Targeted curation of the gut microbial gene content modulating human cardiovascular disease.

IMPORTANCE: One of the most-cited examples of the gut microbiome modulating human disease is the microbial metabolism of quaternary amines from protein-rich foods. By-products of this microbial processing promote atherosclerotic heart disease, a leading cause of human mortality globally. Our research addresses current knowledge gaps in our understanding of this microbial metabolism by holistically inventorying the microorganisms and expressed genes catalyzing critical atherosclerosis-promoting and -ameliorating reactions in the human gut. This led to the creation of an open-access resource, the Methylated Amine Gene Inventory of Catabolism database, the first systematic inventory of gut methylated amine metabolism. More importantly, using this resource we deliver here, we show for the first time that these gut microbial genes can predict human disease, paving the way for microbiota-inspired diagnostics and interventions.

Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum.

The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence.

HcZrt2, a zinc responsive gene, is indispensable for the survival of Histoplasma capsulatum in vivo.

Histoplasma capsulatum (Hc) exists in the soil and is capable of adapting to the shift in environment during infection to ensure survival. Yeast encounter a restrictive host environment low in nutrients such as zinc. In this study we functionally analyzed a putative zinc regulated transporter, HcZrt2, in zinc limiting conditions by complementation of HcZrt2 and gene knockdown through RNA interference (RNAi). Complementation analysis demonstrated HcZrt2's ability to functionally replace the characterized Saccharomyces cerevisiae zinc plasma membrane transporters Zrt1 and Zrt2 in zinc deficient medium. Gene silencing revealed that HcZrt2 is essential for growth in zinc deficient medium and plays a role in zinc accumulation. Fungal burden was reduced in mice infected with HcZrt2 silenced strains compared to a control strain. Sixty-seven percent of mice infected with a lethal dose of HcZrt2-RNAi#1 survived, and 100% of mice infected with HcZrt2-RNAi#2 withstood lethal infection. Our data suggest that HcZrt2 is a vital part of zinc homeostasis and essential for the pathogenesis of histoplasmosis.

Yeast Transcriptome and In Vivo Hypoxia Detection Reveals Histoplasma capsulatum Response to Low Oxygen Tension.

Although there is growing understanding of the microenvironmental conditions fungal pathogens encounter as they colonize their host, nothing is known about Histoplasma capsulatum's response to hypoxia. Here we characterized hypoxia during murine histoplasmosis using an in vivo hypoxia detection agent, Hypoxyprobe-2 (HP-2); and analyzed H. capsulatum's transcriptional profile in response to in vitro hypoxia. Immunohistopathology and flow cytometry analyses revealed distinct regions of hypoxia during infection. Granuloma cells, enriched with macrophages and T-cells isolated from infected livers were 66-76% positive for HP-2, of which, 95% of macrophages and 55% of T-cells were hypoxic. Although inhibited, H. capsulatum was able to survive under in vitro hypoxic conditions (<1% O2), and restored growth when replaced in normoxia. Next-generation sequencing (RNA-seq) analysis after 24 hours of hypoxia demonstrated a significant increase in NIT50 (swirm domain DNA binding protein), a predicted ABC transporter (ABC), NADPH oxidoreductase (NADP/FAD), and guanine nucleotide exchange factor (RSP/GEF); and other genes with no known designated function. Computational transcription factor binding site analysis predicted human sterol regulatory element binding protein (SREBP) binding sites upstream of NIT50, ABC, NADP/FAD and RSP/GEF. Hypoxia resulted in a time-dependent increase in the H. capsulatum homolog of SREBP, here named Srb1. Srb1 peaked at 8 hours and returned to basal levels by 24 hours. Our findings demonstrate that H. capsulatum encounters and survives severe hypoxia during infection. Additionally, the hypoxic response may be regulated at the level of transcription, and these studies contribute to the understanding of hypoxic regulation and adaptation in H. capsulatum.

Phase I study demonstrates safety and tolerability of radiofrequency ablation (RFA) of the anal mucosa.

BACKGROUND: Anal carcinoma is increasing in high-risk populations. Dysplasia is often distributed throughout the anal mucosa, and focal ablative techniques have high rates of recurrence. METHODS: With the goal of eradicating dysplasia from the entire anal mucosa, we conducted a phase I dose-ranging study to determine the safety and tolerability of radiofrequency ablation (RFA). HIV-infected individuals with high-grade anal intraepithelial neoplasia underwent RFA of the anal mucosa. Patient-reported procedural and postprocedural symptoms were recorded, and mucosal healing was visually assessed. RESULTS: Four groups of 3 subjects each were treated with incrementally increasing numbers of RF pulses (1-3) applied to a single area of anal mucosa. Two or three doses of 12 J/cm2 were found to have acceptable patient tolerance and healing of the mucosa within 4 weeks of ablation. Using these doses, 2 groups underwent ablation of 180° of contiguous mucosa. Subjects experienced a loss of 1 to 3 days of daily activities of living, 7 to 14 days of postprocedure symptoms, and mucosal healing within 4 weeks. One subject in the first treatment group had the procedure aborted due to severe procedural pain. CONCLUSIONS: The study provides evidence of the safety and tolerability of anal RFA of 180° of contiguous mucosa in a single procedure and will allow future RFA efficacy studies in the treatment of anal dysplasia.

