Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the acrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed

Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of Cryptococcus neoformans

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication

Role of ficolin-A and lectin complement pathway in the innate defense against pathogenic Aspergillus species

Aspergillus species are saprophytic molds causing life-threatening invasive fungal infections in the immunocompromised host. Innate immune recognition, in particular, the mechanisms of opsonization and complement activation, has been reported to be an integral part of the defense against fungi. We have shown that the complement component ficolin-A significantly binds to Aspergillus conidia and hyphae in a concentration-dependent manner and was inhibited by N-acetylglucosamine and N-acetylgalactosamine. Calcium-independent binding to Aspergillus fumigatus and A. terreus was observed, but binding to A. flavus and A. niger was calcium dependent. Ficolin-A binding to conidia was increased under low-pH conditions, and opsonization led to enhanced binding of conidia to A549 airway epithelial cells. In investigations of the lectin pathway of complement activation, ficolin-A-opsonized conidia did not lead to lectin pathway-specific C4 deposition. In contrast, the collectin mannose binding lectin C (MBL-C) but not MBL-A led to efficient lectin pathway activation on A. fumigatus in the absence of ficolin-A. In addition, ficolin-A opsonization led to a modulation of the proinflammatory cytokine interleukin-8. We conclude that ficolin-A may play an important role in the innate defense against Aspergillus by opsonizing conidia, immobilizing this fungus through enhanced adherence to epithelial cells and modulation of inflammation. However, it appears that other immune pattern recognition molecules, i.e., those of the collectin MBL-C, are involved in the Aspergillus-lectin complement pathway activation rather than ficolin-A

Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak

Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae, the cause of leprosy(1). In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii(2). However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii(3). Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a - alpha sexual cycle, the majority outbreak clone appears to have descended from two alpha mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms