Virulence Factors Identified by Cryptococcus neoformans Mutant Screen Differentially Modulate Lung Immune Responses and Brain Dissemination

Division of Pulmonary & Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan.
American Journal Of Pathology (Impact Factor: 4.59). 07/2012; 181(4):1356-66. DOI: 10.1016/j.ajpath.2012.06.012
Source: PubMed


Deletions of cryptococcal PIK1, RUB1, and ENA1 genes independently rendered defects in yeast survival in human CSF and within macrophages. We evaluated virulence potential of these genes by comparing wild-type Cryptococcus neoformans strain H99 with deletant and complement strains in a BALB/c mouse model of pulmonary infection. Survival of infected mice; pulmonary cryptococcal growth and pathology; immunological parameters; dissemination kinetics; and CNS pathology were examined. Deletion of each PIK1, RUB1, and ENA1 differentially reduced pulmonary growth and dissemination rates of C. neoformans and extended mice survival. Furthermore, pik1Δ induced similar pathologies to H99, however, with significantly delayed onset; rub1Δ was more efficiently contained within pulmonary macrophages and was further delayed in causing CNS dissemination/pathology; whereas ena1Δ was progressively eliminated from the lungs and did not induce pathological lesions or disseminate into the CNS. The diminished virulence of mutant strains was associated with differential modulation of pulmonary immune responses, including changes in leukocyte subsets, cytokine responses, and macrophage activation status. Compared to H99 infection, mutants induced more hallmarks of a protective Th1 immune response, rather than Th2, and more classical, rather than alternative, macrophage activation. The magnitude of immunological effects precisely corresponded to the level of virulence displayed by each strain. Thus, cryptococcal PIK1, RUB1, and ENA1 differentially contribute to cryptococcal virulence, in correlation with their differential capacity to modulate immune responses.

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Available from: Michal A Olszewski,
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    • "Piehler et al. at the 9th ICCC showed that IL-4 producing Th2 cells are directly responsible for the induction of alternative macrophage activation and the pathology associated with progressive pulmonary cryptococcosis in mice (Piehler et al. poster presentations 123 and 124, 9th ICCC). These observations were complemented by studies which demonstrated that changes in cytokine environment can plastically alter macrophage polarization and, thus, their fungicidal properties (Arora et al., 2011; Davis et al., 2013; He et al., 2012) even in macrophages that were previously polarized (Davis et al., 2013). Collectively these studies demonstrated that (1) macrophages are a distal effector cell, ultimately responsible for clearance or persistence of C. neoformans, (2) cytokine microenvironment is a major factor driving macrophage polarization and the resultant macrophage fungicidal activity, and (3) manipulation of cytokine environment and/or interference with cytokine receptors on macrophages creates unique therapeutic opportunities that may be highly effective, even in advanced stages of cryptococcosis (Olszewski et al. seminar presentation S. 81 and Piehler et al. poster presentations 123 and 124, 9th ICCC). "
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    ABSTRACT: Cryptococcus neoformans and Cryptococcus gattii, the predominant etiological agents of cryptococcosis, are fungal pathogens that cause disease ranging from a mild pneumonia to life-threatening infections of the central nervous system (CNS). Resolution or exacerbation of Cryptococcus infection is determined following complex interactions of several host and pathogen derived factors. Alternatively, interactions between the host and pathogen may end in an impasse resulting in the establishment of a sub-clinical Cryptococcus infection. The current review addresses the delicate interaction between the host and Cryptococcus-derived molecules that determine resistance or susceptibility to infection. An emphasis will be placed on data highlighted at the recent 9th International Conference on Cryptococcus and Cryptococcosis (ICCC).
    Fungal Genetics and Biology 11/2014; 78. DOI:10.1016/j.fgb.2014.10.013 · 2.59 Impact Factor
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    • "For example, laccase might be immunomodulatory by catalyzing the formation of PGE2 (Erb-Downward & Huffnagle, 2007). In fact, studies with laccasedeficient yeasts (Huffnagle et al., 1995; Mednick, Nosanchuk, & Casadevall, 2005, Qiu et al., 2012) and/or urease-deficient yeasts (Osterholzer et al., 2009), among many other candidates (He et al., 2012) show that in the absence of certain enzymes there is an increased Th1-type immune response resulting in improved survival of the mouse host. However , these studies could not distinguish whether the effects were due to a direct interference with host immune system or an indirect role by hampering C. neoformans survival. "
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    ABSTRACT: Cryptococcus neoformans is a fungal pathogen that causes almost half a million deaths each year. It is believed that most humans are infected with C. neoformans, possibly in a form that survives through latency in the lung and can reactivate to cause disease if the host becomes immunosuppressed. C. neoformans has a remarkably sophisticated intracellular survival capacities yet it is a free-living fungus with no requirement for mammalian virulence whatsoever. In this review, we discuss the tools that C. neoformans possesses to achieve survival, latency and virulence within its host. Some of these tools are mechanisms to withstand starvation and others aim to protect against microbicidal molecules produced by the immune system. Furthermore, we discuss how these tools were acquired through evolutionary pressures and perhaps accidental stochastic events, all of which combined to produce an organism with an unusual and unique intracellular pathogenic strategy.
    Advances in applied microbiology 03/2014; 87:1-41. DOI:10.1016/B978-0-12-800261-2.00001-3 · 2.74 Impact Factor
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    ABSTRACT: The fungus Cryptococcus neoformans (Cn) has emerged as a major cause of meningoencephalitis worldwide. Host response to the fungus involves both innate and adaptive immunity, but fungal genes that modulate these processes are poorly understood. Previous studies demonstrated attenuated virulence of a mutant of a Virulence Associated DEAD-box protein (VAD1) in mice, despite normal growth at host temperatures, suggesting modulation of the immune response. In the present study, the Δvad1 mutant demonstrated progressive clearance from lung, and was unable to induce pathologic lesions or to cause extra-pulmonary disease, despite retained ability to grow in mouse serum and a J774.16 macrophage cell line. Pulmonary clearance occurred with a minimal cellular infiltrate, marked by reduced CD4 cells, CD11b+Ly6C(high) monocytes and F4/80+ macrophages, but retained recruitment of CD8 cells, compared to infections with wild-type fungi. Adaptive cytokine responses were reduced, including Th1, Th2 and Th17 cytokines; however, early IFN-γ and TNF-α responses were retained while non-protective IL-4 and Il-5 were diminished. Furthermore, the Δvad1 mutant was controlled in lungs despite CD4/CD8-cell depletion. These data, along with improved phagocytosis by macrophages, and increases in early/innate IL-1a, IFN-γ and chemokines elicited in the lungs within 3 days of infection with Δvad1 mutant, indicates that VAD1 expression reduces innate recognition of C. neoformans, rendering the yeast resistant to elimination by the innate mechanisms of host defense. Thus, our studies define a novel role of the cryptococcal Vad1 protein as a central regulator of cryptococcal virulence and illustrate that Vad1 promotes microbe resistance to innate host defenses.
    Infection and immunity 12/2012; 81(3). DOI:10.1128/IAI.00821-12 · 3.73 Impact Factor
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