Dual Transcriptomics To Determine Gamma Interferon-Independent Host Response to Intestinal Cryptosporidium parvum Infection.

Animals with a chronic infection of the parasite Toxoplasma gondii are protected against lethal secondary infection with other pathogens. Our group previously determined that soluble T. gondii antigens (STAg) can mimic this protection and be used as a treatment against several lethal pathogens. Because treatments are limited for the parasite Cryptosporidium parvum, we tested STAg as a C. parvum therapeutic. We determined that STAg treatment reduced C. parvum Iowa II oocyst shedding in gamma interferon knockout (IFN-γ-KO) mice. Murine intestinal sections were then sequenced to define the IFN-γ-independent transcriptomic response to C. parvum infection. Gene Ontology and transcript abundance comparisons showed host immune response and metabolism changes. Transcripts for type I interferon-responsive genes were more abundant in C. parvum-infected mice treated with STAg. Comparisons between phosphate-buffered saline (PBS) and STAg treatments showed no significant differences in C. parvum gene expression. C. parvum transcript abundance was highest in the ileum and mucin-like glycoproteins and the GDP-fucose transporter were among the most abundant. These results will assist the field in determining both host- and parasite-directed future therapeutic targets.

Proteomic and transcriptomic analyses of early and late-chronic Toxoplasma gondii infection shows novel and stage specific transcripts.

BACKGROUND: The protozoan pathogen Toxoplasma gondii has the unique ability to develop a chronic infection in the brain of its host by transitioning from the fast growing tachyzoite morphology to latent bradyzoite morphology. A hallmark of the bradyzoite is the development of neuronal cysts that are resilient against host immune response and current therapeutics. The bradyzoite parasites within the cyst have a carbohydrate and protein-rich wall and a slow-replication cycle, allowing them to remain hidden from the host. The intracellular, encysted lifestyle of T. gondii has made them recalcitrant to molecular analysis in vivo. RESULTS: Here, we detail the results from transcriptional and proteomic analyses of bradyzoite-enriched fractions isolated from mouse brains infected with T. gondii over a time course of 21 to 150 days. The enrichment procedure afforded consistent identification of over 2000 parasitic peptides from the mixed-organism sample, representing 366 T. gondii proteins at 28, 90, and 120 day timepoints. Deep sequencing of transcripts expressed during these three timepoints revealed that a subpopulation of genes that are transcriptionally expressed at a high level. Approximately one-third of these transcripts are more enriched during bradyzoite conditions compared to tachyzoites and approximately half are expressed at similar levels during each phase. The T. gondii transcript which increased the most over the course of chronic infection, sporoAMA1, shows stage specific isoform expression of the gene. CONCLUSIONS: We have expanded the transcriptional profile of in vivo bradyzoites to 120 days post-infection and provided the first in vivo proteomic profile of T. gondii bradyzoites. The RNA sequencing depth of in vivo bradyzoite T. gondii was over 250-fold greater than previous reports and allowed us to identify low level transcripts and a novel bradyzoite-specific isoform of sporoAMA1.

Transcriptional Analysis Shows a Robust Host Response to Toxoplasma gondii during Early and Late Chronic Infection in Both Male and Female Mice.

The long-term host effects caused by the protozoan parasite Toxoplasma gondii are poorly understood. High-throughput RNA sequencing analysis previously determined that the host response in the brain was greater and more complex at 28 days than at 10 days postinfection. Here, we analyzed the host transcriptional profile of age- and sex-matched mice during very early (21 days), early (28 days), mid (3 months), and late (6 months) chronic infection. We found that a majority of the host genes which increase in abundance at day 21 postinfection are still increased at 6 months postinfection for both male and female mice. While most of the differentially expressed genes were similar between sexes, females had far fewer genes that were significantly less abundant, which may have led to the slightly increased cyst burden in males. Transcripts for C-X-C motif chemokine ligand 13 and a C-C motif chemokine receptor 2 (CCR2) were significantly higher in females than in males during infection. As T. gondii chronic infection and profilin (PRF) confer resistance to Listeria monocytogenes infection in a CCR2-dependent manner, the differences in CCR2 expression led us to retest the protection of PRF in both sexes. Male mice were nearly as effective as female mice at reducing the bacterial burden either with a chronic infection or when treated with PRF. These data show that most of the host genes differentially expressed in response to T. gondii infection are similar between males and females. While differences in transcript abundance exist between the sexes, the infection phenotypes tested here did not show significant differences.