Iron uptake and virulence in Histoplasma capsulatum.

Histoplasma capsulatum (Hc) is the causative organism of a spectrum of disease affecting both the immunocompetent and the immunocompromised host. Hc is a dimporhic fungus that converts from conidia to the pathogenic yeast phase after entry into the mammalian host. Despite rapid ingestion by macrophages, it survives intracellularly within the macrophage. The intracellular survival strategy of Hc yeasts focuses on regulating the phagosomal compartment by modulating the intraphagosomal pH to 6.5. As an intracellular pathogen of MΦ, Hc obtains iron from Fe-transferrin, ferritin, or both, via the production of hydroxamate siderophores, and the production of ferric reductases. A better understanding of the mechanisms by which Hc yeasts acquire iron from the host may lead to novel therapeutics for histoplasmosis.

Analysis of current antifungal agents and their targets within the Pneumocystis carinii genome.

Pneumocystis pneumonia (PCP) remains a leading opportunistic infection in patients with weakened immune systems. The fungus causing the infection belongs to the genus, Pneumocystis, and its members are found in a large variety of mammals. Adaptation to the lung environment of a host with an intact immune system has been a key to its successful survival. Unfortunately, the metabolic strategies used by these fungi to grow and survive in this context are largely unknown. There were considerable impediments to standard approaches for investigation of this unique pathogen, the most problematic being the lack of a long term in vitro culture system. The absence of an ex vivo cultivation method remains today, and many fundamental scientific questions about the basic biology, metabolism, and life cycle of Pneumocystis are unanswered. Recent progress in sequencing of the Pneumocystis carinii genome, a species infecting rats, permitted a more informative search for genes and biological pathways within this pathogen that are known to be targets for existing antifungal agents. In this work, we review the classes of antifungal drugs with respect to their potential applicability to the treatment of PCP. Classes covered in the review are the azoles, polyenes, allylamines, and echinocandins. Factors limiting the use of standard antifungal treatments and the currently available alternatives (trimethoprim-sulfamethoxazole, atovaquone, and pentamidine) are discussed. A summary of genomic sequences within Pneumocystis carinii associated with the corresponding targeted biological pathways is provided. All sequences are available via the Pneumocystis Genome Project at http://pgp.cchmc.org/.

VEA1 is required for cleistothecial formation and virulence in Histoplasma capsulatum.

Histoplasma capsulatum is a pathogenic fungus dependent on dimorphism for virulence. Among the four described Velvet family genes, two of them, Ryp2 and Ryp3, have been shown to be required for dimorphism. It is known that Velvet A (VeA) is necessary for sexual development and toxin production in Aspergillus nidulans. However, the role of the VeA ortholog in H. capsulatum has not yet been explored. Vea1, H. capsulatum homolog of VeA, was studied to determine its role in cleistothecial formation, dimorphism, and virulence. H. capsulatum Vea1 restores cleistothecial formation and partially restores sterigmatocystin production in an A. nidulans veA deletion strain. Furthermore, silencing VEA1 in an H. capsulatum strain capable of forming cleistothecia abolishes cleistothecial formation. Silenced strains also switch to mycelial phase faster, and show impaired switching to the yeast phase once in mycelial phase. Virulence in mice and macrophages is attenuated in VEA1 silenced strains and silenced strains demonstrate increased sensitivity during growth under acidic conditions. These results indicate that H. capsulatum Vea1 shares a similar role in development as VeA. H. capsulatum is also more susceptible to growth in acidic conditions when VEA1 is silenced, which may contribute to the silenced strains' attenuated virulence in mice and macrophages.

Invasive models of histoplasmosis.

Histoplasmosis results from infection with the fungal organism Histoplasma capsulatum. Most Histoplasma research today uses models based on primary pulmonary infection. Following inhalation, conidia are rapidly ingested and intracellularly convert to the yeast form. After initial pulmonary infection, organisms are carried throughout the body giving rise to disseminated infection. Three murine infection models have been developed which mimic human disease. These comprise models of primary infection following initial exposure to the organism, secondary exposure to Histoplasma following spontaneous recovery from primary disease and a model of reactivated infection following spontaneous clearance of primary disease. This chapter describes these models and explores variability introduced with the use of mice of varied genetic backgrounds and different H. capsulatum strains.

Sequence and structure of the linear mitochondrial genome of Pneumocystis carinii.

