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Background: Protective immune responses to Cryptosporidium parvum, a zoonotic, gastrointestinal parasite, are primarily dependent on the presence of interferon-gamma (IFNγ). We discovered that treatment with soluble T. gondii antigen (STAg) reduces Cryptosporidium parvum shedding in the absence of IFNγ. To identify the protective IFNγ independent responses elicited by STAg, we conducted a transcriptomic analysis of intestinal sections of IFNγ-deleted, C. parvum infected or uninfected mice treated with STAg or PBS. Results: STAg treatment reduced oocyst shedding in C. parvum infected IFNγ deleted mice. Gene ontology analysis of the intestinal transcriptomes suggested that both C. parvum infection and STAg treatment changes the transcript abundance of genes involved in the host cell membrane, intracellular and extracellular transport, and immune responses. We found in high abundance 37 genes related to IFN type I response in infected mice treated with STAg. Among these genes, members of the oligoadenylate synthetase and Schlafen family were identified. Conclusions: STAg treatment of C. parvum infected mice induced both host immune and metabolism changes associated with a reduction in shedding. Several components of the type I interferon immune response were more abundant in the ileum of C. parvum infected in IFNγ-deleted mice. Future studies will explore the role of type I IFN mediated immune responses in controlling C. parvum infections. STAg treatment appears to only affects the host transcriptome while the parasite transcriptome remains unaffected. Several C. parvum genes, including mucin genes, are more abundant during infection of animals, which opens new avenues in C. parvum research.

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 β-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.

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 β-integrin family of phagocytic receptors to promote entry into macrophages. In addition, H. capsulatum yeasts conceal immunostimulatory β-glucans to avoid triggering signaling receptors such as the β-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.

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 β-(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 β-glucans, which results in enhanced binding to the Dectin-1 β-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 β-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 β-glucan exposure: α-glucan provides a masking function by covering the β-glucan-rich cell wall, while Eng1 removes any remaining exposed β-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed β-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 β-glucanase, Eng1, which reduces recognition of fungal cell wall β-glucans. We demonstrated that the Eng1 β-glucanase promotes Histoplasma virulence by reducing levels of surface-exposed β-glucans on yeast cells, thereby enabling Histoplasma yeasts to escape detection by the host β-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 β-glucans by α-glucan and enzymatic removal of exposed β-glucans by the Eng1 β-glucanase.

Polysorbate 80 (PS80) is a nonionic surfactant and detergent that inhibits biofilm formation by Pseudomonas aeruginosa at concentrations as low as 0.001% and is well tolerated in human tissues. However, certain clinical and laboratory strains (PAO1) of P. aeruginosa are able to form biofilms in the presence of PS80. To better understand this resistance, we performed transposon mutagenesis with a PS80-resistant clinical isolate, PA738. This revealed that mutation of algC rendered PA738 sensitive to PS80 biofilm inhibition. AlgC contributes to the biosynthesis of the exopolysaccharides Psl and alginate, as well as lipopolysaccharide and rhamnolipid. Analysis of mutations downstream of AlgC in these biosynthetic pathways established that disruption of the psl operon was sufficient to render the PA738 and PAO1 strains sensitive to PS80-mediated biofilm inhibition. Increased levels of Psl production in the presence of arabinose in a strain with an arabinose-inducible psl promoter were correlated with increased biofilm formation in PS80. In P. aeruginosa strains MJK8 and ZK2870, known to produce both Pel and Psl, disruption of genes in the psl but not the pel operon conferred susceptibility to PS80-mediated biofilm inhibition. The laboratory strain PA14 does not produce Psl and does not form biofilms in PS80. However, when PA14 was transformed with a cosmid containing the psl operon, it formed biofilms in the presence of PS80. Taken together, these data suggest that production of the exopolysaccharide Psl by P. aeruginosa promotes resistance to the biofilm inhibitor PS80.