O-Mannosylation of Proteins Enables Histoplasma Yeast Survival at Mammalian Body Temperatures.

The ability to grow at mammalian body temperatures is critical for pathogen infection of humans. For the thermally dimorphic fungal pathogen Histoplasma capsulatum, elevated temperature is required for differentiation of mycelia or conidia into yeast cells, a step critical for invasion and replication within phagocytic immune cells. Posttranslational glycosylation of extracellular proteins characterizes factors produced by the pathogenic yeast cells but not those of avirulent mycelia, correlating glycosylation with infection. Histoplasma yeast cells lacking the Pmt1 and Pmt2 protein mannosyltransferases, which catalyze O-linked mannosylation of proteins, are severely attenuated during infection of mammalian hosts. Cells lacking Pmt2 have altered surface characteristics that increase recognition of yeast cells by the macrophage mannose receptor and reduce recognition by the ß-glucan receptor Dectin-1. Despite these changes, yeast cells lacking these factors still associate with and survive within phagocytes. Depletion of macrophages or neutrophils in vivo does not recover the virulence of the mutant yeast cells. We show that yeast cells lacking Pmt functions are more sensitive to thermal stress in vitro and consequently are unable to productively infect mice, even in the absence of fever. Treatment of mice with cyclophosphamide reduces the normal core body temperature of mice, and this decrease is sufficient to restore the infectivity of O-mannosylation-deficient yeast cells. These findings demonstrate that O-mannosylation of proteins increases the thermotolerance of Histoplasma yeast cells, which facilitates infection of mammalian hosts.IMPORTANCE For dimorphic fungal pathogens, mammalian body temperature can have contrasting roles. Mammalian body temperature induces differentiation of the fungal pathogen Histoplasma capsulatum into a pathogenic state characterized by infection of host phagocytes. On the other hand, elevated temperatures represent a significant barrier to infection by many microbes. By functionally characterizing cells lacking O-linked mannosylation enzymes, we show that protein mannosylation confers thermotolerance on H. capsulatum, enabling infection of mammalian hosts.

Eng1 and Exg8 Are the Major ß-Glucanases Secreted by the Fungal Pathogen Histoplasma capsulatum.

Fungal cell walls contain ß-glucan polysaccharides that stimulate immune responses when recognized by host immune cells. The fungal pathogen Histoplasma capsulatum minimizes detection of ß-glucan by host cells through at least two mechanisms: concealment of ß-glucans beneath α-glucans and enzymatic removal of any exposed ß-glucan polysaccharides by the secreted glucanase Eng1. Histoplasma yeasts also secrete the putative glucanase Exg8, which may serve a similar role as Eng1 in removing exposed ß-glucans from the yeast cell surface. Here, we characterize the enzymatic specificity of the Eng1 and Exg8 proteins and show that Exg8 is an exo-ß1,3-glucanase and Eng1 is an endo-ß1,3-glucanase. Together, Eng1 and Exg8 account for nearly all of the total secreted glucanase activity of Histoplasma yeasts. Both Eng1 and Exg8 proteins are secreted through a conventional secretion signal and are modified post-translationally by O-linked glycosylation. Both glucanases have near maximal activity at temperature and pH conditions experienced during infection of host cells, supporting roles in Histoplasma pathogenesis. Exg8 has a higher specific activity than Eng1 for ß1,3-glucans; yet despite this, Exg8 does not reduce detection of yeasts by the host ß-glucan receptor Dectin-1. Exg8 is largely dispensable for virulence in vivo, in contrast to Eng1. These results show that Histoplasma yeasts secrete two ß1,3-glucanases and that Eng1 endoglucanase activity is the predominant factor responsible for removal of exposed cell wall ß-glucans to minimize host detection of Histoplasma yeasts.

The Eng1 ß-Glucanase Enhances Histoplasma Virulence by Reducing ß-Glucan Exposure.

