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Animal models are frequently used as surrogates to understand human disease. In the fungal pathogen Cryptococcus species complex, several variations of a mouse model of disease were developed that recapitulate different aspects of human disease. These mouse models have been implemented using various inbred and outbred mouse backgrounds, many of which have genetic differences that can influence host response and disease outcome. In this review, we will discuss the most commonly used inbred mouse backgrounds in C. neoformans infection models.

Studies across various pathogens highlight the importance of pathogen genetic differences in disease manifestation. In the human fungal pathogen Cryptococcus neoformans , sequence type (ST) associates with patient outcome. We performed a meta-analysis of four genomic studies and identified overlapping gene regions associated with virulence, suggesting the importance of these gene regions in cryptococcal disease in diverse clinical isolates. We explored the relationship between virulence and strain genetic differences using the cryptococcosis mouse model and a closely related library of ST93 clinical isolates. We identified four in vivo virulence phenotypes: hypervirulence, typical virulence with CNS disease, typical virulence with non-CNS disease, and latent disease. Hypervirulent isolates were clade specific and associated with an interferon gamma (IFNγ) dominated immune response. Using a genome wide association study (GWAS), we identified nine genes with polymorphisms associated with IFNγ production, including the inositol sensor ITR4 . The itr4Δ mutant recapitulated the hypervirulence phenotype and ITR4 affects expression of two IFNγ associated genes. Finally, we showed that IFNγ production is associated with SNPs that downregulate ITR4 and with SNP accumulation in other IFNγ associated genes. These data highlight the complex role of pathogen genetics in virulence and identify genes associated with hypervirulence and IFNγ in Cryptococcus neoformans . Identification of virulence-associated genes in pathogens is important to understand mechanisms of disease. Here, Jackson et al. use a mouse model and clinical isolates of Cryptococcus neoformans to identify novel gene networks that impact virulence.

The human pathogenic fungus Cryptococcus neoformans is a global health concern. Previous research in the field has focused on studies using reference strains to identify virulence factors, generate mutant libraries, define genomic structures, and perform functional studies. In this review, we discuss the benefits and drawbacks of using reference strains to study C. neoformans, describe how the study of clinical isolates has expanded our understanding of pathogenesis, and highlight how studies using clinical isolates can further develop our understanding of the host–pathogen interaction during C. neoformans infection.

Cryptococcus neoformans is a fungal pathogen that disseminates from the lungs to the brain to cause fatal disease. Latent C. neoformans infection in the lungs is controlled by organized collections of immune cells called granulomas. The formation and structure of Cryptococcus granulomas are poorly understood due to inconsistent human pathology results and disagreement between necrotic granuloma-forming rat models and non-necrotic granuloma-forming mouse models. To overcome this, we investigated granuloma formation during latent C. neoformans infection in the C3HeB/FeJ mouse strain which forms necrotic lung granulomas in response to other pathogens. We found that latent C. neoformans granuloma formation progresses through phases that we described as early, intermediate, and late with different immune response profiles and granulomatous characteristics. Ultimately, we show that C3HeB/FeJ mice latently infected with C. neoformans form non-necrotic granulomas and could provide a novel mouse model to investigate host immune response profiles.

The mechanisms of latency in the context of C. neoformans infection remain poorly understood. Two reasons for this gap in knowledge are: 1) the lack of standardized criteria for defining latent cryptococcosis in animal models and 2) limited genetic and immunological tools available for studying host parameters against C. neoformans in non-murine models of persistent infection. In this study, we defined criteria required for latency in C. neoformans infection models and used these criteria to develop a murine model of persistent C. neoformans infection using clinical isolates. We analyzed infections with two clinical C. neoformans strains, UgCl223 and UgCl552, isolated from advanced HIV patients with cryptococcal meningitis. Our data show that the majority of C57BL/6 mice infected with the clinical C. neoformans isolates had persistent, stable infections with low fungal burden, survived beyond 90 days-post infection, exhibited weight gain, had no clinical signs of disease, and had yeast cells contained within pulmonary granulomas with no generalized alveolar inflammation. Infected mice exhibited stable relative frequencies of pulmonary immune cells during the course of the infection. Upon CD4+ T-cell depletion, the CD4 DTR mice had significantly increased lung and brain fungal burden that resulted in lethal infection, indicating that CD4+ T-cells are important for control of the pulmonary infection and to prevent dissemination. Cells expressing the T bet transcription factor were the predominant activated CD4 T-cell subset in the lungs during the latent infection. These T bet -expressing T-cells had decreased IFNγ production, which may have implications in the capacity of the cells to orchestrate the pulmonary immune response. Altogether, these results indicate that clinical C. neoformans isolates can establish a persistent controlled infection that meets most criteria for latency; highlighting the utility of this new mouse model system for studies of host immune responses that control C. neoformans infections.

Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G 2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro . Using a copper-repressible promoter P CTR4 -CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G 2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G 2 arrest. Release from this G 2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G 2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

Cryptococcal meningitis, caused by the opportunistic fungal pathogen Cryptococcus neoformans, is a leading cause of HIV-related mortality worldwide, and a frequent cause of morbidity and mortality in other immunocompromised patient populations. In healthy individuals, exposure to C. neoformans in early childhood results in a latent pulmonary infection that is asymptomatic, but leads to the formation of lung granulomas. When the immune system fails, due to HIV or medical interventions that suppress immunity, immune control in the pulmonary granuloma is lost and the latent C. neoformans infection disseminates to cause meningitis. Treatment of cryptococcal meningitis requires appropriate antifungal therapy, management of intracranial pressure, and supportive care. In the case of HIV infection, antiretroviral therapy is initiated following successful resolution of cryptococcal meningitis. However, in a subset of individuals, the restoration of an intact immune system (i.e., via antiretroviral therapy), will trigger a dysregulated hyper-inflammatory response to C. neoformans antigen, known as immune reconstitution inflammatory syndrome (IRIS). The host immune cells and effector functions critical for controlling latency or promoting a dysregulated hyper-inflammatory response in IRIS have yet to be fully characterized.Currently, there are very few experimental mouse models for studying latent C. neoformans infection and cryptococcal IRIS. However, recent advances in the characterization and categorization of clinical isolates based on virulence phenotype and genetic background, allowed us to develop novel mouse models that recapitulate latency and certain aspects of cryptococcal IRIS. In this thesis, two such models are presented – a mouse inhalation model of latent C. neoformans infection and a mouse inhalation model of hypervirulent C. neoformans infection. Thus, the aims of this thesis are to 1) define and characterize novel mouse models of C. neoformans infection using clinical isolates, and 2) determine how the pulmonary immune response is able to control or become perturbed by the fungal pathogen.

The human pathogenic fungus Cryptococcus neoformans is a global health concern. Previous research in the field has focused on studies using reference strains to identify virulence factors, generate mutant libraries, define genomic structures, and perform functional studies. In this review, we discuss the benefits and drawbacks of using reference strains to study C. neoformans, describe how the study of clinical isolates has expanded our understanding of pathogenesis, and highlight how studies using clinical isolates can further develop our understanding of the host-pathogen interaction during C. neoformans infection.