The Mouse Inhalation Model of Cryptococcus neoformans Infection Recapitulates Strain Virulence in Humans and Shows that Closely Related Strains Can Possess Differential Virulence

Abstract

Cryptococcal meningitis (CM) causes high rates of HIV-related mortality, yet the Cryptococcus factors influencing patient outcome are not well understood. Pathogen-specific traits, such as the strain genotype and degree of antigen shedding, are associated with the clinical outcome, but the underlying biology remains elusive. In this study, we examined factors determining disease outcome in HIV-infected cryptococcal meningitis patients infected with Cryptococcus neoformans strains with the same multilocus sequence type (MLST). Both patient mortality and survival were observed during infections with the same sequence type. Disease outcome was not associated with the patient CD4 count. Patient mortality was associated with higher cryptococcal antigen levels, the cerebrospinal fluid (CSF) fungal burden by quantitative culture, and low CSF fungal clearance. The virulence of a subset of clinical strains with the same sequence type was analyzed using a mouse inhalation model of cryptococcosis. We showed a strong association between human and mouse mortality rates, demonstrating that the mouse inhalation model recapitulates human infection. Similar to human infection, the ability to multiply in vivo, demonstrated by a high fungal burden in lung and brain tissues, was associated with mouse mortality. Mouse survival time was not associated with single C. neoformans virulence factors in vitro or in vivo; rather, a trend in survival time correlated with a suite of traits. These observations show that MLST-derived genotype similarities between C. neoformans strains do not necessarily translate into similar virulence either in the mouse model or in human patients. In addition, our results show that in vitro assays do not fully reproduce in vivo conditions that influence C. neoformans virulence.

Keywords: Cryptococcus; Cryptococcus neoformans; cryptococcosis; human; meningitis; model; mouse; pathogenesis; sequence type; virulence.

FIG 1

C. neoformans sequence types and human mortality. C. neoformans clinical strains were isolated from the CSF of HIV-infected patients with CM, grown on YPD agar medium at 30°C, and stored at −80°C before their sequence types were identified. (A) Cryptococcus strains from all participants have different sequence types and can be classified as early- versus late-mortality strains. (B) To test the association between human and mouse infections, 11 strains with various sequence types and different degrees of human mortality were chosen to be tested in the mouse model of cryptococcosis. The sequence type and human mortality for this subset of strains are presented. Early mortality indicates that the patient died within 10 weeks from the time of diagnosis, late mortality indicates that the patient died after 10 weeks, and survival indicates that the patient survived the infection. Unique ST, sequence types infecting only one patient.

FIG 2

Human mortality is not determined by patient parameters. The clinical outcome was compared to baseline CM patient characteristics. (A to C) Data from individual participants (circles); (D to F) patient survival, classified as died (mortality by 10 weeks postdiagnosis) or lived (survival past 10 weeks postdiagnosis). (A, D) CD4⁺ cell counts (for all participants, 36 died and 70 lived; for ST93-infected patients, 20 died and 30 lived). (B, E) White blood cell counts in the CSF (for all participants, 34 died and 68 lived; for ST93-infected patients, 18 died and 29 lived). (C, F) HIV viral load (for all participants, 35 died and 70 lived; for ST93-infected patients, 20 died and 30 lived). Error bars represent the standard error of the mean. Patients who died and those who survived the infection were compared by the Mann-Whitney test. ***, P < 0.001. CD4 cells, CD4⁺ T helper cells; CM, cryptococcal meningitis; CSF, cerebrospinal fluid; HIV, human immunodeficiency virus; ST93, sequence type 93; WBC, white blood cells.

FIG 3

Patient survival is partly associated with fungal parameters. The clinical outcome was compared to fungal parameters. (A to C) Data from individual participants (circles); (D to F) patient survival classified as died (mortality by 10 weeks postdiagnosis) or lived (survival past 10 weeks postdiagnosis). (A, D) CSF fungal burden determined by enumeration of CFU (for all participants, 36 died and 70 lived; for ST93-infected patients, 20 died and 30 lived). (B, E) Cryptococcal antigen titers in the CSF (for all participants, 31 died and 55 lived; for ST93-infected patients, 16 died and 23 lived). (C, F) Rate of fungal clearance determined by the early fungicidal activity (for all participants, 32 died and 69 lived; for ST93-infected patients, 18 died and 29 lived). Patients who died and those who survived the infection were compared by Student’s t test or the Mann-Whitney test. *, P < 0.05; **, P < 0.01. Error bars represent the standard error of the mean. CSF, cerebrospinal fluid; CrAg LFA, cryptococcal antigen lateral flow assay; EFA, early fungicidal activity; ST93, sequence type 93.

FIG 4

C. neoformans clinical strains show differential virulence in mice. Groups of 10 6- to 8-week-old A/J mice were infected intranasally with 5 × 10⁴ cells from C. neoformans clinical strains isolated from the CSF of HIV-infected patients with CM. Progression to severe morbidity was monitored for 150 days, and mice were sacrificed when the endpoint criteria were reached. (A) High-virulence strains; (B) strains with intermediate virulence; (C) low-virulence strains.

FIG 5

Disease outcome in humans is associated with disease outcome in mice. The clinical outcome in human participants was compared to the infection outcome in mice infected with the same C. neoformans strain. (A) Correlation between human and mouse survival for all 10 clinical strains. (B) Correlation between human and mouse survival when outliers (the SACl010 and UgCl552 strains) were removed. *, the SACl010 strain was associated with low virulence in mice, but the human participant died of IRIS at 41 days postdiagnosis; #, the participant infected with UgCl552 died at home after clearing the initial C. neoformans infection. The cause of death was not determined.

FIG 6

Mouse survival correlates with organ fungal burden. Groups of 10 6- to 8-week-old A/J mice were infected intranasally with 11 C. neoformans clinical strains. Progression to severe morbidity was monitored for 150 days, and mice were sacrificed at 150 days or when the endpoint criteria were reached. Lungs (A), spleen (B), and brain (C) were harvested and homogenized, and serial dilutions were plated on agar plates to determine tissue fungal burdens. Strains were classified into three groups as high-, intermediate-, and low-virulence strains, as determined in the mouse survival experiment. (Left) Individual C. neoformans strains plotted as the tissue fungal burden (number of CFU) against time to 80% mortality of infected mice. Each dot represents the average for mice infected with the same strain (n = 3 to 8 mice). (Right) Average number of CFU of high-virulence (n = 4 strains), intermediate-virulence (n = 4 strains), and low-virulence (n = 3 strains) strains. Error bars represent the standard error of the mean. The difference between high-, intermediate-, and low-virulence strains was compared by Student’s t test or the Mann-Whitney U test. *, P < 0.05; NS, not statistically significant.