UNLABELLED: The fungal pathogen Histoplasma capsulatum parasitizes host phagocytes. To avoid antimicrobial immune responses, Histoplasma yeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. Pathogenic Histoplasma yeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show that Histoplasma Eng1 is a glucanase that hydrolyzes ß-(1,3)-glycosyl linkages but is not required for Histoplasma growth in vitro or for cell separation. However, Histoplasma yeasts lacking Eng1 function have attenuated virulence in vivo, particularly during the cell-mediated immunity stage. Histoplasma yeasts deficient for Eng1 show increased exposure of cell wall ß-glucans, which results in enhanced binding to the Dectin-1 ß-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed ß-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In α-glucan-producing Histoplasma strains, Eng1 acts in concert with α-glucan to minimize ß-glucan exposure: α-glucan provides a masking function by covering the ß-glucan-rich cell wall, while Eng1 removes any remaining exposed ß-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed ß-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes. IMPORTANCE: The success of Histoplasma capsulatum as an intracellular pathogen results, in part, from an ability to minimize its detection by receptors on phagocytic cells of the immune system. In this study, we showed that Histoplasma pathogenic yeast cells, but not avirulent mycelia, secrete a ß-glucanase, Eng1, which reduces recognition of fungal cell wall ß-glucans. We demonstrated that the Eng1 ß-glucanase promotes Histoplasma virulence by reducing levels of surface-exposed ß-glucans on yeast cells, thereby enabling Histoplasma yeasts to escape detection by the host ß-glucan receptor, Dectin-1. As a consequence, phagocyte recognition of Histoplasma yeasts is reduced, leading to less proinflammatory cytokine production by phagocytes and less control of Histoplasma infection in vivo Thus, Histoplasma yeasts express two mechanisms to avoid phagocyte detection: masking of cell wall ß-glucans by α-glucan and enzymatic removal of exposed ß-glucans by the Eng1 ß-glucanase.

Histoplasma capsulatum surmounts obstacles to intracellular pathogenesis.

The fungal pathogen Histoplasma capsulatum causes respiratory and disseminated disease, even in immunocompetent hosts. In contrast to opportunistic pathogens, which are readily controlled by phagocytic cells, H. capsulatum yeasts are able to infect macrophages, survive antimicrobial defenses, and proliferate as an intracellular pathogen. In this review, we discuss some of the molecular mechanisms that enable H. capsulatum yeasts to overcome obstacles to intracellular pathogenesis. H. capsulatum yeasts gain refuge from extracellular obstacles such as antimicrobial lung surfactant proteins by engaging the ß-integrin family of phagocytic receptors to promote entry into macrophages. In addition, H. capsulatum yeasts conceal immunostimulatory ß-glucans to avoid triggering signaling receptors such as the ß-glucan receptor Dectin-1. H. capsulatum yeasts counteract phagocyte-produced reactive oxygen species by expression of oxidative stress defense enzymes including an extracellular superoxide dismutase and an extracellular catalase. Within the phagosome, H. capsulatum yeasts block phagosome acidification, acquire essential metals such as iron and zinc, and utilize de novo biosynthesis pathways to overcome nutritional limitations. These mechanisms explain how H. capsulatum yeasts avoid and negate macrophage defense strategies and establish a hospitable intracellular niche, making H. capsulatum a successful intracellular pathogen of macrophages.

Histoplasma capsulatum depends on de novo vitamin biosynthesis for intraphagosomal proliferation.

During infection of the mammalian host, Histoplasma capsulatum yeasts survive and reside within macrophages of the immune system. Whereas some intracellular pathogens escape into the host cytosol, Histoplasma yeasts remain within the macrophage phagosome. This intracellular Histoplasma-containing compartment imposes nutritional challenges for yeast growth and replication. We identified and annotated vitamin synthesis pathways encoded in the Histoplasma genome and confirmed by growth in minimal medium that Histoplasma yeasts can synthesize all essential vitamins with the exception of thiamine. Riboflavin, pantothenate, and biotin auxotrophs of Histoplasma were generated to probe whether these vitamins are available to intracellular yeasts. Disruption of the RIB2 gene (riboflavin biosynthesis) prevented growth and proliferation of yeasts in macrophages and severely attenuated Histoplasma virulence in a murine model of respiratory histoplasmosis. Rib2-deficient yeasts were not cleared from lung tissue but persisted, consistent with functional survival mechanisms but inability to replicate in vivo. In addition, depletion of Pan6 (pantothenate biosynthesis) but not Bio2 function (biotin synthesis) also impaired Histoplasma virulence. These results indicate that the Histoplasma-containing phagosome is limiting for riboflavin and pantothenate and that Histoplasma virulence requires de novo synthesis of these cofactor precursors. Since mammalian hosts do not rely on vitamin synthesis but instead acquire essential vitamins through diet, vitamin synthesis pathways represent druggable targets for therapeutics.