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

Abstract

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 (ena1⌬), a putative NEDD8 ubiquitin-like protein (rub1⌬), and a phosphatidylinositol 4-kinase (pik1⌬). 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.

The Cryptococcus neoformans Rim101 Transcription Factor Directly Regulates Genes Required for Adaptation to the Host

“…The CDA1 gene regulates chitosan levels in the cell, and KRE6 is involved in ␤-glucan synthesis, consistent with the role of Rim101 in the regulation of cell wall remodeling. ENA1 is also required for full proliferation within the cerebrospinal fluid (CSF), although we have not observed a difference in neurotropism or proliferation in the CSF between the rim101⌬ mutant and the wild type (67). Interestingly, the wild-type strain expressed more CTR4 transcripts than the rim101⌬ strain under both in vitro growth conditions but 2.5-fold fewer in the context of the host lung.…”

Chronological Aging Is Associated with Biophysical and Chemical Changes in the Capsule of Cryptococcus neoformans

“…These broad structural changes were associated with altered antibody reactivity to the PS capsule and complement deposition and an increased resistance to complement-mediated phagocytosis by macrophages. Overall, our results demonstrate that the C. neoformans PS capsule is highly dynamic and can undergo substantial structural and physicochemical transformations upon prolonged stationary-phase growth, which mimics some of the growth conditions that C. neoformans cells encounter during chronic infection on cerebrospinal fluid of patients, which is not a rich growth medium and does not support logarithmic growth (29). …”

Efficient phagocytosis and laccase activity affect the outcome of HIV-associated cryptococcosis

“…Cultures were then incubated at 37°C for 96 hours with shaking at 150 rpm. Aliquots were collected at time point 0 and subsequently at 12, 24, 36, 72, and 96 hours after inoculation and plated on SDA for CFU counts (42). In parallel, 1% saline at a different pH, PBS, and SDB were inoculated and monitored as pH, nutrient-deplete and nutrient-rich growth media controls, respectively.…”