Research

Cryptococcus neoformans is a eukaryotic organism with a complex sexual development pathway that can be investigated to elucidate the molecular mechanisms of fungal pathogenesis and developmental control. Fungi cause devastating disease in humans, particularly in immunocompromised patients. Up to 9% of very low birth weight pre-term babies contract fungal infections and ~28% of these systemic infections are fatal. In addition, the fourth most common cause of hospital-acquired infection in all age groups is fungal in origin, and ~75% of all women will experience at least one vaginal yeast infection. Exploiting a genetic system in which to identify disease-causing properties of fungi promises to lead to the development of novel drug targets. This elegant system also allows the study of the basic processes involved in regulating eukaryotic development.

The long-term goal of the Hull Laboratory is to identify and characterize the factors associated with sexual development in C. neoformans and understand their contributions to infectious particle production. C. neoformans is a fungal pathogen that causes meningitis primarily in immunocompromised individuals. Infection with C. neoformans results from environmental exposure, and a pulmonary route of infection is generally accepted. The organism is inhaled into the alveoli of the lungs, and the infectious propagule is widely presumed to be the spore. Once inhaled, spores can grow in the lung and cause respiratory disease, but cryptococcal disease is most often recognized after dissemination to the central nervous system (Figure 1).

Figure 1. The C. neoformans infectious cycle. C. neoformans resides in the environment and has been found associated primarily with pigeon droppings and Eucalyptus trees. It is thought that infection of humans generally occurs when basidiospores produced by C. neoformans in nature are inhaled into the lungs. Inhaled spores are deposited into the alveoli and germinate to establish a dormant infection or disseminate to the central nervous system. Once dissemination has occurred, viable cells can be cultured from the cerebrospinal fluid of affected individuals. Figure 1. The C. neoformans infectious cycle. C. neoformans resides in the environment and has been found associated primarily with pigeon droppings and Eucalyptus trees. It is thought that infection of humans generally occurs when basidiospores produced by C. neoformans in nature are inhaled into the lungs. Inhaled spores are deposited into the alveoli and germinate to establish a dormant infection or disseminate to the central nervous system. Once dissemination has occurred, viable cells can be cultured from the cerebrospinal fluid of affected individuals.

Spore formation is the result of sexual development. When haploid cells of opposite mating types (a and a) encounter one another and fuse, the resulting binucleate a+a cell is competent to initiate a new developmental program. Sexual development is defined by a change from mononucleate, budding yeast growth to hyphal, dikaryotic filament formation, followed by nuclear fusion, meiosis, and spore production (Figure 2).

Figure 2. C. neoformans sexual development. When haploid a cells encounter haploid αcells at 25°C in the presence of soluble plant factor, the cells fuse with one another (stage 1 – mate recognition and cell fusion). The fused cells adopt a filamentous growth pattern with distinct nuclei (stage 2 – dikaryotic filament formation). This filament grows in a polar manner until a sphere forms on the end of the terminal cell (stage 3 – basidium formation). In the basidium, nuclear fusion and meiosis occur (stage 4 – nuclear fusion and meiosis). Meiotic products are then packaged, and spores are deposited on the surface of the basidium (stage 5 - sporulation). Figure 2. C. neoformans sexual development. When haploid a cells encounter haploid α cells at 25°C in the presence of soluble plant factor, the cells fuse with one another (stage 1 – mate recognition and cell fusion). The fused cells adopt a filamentous growth pattern with distinct nuclei (stage 2 – dikaryotic filament formation). This filament grows in a polar manner until a sphere forms on the end of the terminal cell (stage 3 – basidium formation). In the basidium, nuclear fusion and meiosis occur (stage 4 – nuclear fusion and meiosis). Meiotic products are then packaged, and spores are deposited on the surface of the basidium (stage 5 - sporulation).

Sexual development is controlled by the SexInducer genes, SXI1a and SXI2a. These key regulators are specific to a and a cells, respectively, and although both factors are essential for sexual development, their mechanisms of action are unknown. Using a combination of genetic, biochemical, bioinformatic, and cell biological approaches, we will test our hypothesis that Sxi1a and Sxi2a control sexual development in Cryptococcus neoformans by directly regulating the transcription of key targets to specify the dikaryotic state (Figure 3).

Figure 3. Model for the roles of Sxi1α and Sxi2a in controlling sexual development. Large ovals represent cells. Circles represents the a and α nuclei. The shaded oval represents Sxi1α and the star represents Sxi2a. Top: SXI1α and SXI2a are expressed at very low levels in haploid cells and play no apparent role in this cell type. Middle: Following cell fusion, SXI1α and SXI2a are expressed. Bottom: After induction, Sxi1α and Sxi2a form a heterodimeric complex that establishes the dikaryotic state and induces sexual development. Figure 3. Model for the roles of Sxi1α and Sxi2a in controlling sexual development. Large ovals represent cells. Circles represents the a and α nuclei. The shaded oval represents Sxi1α and the star represents Sxi2a. Top: SXI1α and SXI2a are expressed at very low levels in haploid cells and play no apparent role in this cell type. Middle: Following cell fusion, SXI1α and SXI2a are expressed. Bottom: After induction, Sxi1α and Sxi2a form a heterodimeric complex that establishes the dikaryotic state and induces sexual development.

We are currently focusing on three distinct areas of study:

  1. Mapping the regulatory circuit controlled by Sxi1a and Sxi2a by identifying and characterizing their downstream targets.
  2. Establishing the properties of the Sxi1a and Sxi2a proteins required to control sexual development by evaluating the relationships between their structures and functions.
  3. Investigating the cellular processes required for sexual development with an initial focus on dikaryotic filamentation.

Understanding the biology of sexual development and spore production in C. neoformans promises to provide information useful in the prevention and/or treatment of cryptococcal disease. Understanding this basic fungal biology also sets the stage for the development of more effective methods of preventing and treating fungal disease in general.