in macrophages. Within HTE cells inoculated with parental strains, some organisms retained a thick wall and alpha-(1,3)-glucan but appeared to be degrading. In contrast, apparently intact intracellular yeasts had thin, wavy walls lacking alpha-(1,3)-glucan. A microenvironment within HTE cells that is unfavorable for the parental phenotype may trigger this ultrastructural change, potentially explaining why only variant yeasts are harvested from such cultures.

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.

Autotoxicity of nitric oxide in airway disease.

Though nitric oxide (NO) plays a role in many normal pulmonary functions and is involved in inflammatory and immune responses, it also has cytopathologic potential if not tightly controlled. In Bordetella pertussis infection, NO mediates the respiratory epithelial pathology that is a hallmark of the pertussis syndrome. Tracheal cytotoxin (TCT) released by B. pertussis triggers the production of an inducible NO synthase (iNOS) within tracheal epithelial cells, which produce the NO ultimately responsible for their destruction. The induction of iNOS is most likely due to the cytokine interleukin-1, which is generated intracellularly in response to TCT; this cytokine, like TCT, can reproduce the pathology caused by B. pertussis infection. Similar epithelial destruction is observed in asthma, but the precise mechanism of damage remains incompletely defined. It is possible that NO induced by proinflammatory cytokines in the asthmatic respiratory epithelium plays a central role in the observed epithelial damage in asthma as it does in pertussis.

An isogenic haemolysin-deficient mutant of Haemophilus ducreyi lacks the ability to produce cytopathic effects on human foreskin fibroblasts.

The haemolysin of Haemophilus ducreyi is the newest member of the Proteus/Serratia family of pore-forming toxins. In order to assess the role of the haemolysin in virulence, we constructed an isogenic haemolysin-deficient mutant of H. ducreyi strain 35000 This strain, designated 35000-3, lacks detectable haemolytic activity. We tested H. ducreyi strains 35000 and 35000-3 for their cytopathic activity against human foreskin fibroblasts (HFFs). We observed strong cytopathic activity when strain 35000 was co-cultured with HFFs. In contrast, cytopathic activity was not observed when strain 35000-3 was co-cultured with HFF cells. We also analysed the isogenic pair of H. ducreyi strains for cytopathic activity against HeLa cells and the keratinocyte cell line HaCaT. Strains 35000 and 35000-3 were strongly cytotoxic when co-cultured with HeLa cells. HaCaT monolayers were slightly damaged by cocultivation with strain 35000-3 but this damage was much less than that observed when HaCaT cells were cocultured with strain 35000. These results indicate that the H. ducreyi haemolysin is responsible for the previously observed cytotoxic activity against HFF cells and is partially responsible for the activity observed with HaCaT cells. The haemolysin, however, is not responsible for the activity observed with HeLa cells.