Virulence factors identified by Cryptococcus neoformans mutant screen differentially modulate lung immune responses and brain dissemination

Abstract

Deletions of cryptococcal PIK1, RUB1, and ENA1 genes independently rendered defects in yeast survival in human CSF and within macrophages. We evaluated virulence potential of these genes by comparing wild-type Cryptococcus neoformans strain H99 with deletant and complement strains in a BALB/c mouse model of pulmonary infection. Survival of infected mice; pulmonary cryptococcal growth and pathology; immunological parameters; dissemination kinetics; and CNS pathology were examined. Deletion of each PIK1, RUB1, and ENA1 differentially reduced pulmonary growth and dissemination rates of C. neoformans and extended mice survival. Furthermore, pik1Δ induced similar pathologies to H99, however, with significantly delayed onset; rub1Δ was more efficiently contained within pulmonary macrophages and was further delayed in causing CNS dissemination/pathology; whereas ena1Δ was progressively eliminated from the lungs and did not induce pathological lesions or disseminate into the CNS. The diminished virulence of mutant strains was associated with differential modulation of pulmonary immune responses, including changes in leukocyte subsets, cytokine responses, and macrophage activation status. Compared to H99 infection, mutants induced more hallmarks of a protective Th1 immune response, rather than Th2, and more classical, rather than alternative, macrophage activation. The magnitude of immunological effects precisely corresponded to the level of virulence displayed by each strain. Thus, cryptococcal PIK1, RUB1, and ENA1 differentially contribute to cryptococcal virulence, in correlation with their differential capacity to modulate immune responses.

Figure 1

Effect of cryptococcal PIK1, RUB1, and ENA1 deletions on mice survival following pulmonary C. neoformans infection. BALB/c mice were infected intranasally with 2 × 10⁴ cells of three mutant strains, pik1Δ, rub1Δ, and ena1Δ, matching complement strains pik1Δ::PIK1, rub1Δ::RUB1, and ena1Δ::ENA1, or H99 wild-type strain. Mice were observed daily, and moribund animals were humanely euthanized and survival data recorded. Note improvement of survival time in the infected mice with pik1Δ showing the least attenuated virulence (A); rub1Δ showing intermediate virulence (B); and ena1Δ showing the most profound effect with no mortality over 60 days (C). All of the survival curves of complement strains are significantly different from their corresponding deletant strains, demonstrating that observed attenuation was gene specific for each mutant. The plots show data pulled from at least two independent experiments expressed as percentages of surviving mice out of n = 10 mice subjected to infection with each gene-deleted strain and H99, and n = 5 or above for the complements. *Significant difference in survival of mice infected with gene deletion mutant versus the wild type or the complement strain; ^†Significant difference in survival of mice infected with mutant strains compared to ena1Δ-infected mice.

Figure 2

Effect of cryptococcal PIK1, RUB1, and ENA1 deletions on pulmonary growth of C. neoformans. Lungs were harvested at days 3, 7, 14, and 21 postinfection with C. neoformans H99 (black bars in A, B, and C) or pik1Δ (A), rub1Δ (B), or ena1Δ (C) mutants, respectively. Panels B and C additionally show fungal burden in surviving mice at the conclusion of the survival study (day 60). Note that all strains showed significantly decreased fungal burden compared to H99 at all time points. Fungal burden of pik1Δ and rub1Δ were significantly higher than ena1Δ at days 14 and 21. Data are expressed as the mean colony forming units (CFU) per lung ± SEM; n = 5 mice and above per time point per group. Mutant-infected groups significantly different from *H99-, ^†ena1Δ-, and ^‡rub1Δ-infected groups at the matching time points.

Figure 3

Effect of cryptococcal PIK1, RUB1, and ENA1 deletions on development of pulmonary pathology in C. neoformans–infected mice. Lungs were collected at day 21 from H99-infected mice (A and B) and at day 60 from pik1Δ-infected (C and D), rub1Δ-infected (E and F), and ena1Δ-infected (G and H) mice and processed for histology (H&E and mucicarmine stains). The photographs were taken at ×10 and ×40 objective power. Note that lungs infected with H99 and pik1Δ show similar pathological lesions (A versus C at ×10 power, and B versus D at ×40 power), including wide-spread C. neoformans organisms in the lungs (black arrows) and tissue displacement by its rapid growth, and improved containment of the yeast within the macrophages in lungs infected with rub1Δ (E and F), including robust leukocyte infiltration (yellow arrows) with large macrophages. Note that these macrophages are heavily laden with YM1/2 crystals (red arrows) containing strongly stained fungal cells, characteristics of C. neoformans harbored in alternatively activated macrophages. Note the absence of a severe pathology or fungus in lungs infected with ena1Δ (G and H) and focused mononuclear cell infiltrates consistent with a protective immune response to C. neoformans.