With the exception of a few genes, most of the mitochondrial (mt) genome of Pneumocystis carinii has not previously been sequenced. Shotgun sequences generated as a result of the Pneumocystis Genome Project (PGP) were assembled with the gap4 assembly program into a 23-kb contig. Annotation of the mt genome identified 4 open reading frames and 20 tRNAs in addition to 17 other genes: ATP synthase, subunits 6, 8, and 9; cytochrome c oxidase, subunits 1, 2, and 3; NADH dehydrogenase, subunits 1, 2, 3, 4, 4L, 5, and 6; apocytochrome b; RNase P RNA gene; and the mitochondrial large and small ribosomal RNA subunits. A 24-bp unit that repeated from one to five times was identified interior to the ends of the mt genome. Migration of the genome on CHEF gels was consistent with that of linear DNA and digestion with BAL31 showed a concomitant reduction in size of the genome, a characteristic of linear DNA. Together with the identification of terminal repeats similar to those found in other linear fungal mt genomes and the inability to join the ends by PCR, these data provide strong evidence that the mt genome of P. carinii is linear.

The Histoplasma capsulatum vacuolar ATPase is required for iron homeostasis, intracellular replication in macrophages and virulence in a murine model of histoplasmosis.

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mphi). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mphi. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron-replete medium, but not on iron-deficient media. On iron-deficient medium, the growth of the vma1 mutant was restored in the presence of wild-type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mphi was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28 degrees C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mphi, grow on iron-poor medium and grow as a mold at 28 degrees C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis and in fungal dimorphism.

Histoplasma capsulatum manifests preferential invasion of phagocytic subpopulations in murine lungs.

Numerous in vitro studies have demonstrated that Histoplasma capsulatum is engulfed by the diverse populations of phagocytic cells including monocytes/macrophages (Mphi), immature dendritic cells (DC), and neutrophils. The in vivo distribution of H. capsulatum has yet to be examined following an intrapulmonary challenge. To accomplish this goal, we engineered GFP into two genetically dissimilar strains of H. capsulatum, G217B and 186R. C57BL/6 mice were infected with each of these strains, and we analyzed the distribution of this fungus in the three major phagocytic populations on successive days. Yeast cells were found in all three populations of cells from Days 1 through 7. Proportionally, DC dominated at Day 1, whereas the majority of yeast cells was detected in neutrophils thereafter. Yeast cells were present in inflammatory and resident Mphi on Day 3, but on Day 7, they were chiefly in inflammatory Mphi. Yeast cells were predominantly in a CD11c(+intermediate/high), F4/80(-), CD11b(+), Ly-6C(+), CD205(+) DC population. Neutralization of TNF-alpha or IFN-gamma produced a significant redistribution of yeast cells. These results reveal the complex nature of intracellular residence of this fungus. Moreover, the findings demonstrate that there is a skewing in the subpopulations of cells that are infected, especially DC.

Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum.

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

Transcriptome of Pneumocystis carinii during fulminate infection: carbohydrate metabolism and the concept of a compatible parasite.

Members of the genus Pneumocystis are fungal pathogens that cause pneumonia in a wide variety of mammals with debilitated immune systems. Little is known about their basic biological functions, including life cycle, since no species can be cultured continuously outside the mammalian lung. To better understand the pathological process, about 4500 ESTS derived from sequencing of the poly(A) tail ends of P. carinii mRNAs during fulminate infection were annotated and functionally characterized as unassembled reads, and then clustered and reduced to a unigene set with 1042 members. Because of the presence of sequences from other microbial genomes and the rat host, the analysis and compression to a unigene set was necessarily an iterative process. BLASTx analysis of the unassembled reads (UR) vs. the Uni-Prot and TREMBL databases revealed 56% had similarities to existing polypeptides at E values of

The MAT1 locus of Histoplasma capsulatum is responsive in a mating type-specific manner.

Recombination events associated with sexual replication in pathogens may generate new strains with altered virulence. Histoplasma capsulatum is a mating-competent, pathogenic fungus with two described phenotypic mating types, + and -. The mating (MAT) locus of H. capsulatum was identified to facilitate molecular studies of mating in this organism. Through syntenic analysis of the H. capsulatum genomic sequence databases, a MAT1-1 idiomorph region was identified in H. capsulatum strains G217B and WU24, and a MAT1-2 idiomorph region was identified in the strain G186AR. A mating type-specific PCR assay was developed, and two clinical isolates of opposite genotypic mating type, UH1 and VA1, were identified. A known--mating type strain, T-3-1 (ATCC 22635), was demonstrated to be of MAT1-2 genotypic mating type. The clinical isolates UH1 and VA1 were found to be mating compatible and also displayed mating-type-dependent regulation of the MAT transcription factors in response to extracts predicted to contain mating pheromones. These studies support a role for the identified MAT1 locus in determining mating type in H. capsulatum.