No abstract

Our understanding of normal immunity to opportunistic fungal infection is limited by a lack of species-specific commensal/host/pathogen models. There are many species of opportunistic fungal pathogens (such as Candida spp. and Cryptococcus spp.) which exist as commensal organisms or are common in our environment. Therefore, we sought to develop a model of commensal opportunistic infection within zebrafish by identification of a normal commensal fungus that could act as an opportunistic pathogen. Using dissections of D. rerio guts we isolated a monoculture of a commensal yeast. The strain was identified as Candida famata via DNA sequencing and phenotypic analysis (e.g. halotolerance). C. famata was isolated from six individual animals and three strains (DJ1-3) have been deposited in the CBS collection. C. famata is a common commensal of animals, including humans and is a rare opportunistic pathogen. Our DJ1 strain was thermotolerant and grows at mammalian body temperature. We found it was easily phagocytosed by mouse macrophages, in some cases to the extent of host cell destruction. Infection of zebrafish larval model of opportunistic infection demonstrated C. famata could act as an opportunistic pathogen of its commensal host. Thus, we have characterised the first commensal fungus to be isolated from zebrafish and demonstrated that it may be a potential opportunistic pathogen, as in humans. The study of C. famata in zebrafish therefore represents an unparalleled opportunity to understand the evolution of fungal immunology and the switch between commensal and pathogen.

Cryptococcus neoformans causes life-threatening infection in the immunocompromised. This and other opportunistic pathogens are an increasing threat as immunosuppression increases globally. To counter antibiotic resistance, there is precedent for developing immune enhancing therapy. However, our understanding of how immunocompetent patients resolve these infections is poor as opportunistic infections typically resolve subclinically. Because this has led to a lack of clinical data, we rely on animal models. Current in vivo infection models either lack mammalian immunity or are not compatible with long term high content imaging required to model the complexities of human host-pathogen interactions. Therefore, we have developed an ex vivo murine precision cut lung slice (PCLS) model to understand innate immunity in cryptococcosis. C57BL/6 mice were sacrificed 0 or 24 hours post infection with KN99α cryptococci. Lungs were inflated with 37 o C agarose, 300μm thick PCLS were prepared on a vibratome and imaged by confocal or wide-field fluorescence microscopy.Using PCLS and immunofluorescence, we demonstrate cryptococcal replication and clearance rates are balanced over the first 24 hours of infection. Cell-mediated immunity is alveolar macrophage centric, although alveolar macrophages demonstrate limited phagocytosis of cryptococci and enable intracellular cryptococcal replication. Cryptococcus neoformans responded to the lung environment by forming enlarged cells, although these were not large enough to be titan cells. To further understand cryptococcal proliferation in vivo, we also infected animals with plb1 mutant Cryptococcus neoformans that has been shown to exhibit proliferation defects in vivo. We found no difference in fungal burden with plb1 infected animals 24 hours post infection, but observed significantly larger fungal cells and no incidences of phagocytosis. Thus, the PCLS model can be used to assess the lung immune response early in cryptococcal infection, demonstrating that resident lung macrophages cannot control cryptococcal infection and offer an intracellular niche for Cryptococcus neoformans growth.