Survival Defects of iCryptococcus neoformans/i Mutants Exposed to Human Cerebrospinal Fluid Result in Attenuated Virulence in an Experimental Model of Meningitis

Cryptococcus neoformans is a fungal pathogen that encounters various microenvironments during growth in the mammalian host, including intracellular vacuoles, blood, and cerebrospinal fluid (CSF). Because the CSF is isolated by the blood-brain barrier, we hypothesize that CSF presents unique stresses that C. neoformans must overcome to establish an infection. We assayed 1,201 mutants for survival defects in growth media, saline, and human CSF. We assessed CSF-specific mutants for (i) mutant survival in both human bronchoalveolar lavage (BAL) fluid and fetal bovine serum (FBS), (ii) survival in macrophages, and (iii) virulence using both Caenorhabditis elegans and rabbit models of cryptococcosis. Thirteen mutants exhibited significant survival defects unique to CSF. The mutations of three of these mutants were recreated in the clinical serotype A strain H99: deletions of the genes for a cation ATPase transporter ( ena1Δ ), a putative NEDD8 ubiquitin-like protein ( rub1Δ ), and a phosphatidylinositol 4-kinase ( pik1Δ ). Mutant survival rates in yeast media, saline, and BAL fluid were similar to those of the wild type; however, survival in FBS was reduced but not to the levels in CSF. These mutant strains also exhibited decreased intracellular survival in macrophages, various degrees of virulence in nematodes, and severe attenuation of survival in a rabbit meningitis model. We analyzed the CSF by mass spectrometry for candidate compounds responsible for the survival defect. Our findings indicate that the genes required for C. neoformans survival in CSF ex vivo are necessary for survival and infection in this unique host environment.

Cryptococcus neoformans is a basidiomycete fungal pathogen that frequently invades the central nervous system, causing meningoencephalitis in severely immunocompromised patients. Among these patients, such as those with HIV or solid-organ transplants, cryptococcosis results in one of the largest burdens of mortality and morbidity (10). There are now over 1 million cases of cryptococcosis worldwide, with estimated deaths of 700,000 per year (38). However, recent outbreaks of cryptococcosis by a newly identified genotype of Cryptococcus gattii (a sister species of C. neoformans ) in the Pacific Northwest have emphasized that apparently immunocompetent individuals are susceptible to this disease as well (17, 18, 32, 48).

Although the pathobiology of cryptococcosis within each body site is not fully understood, the ability of C. neoformans to survive in the cerebrospinal fluid (CSF) is critical for its production of life-threatening disease in the central nervous system. CSF performs a variety of essential functions: cushions the brain from physical impact, transports nutrients and wastes to and from cells, and circulates various signaling molecules, such as regulatory peptides (29). Due to the relative isolation of the CSF by the blood-brain and blood-CSF barriers, recent proteomic and metabolomic analyses support the hypothesis that the CSF is a unique environment compared to other body fluids and sites, such as the alveolar spaces, intracellular vacuoles, and blood, that C. neoformans encounters in the course of an infection (55, 57, 60).

Three well-established virulence factors essential for cryptococcal disease have been identified, namely, the production of a polysaccharide capsule, melanin, and survival at 37°C. Many genes controlling these phenotypes have also been identified (40). The use of random insertion mutagenesis has led to the discovery of many essential virulence-associated genes, such as CNA1 , VPH1 , and CLC1 (14, 26, 27, 59), and site-directed techniques have revealed the importance of urease and phospholipase genes for virulence (12, 13). Furthermore, the recent completion of the C. neoformans var. grubii genome presents an exciting opportunity for accelerated discovery of genes encoding virulence factors through the use of targeted genetic screens. With these genomic tools, forward genetics can be a powerful strategy to better understand how C. neoformans copes with various microenvironments within the host.

We hypothesized that the CSF presents unique stresses to C. neoformans in the host and that determination of specific factors required for survival in CSF could shed light on novel disease-producing mechanisms. Using a library of signature-tagged, site-directed mutant strains, we employed a forward genetics screen to assay for mutant-cell survival defects in human CSF. A series of mutants that displayed significant deficiencies in survival when exposed to CSF were found. Extensive characterization of these mutants suggests that CSF presents a unique stress composite to this yeast, and the inability of certain C. neoformans mutants to survive CSF exposure in vitro correlates with attenuated virulence in the mammalian host.