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bioRxiv · 10.1101/186668

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

Denham, S. T.·Verma, S.·Reynolds, R. C.·Worne, C. L.·Daugherty, J. M.·Lane, T. E.·Brown, J. C. S.
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Abstract

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.

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Denham, S. T., Verma, S., Reynolds, R. C., Worne, C. L., Daugherty, J. M., Lane, T. E., Brown, J. C. S.. 2017-09-10. Regulated release of cryptococcal polysaccharide drives virulence and suppresses immune cell infiltration into the central nervous system. https://doi.org/10.1101/186668

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