Figure 4

Effect of cryptococcal PIK1, RUB1, and ENA1 deletions on CD45⁺ cells and T lymphocyte recruitment in the lungs. Leukocytes were isolated from the lungs of H99-, pik1Δ-, rub1Δ-, or ena1Δ-infected mice at week 3 postinfection and then stained with specific antibodies. A: APC-labeled anti-CD45 was used for leukocytes staining. B and C: The lymphocytes in the preselected CD45⁺ cell population were gated on forward and side scatter plots, and the CD3⁺ T cells were selected (Percp-Cy5.5); CD4⁺ and CD8⁺ subsets were identified using fluorescein isothiocyanate–labeled anti-CD8 (for Tc cells) or anti-CD4 antibodies (for Th cells) and calculated to express a total number per lung. Note that pik1Δ-, rub1Δ-, or ena1Δ-infected mice had less inflammation in the lungs compared to the H99 group; however, they show increased CD8⁺ T-cell recruitment and equally robust CD4⁺ T-cell recruitment to the infected lungs at week 3. n = 4 or more mice per group. *Significantly different from H99-infected group.

Figure 5

Effect of cryptococcal PIK1, RUB1, and ENA1 deletions on the recruitment of myeloid leukocyte subsets to C. neoformans–infected lungs. Leukocytes were isolated from the lungs of H99-, pik1Δ-, rub1Δ-, or ena1Δ-infected mice at week 3. Frequencies of each subset (left) were determined by microscopic enumeration of cytospun leukocyte isolates and calculated times the total CD45⁺ cells per lung to evaluate total number (right). Note that lungs infected with mutant strains had a significantly higher percentage of mononuclear phagocytes and lower percentages of neutrophils and eosinophils compared to H99-infected mice. All strains induced an equivalent recruitment of mononuclear cells but showed decreasing total numbers of neutrophils and eosinophils in order from highest to lowest virulence. n = 4 mice or more per group; mutant-infected groups significantly different from *H99-, ^†ena1Δ-, and ^‡rub1Δ-infected groups. Mono & Mac, mononuclear cells and macrophages.

Figure 6

Effect of cryptococcal PIK1, RUB1, and ENA1 deletion on cytokine expression by pulmonary leukocytes from C. neoformans–infected mice. RNA was isolated from pulmonary leukocytes at week 3 postinfection, converted to cDNA, and analyzed by quantitative PCR for the expression of “Th polarizing” cytokines. Cytokine mRNA expression level was normalized using a housekeeping gene (GAPDH) and expressed as percent (relative expression). Note a highly Th2-biased phenotype in the H99-infected group, a Th1-biased cytokine profile in rub1Δ- and ena1Δ-infected groups, and pik1Δ showing mixed cytokine phenotype. n = 5 of infected mice for each of the strains; mutant-infected groups significantly different from *H99, ^†ena1Δ, and ^‡rub1Δ groups.

Figure 7

Effect of cryptococcal PIK1, RUB1, and ENA1 deletion on macrophage activation profile in C. neoformans–infected lungs. mRNA was isolated from adherence-enriched pulmonary macrophages at week 3 postinfection and converted to cDNA. Arginase (ARG1) and nitric oxide synthase (iNOS) expression levels were determined as in Figure 6, and the ratio between Arg1/iNOS is shown as readout of M2 versus M1 macrophage activation status. Note the profound M2 bias in macrophages from H99-infected lungs, shift of the balance toward M1 in pik1Δ-, rub1Δ-, and ena1Δ-infected groups, with the ena1Δ group showing the most significant shift toward he M1 phenotype. n = 5 or more infected mice for each of the strains; mutant-infected groups significantly different from *H99- and ^†ena1Δ-infected groups.

Figure 8

Effect of cryptococcal PIK1, RUB1, and ENA1 deletions on the development of brain pathologies. Brains were collected at day 21 from H99-infected mice (A) and at day 60 from pik1Δ-infected (B), rub1Δ-infected (C), and ena1Δ-infected (D) mice and processed for histology (H&E and mucicarmine stains). Mice infected with pik1Δ or rub1Δ show similar pathological lesions in their brains as H99-infected mice, with large lesions in the brain containing proliferating fungi in these mice (A–C); (D) ena1Δ-infected mice without brain pathology.