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 (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.

FIG. 1.

CSF-defective ATCC mutants in a CSF survival assay. The strains were grown to saturation in YPD at 30°C and then diluted 1:10 in CSF or YPD and incubated at 37°C for 4 days. “Neat” represents the CFU concentration in CSF, and strains were diluted serially in 10-fold increments prior to being spotted onto YPD plates. CM018 is the background strain of the targeted mutant library, H99 is the clinical correlate, and the tps2Δ mutant (trehalose-6-phosphotase mutant), which does not survive in CSF at 37°C, serves as a positive control. (YPD spots are diluted from the saturated stock cultures.) Names of genes described in this study are listed in Table 2.

FIG. 2.

CSF-defective mutant strains show a wide range of virulence in a C. elegans killing assay. Fifty 1-day-old adult hermaphroditic nematodes were placed on BHI with 50 μg/ml gentamicin containing a lawn of wild-type or mutant C. neoformans. Worms were monitored for mortality twice daily. Dead nematodes were defined as nematodes that failed to respond to touch. The tps1Δ mutant is a trehalose-6-phosphate synthase mutant that exhibits virulence attenuation in C. elegans (41).

FIG. 3.

Growth defects are specific to mutants in CSF. Selected H99 mutants were grown until saturation and then diluted 1:10 in YPD, CSF, or PBS, in which they were then grown at 37°C for 4 days. Then mutants were serially diluted in 10-fold increments and spotted onto YPD plates. (A) The survival-defective phenotype is seen specifically in CSF and is not the result of nutrient starvation. (B) The ena1Δ mutant exhibits complete cell death after 36 h of incubation in CSF. (C) The rub1Δ and pik1Δ mutants show approximately 10- to 20-fold cell loss compared to WT survival at day 4; the ena1Δ mutant shows complete cell death (P < 0.01). (D) All mutants have a significant decrease in CSF survival when a decrease in H99 levels (P < 0.01) is controlled for. d4/d0, day 4/day 0 ratio; 99% CI, 99% confidence interval. (E) At day 4, survival defects were greatest in CSF compared to in BAL fluid and FBS for all mutants (**, P < 0.01). Error bars represent standard errors.

FIG. 4.

The survival factor in CSF is a small molecule. To determine the survival-inhibiting factor in CSF, we performed a series of assays to assess fungal viability. (A) Incubation in 1 M sorbitol (sorb) shows that osmotic stress is not the survival-inhibiting factor. (B) The ena1Δ mutants are not cation sensitive to 1.5 M KCl or NaCl. The BW31 strain (ena1Δ-ena4Δ nh1Δ) is an S. cerevisiae control strain. (C) Serial fractions of the CSF reveal that the survival-inhibiting factor is smaller than 3 kDa, suggesting a small peptide or unique ion composition. (D) Mutants were grown in proteinase K-treated or heat-inactivated CSF (to denature proteins in CSF). Proteinase K appeared to have no effect on the survival of the ena1Δ and rub1Δ mutants in CSF; however, heated CSF ablated the growth inhibition effect for all mutant strains.

FIG. 5.

Qualitative identification of vancomycin. (A) The measured isotopic distribution of the m/z 724.7190 ([M + 2H]²⁺) signal was overlaid with the theoretical isotope distribution of C₆₆H₇₅Cl₂N₉O₂₄ (vancomycin). (B) High-energy MS/MS spectrum of the m/z 724.7190 (M + 2H]²⁺) signal correlated with the reported MS/MS spectrum at a collision energy of 40 (reported directly from the Metlin database).

FIG. 6.

Vancomycin in heat-treated and nontreated samples. Vancomycin intensity ratios of nonboiled CSF to boiled CSF indicates an average 5.6-fold increase in nonboiled CSF, with a two-tailed t test significance value of 0.03. Intensity ratios are plotted relative to vancomycin levels in heated CSF.

FIG. 7.

Ena1, Rub1, and Pik1 mediate survival in macrophages. Activated J774A.1 macrophages were coincubated with the ena1Δ, rub1Δ, and pik1Δ mutants of C. neoformans for 60 min at 37°C in 5% CO₂. Extracellular yeasts were then removed, and cocultures were incubated overnight under the same conditions as described above. Following coincubation, macrophages were lysed and viable C. neoformans colonies were incubated on YPD plates overnight at 30°C for 2 days for CFU quantification. Strains were assayed in triplicate (P < 0.01).

FIG. 8.

In vitro CSF-specific-survival-defective mutants exhibit an attenuation of virulence in a rabbit model of meningitis. CSF-sensitive mutants were each inoculated into the subarachnoid space of rabbits (n = 3) with 3.3 × 10⁸ cells/ml. CSF was removed from each rabbit via spinal taps at the indicated times, and serial dilutions were plated onto YPD medium and incubated at 30°C for 2 days for CFU quantification. CFU was used as a measure of the burden of infection. Strains that exhibited growth defects in CSF in vitro also showed attenuated virulence in vivo. Note that no animals injected with H99 survived past 10 days of infection.

FIG. 9.

In vitro, the CSF-defective ena1Δ mutant shows severe virulence attenuation in a rabbit model of meningitis, and its reconstituted strain recovers full virulence. The ena1Δ mutant was reconstituted via a 2-step biolistic transformation with the original gene and a neo^R cassette. Strains were then grown to saturation and inoculated into CSF for 4 days at 37°C prior to being plated on YPD medium for CFU quantification. The ena1Δ mutant shows significant attenuation of infection in a rabbit model of meningitis. The ena1Δ::ENA1 mutant recovers the virulence phenotype. All rabbits infected with the reconstituted strain died on day 7 (n = 3).