Cryptococcus neoformans {alpha} strains preferentially disseminate to the central nervous system during coinfection

Abstract

Cryptococcus neoformans is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (gattii, grubii, and neoformans). Each variety manifests differences in epidemiology and disease, and var. grubii strains are responsible for the vast majority of human disease. In previous studies, alpha strains were more virulent than congenic a strains in var. neoformans, whereas var. grubii congenic a and alpha strains exhibited equivalent levels of virulence. Here the role of mating type in the virulence of var. grubii was further characterized in a panel of model systems. Congenic var. grubii a and alpha strains had equivalent survival rates when cultured with amoebae, nematodes, and macrophages. No difference in virulence was observed between a and alpha congenic strains in multiple inbred-mouse genetic backgrounds, and there was no difference in accumulations in the central nervous system (CNS) late in infection. In contrast, during coinfections, a and alpha strains are equivalent in peripheral tissues but alpha cells have an enhanced predilection to penetrate the CNS. These studies reveal the first virulence difference between congenic a and alpha strains in the most common pathogenic variety and suggest an explanation for the prevalence of alpha strains in clinical isolates.

FIG. 1.

Characterization of the C. neoformans var. grubii serotype A congenic strains H99, KN99α, and KN99a. (A) Schematic diagram of the mating scheme used to produce the serotype A congenic strains. (B) Computer-generated AFLPs from the parental α strains H99 and 8-1, the parental a strains 125.91 and KNA14, the congenic-progeny strains KN99a and KN99α, and the fifth-backcross strain KN99-5a. Polymorphic products are indicated by arrows.

FIG. 2.

Survival of C. neoformans var. grubii serotype A congenic strains in heterologous hosts and macrophages. (A) Virulence of C. neoformans in Caenorhabditis elegans. C. elegans animals were allowed to feed on lawns of the a strain KN99a or the α strains KN99α and H99. The animals were examined for survival at 24-h intervals, and log rank tests showed P values of 0.2, 0.2, and 0.97 for H99/KN99a, H99/KN99α, and KN99a/KN99α, respectively. (B) Survival in the presence of Acanthamoeba castellanii. The a strain KN99a and α strains KN99α and H99 were incubated with A. castellanii or in PBS alone at 28°C. At24 and 48 h, the amoebae were lysed, diluted, and plated. Numbers of CFU were determined after incubation overnight at 30°C. P values were 0.89 for all comparisons at 24 h and 0.49, 0.89, and 0.49 at 48 h for H99/KN99a, H99/KN99α, and KN99a/KN99α, respectively. (C) Fungal survival in the presence of stimulated murine macrophages. MH-S murine alveolar macrophages were activated overnight with 50 units/ml gamma interferon and 0.3 mg/ml LPS. The activated macrophages were incubated at 37°C in 5% CO₂ with 10 μg/ml monoclonal antibody 1887 and C. neoformans strain KN99a, KN99α, or H99. After 1 h, the macrophages were washed to remove unengulfed cryptococcal cells. Numbers of CFU were determined. P values were 0.70, 1.0, and 0.40 for H99/KN99a, H99/KN99α, and KN99a/KN99α, respectively.

FIG. 3.

Virulence of C. neoformans var. grubii serotype A congenic strains H99 and KN99a in A/JCr, BALB/c, and C57BL/6 inbred-mouse lines. Animals were infected intranasally with 5 × 10⁴ cells, and progression to severe morbidity was monitored. P values were 0.35, 0.47, and 0.66 for H99AJ/KN99aAJ, H99BALBc/KN99aBALBc, and H99C57BL6/KN99aC57BL6, respectively. P values were 0.008, 0.002, 0.004, 0.009, 0.002, and 0.008 for H99AJ/H99BALBc, H99AJ/H99C57BL6, H99BALBc/H99C57BL6, KN99aAJ/KN99aBALBc, KN99aAJ/KN99aC57BL6, and KN99aBALBc/KN99aC57BL6, respectively.

FIG. 4.

Tissue burden cultures of C. neoformans var. grubii serotype A congenic strains H99 and KN99a. Mice (A/JCr) were infected with 5 × 10⁴ cells intranasally. Organs were extracted and homogenized, and serial dilutions were plated for tissue burden cultures at 7, 14, and 21 days postinfection. The number of yeast cells present is expressed as the geometric mean of the number of CFU per gram of lungs, spleen, or brain. Open circles denote values from individual animals, and the height of each bar represents the geometric mean.

FIG. 5.

C. neoformans var. grubii α strains predominate in the brain during intranasal coinfection with congenic strains. Mice (A/JCr) were inoculated with an approximately 1:1 ratio of (A) KN99aNAT and KN99α or (B) KN99aNAT and KN99a intranasally at a final concentration of 5 × 10⁴ cells. The actual proportion of a cells in the infecting inoculum was determined by growth on selective medium and is plotted as a horizontal dotted line. At 21 days postinoculation, animals were sacrificed. The lungs, brain, and spleen from each animal were homogenized, and serial dilutions were plated. More than 500 colonies per organ were isolated from each animal and assayed for NAT resistance to determine mating type. The proportion of a cells present in each organ is plotted, with open circles denoting values from individual animals and the height of each bar representing the geometric mean.

FIG. 6.

C. neoformans var. grubii intracerebral infections show equivalent levels of growth of congenic strains. Mice (A/JCr) were inoculated intracerebrally with either KN99a or KN99α or coinfected with an approximately 1:1 ratio of KN99aNAT and KN99α at a final concentration of 1.5 × 10³ cells. The actual proportion of a cells in the coinfecting inoculum was determined by growth on selective medium and is plotted as a horizontal dotted line in panel B. At 4 days postinoculation, animals were sacrificed. The brain from each animal was extracted and homogenized. Serial dilutions were plated for tissue burden cultures (A). The number of yeast cells present is expressed as the geometric mean of numbers of CFU per gram of tissue. Open circles denote values from individual animals, and the height of each bar represents the geometric mean. (B) For coinfection experiments, >500 colonies per organ were isolated from each animal and assayed for NAT resistance to determine mating type. The proportion of a cells present in each organ is plotted, with open circles denoting values from individual animals and the height of each bar representing the geometric mean.

FIG. 7.

Proposed models for the role of mating type in the enhanced virulence of α cells in the CNS. (A) Fungal-fungal interactions result in changes in virulence factors; (B) fungal-fungal interactions result in morphological changes which affect virulence; (C) host-fungal interactions result in changes in the virulence potential of the fungus. MF, mating factor/pheromone.