Brown, J. C. S. | bioRxiv Science

bioRxiv2018

A Novel in vivo Model of Anaerobic Infection: The Investigation of Clostridium perfringens in Galleria mellonella Larvae.

ABSTRACTImportant research progress into the mechanisms of Clostridium perfringens associated diseases (CPAD) has been slowed by the lack of a reliable infection model. Wax moth larvae (Galleria mellonella) have emerged as a viable alternative to traditional mammalian organisms since they are economic, survive at 37{degrees}C and require no specialist equipment. This study aims to establish whether G. mellonella larvae can be developed as a viable model for the study of CPAD and their suitability for studying novel treatment strategies. In addition, the study demonstrates a novel time-lapse approach to data collection. Mortality and morbidity rates of larvae challenged with 105 CFU of C. perfringens isolates from various sources were observed over 72h and dose response data obtained using inoculum sizes of 10 - 105 CFU. Phenoloxidase enzyme activity was investigated as a marker for immune response and tissue burden by histopathological techniques. Results show that C. perfringens is pathogenic towards G. mellonella although potency varies between isolates. Infection activates the melanisation pathway resulting in melanin deposition but no increase in enzyme activity was observed. Efficacy of antibiotic therapy (penicillin G, bacitracin, neomycin and tetracycline) administered parenterally loosely correlates with that of in vitro analysis. The findings suggest G. mellonella can be a useful in vivo model of infection when investigating CPAD. Although they are unlikely to replace traditional mammals they may be useful as a pre-screening assay for virulence of C.perfringens strains or as a simple, cheap and rapid in vivo assay in the development and pre-clinical development of novel therapeutics.\n\nHighlightsO_LINovel in vivo model for the study of Clostridium perfringens infection.\nC_LIO_LINovel time-lapse approach to data collection.\nC_LIO_LIFirst report of the use of G. mellonella model for characterizing virulence in C. perfringens strains.\nC_LIO_LIAntibiotic therapy in the model that loosely correlates with in vitro testing.\nC_LI

microbiology↗

bioRxiv2017

Regulated release of cryptococcal polysaccharide drives virulence and suppresses immune cell infiltration into the central nervous system

Cryptococcus neoformans is a common environmental yeast and opportunistic pathogen responsible for 15% of AIDS-related deaths worldwide. Mortality primarily results from meningoencephalitis, which occurs when fungal cells disseminate from the initial pulmonary infection site and spread to the brain. A key C. neoformans virulence trait is the polysaccharide capsule. Capsule shields C. neoformans from immune-mediated recognition and destruction. The main capsule component, glucuronoxylomannan (GXM), is found both attached to the cell surface and free in the extracellular space (as exo-GXM). Exo-GXM accumulates in patient serum and cerebrospinal fluid at g/mL concentrations, has well-documented immunosuppressive properties, and correlates with poor patient outcomes. However, it is poorly understood whether exo-GXM release is regulated or the result of shedding during normal capsule turnover. We demonstrate that exo-GXM release is regulated by environmental cues and inversely correlates with surface capsule levels. We identified genes specifically involved in exo-GXM release that do not alter surface capsule thickness. The first mutant, liv7{triangleup}, released less GXM than wild-type cells when capsule is not induced. The second mutant, cnag_00658{triangleup}, released more exo-GXM under capsule-inducing conditions. Exo-GXM release observed in vitro correlated with polystyrene adherence, virulence, and fungal burden during murine infection. Additionally, we find that exo-GXM reduces cell size and capsule thickness in capsule-inducing conditions, potentially influencing dissemination. Finally, we demonstrated that exo-GXM prevents immune cell infiltration into the brain during disseminated infection and highly inflammatory intracranial infection. Our data suggest that exo-GXM performs a different role from capsule GXM during infection, altering cell size and suppressing inflammation.\n\nImportanceCryptococcus neoformans is a leading cause of life-threatening meningoencephalitis in humans. C. neoformans cells produce an immunosuppressive polysaccharide, glucuronoxylomannan (GXM), that is the main component of a protective surface capsule. GXM is also released free into extracellular space as exo-GXM, although the distinction between cell-attached GXM and exo-GXM has been unclear. Exo-GXM influences the outcome of infection, is the basis for current diagnostic tools, and has potential therapeutic applications. This study increases our basic understanding of the fungal biology that regulates polysaccharide release, suggesting that the release of cell-attached GXM and exo-GXM are distinctly regulated. We also introduce a new concept that exo-GXM may alter cell body and capsule size, thereby influencing dissemination in the host. Finally, we provide experimental evidence to confirm clinical observations that exo-GXM influences inflammation during brain infection.

microbiology↗