Cryptococcus neoformans is a basidiomycetous yeast with clinical importance
due to its role as an opportunistic pathogen in immunocompromised individuals. Upon
exposure to C. neoformans patients may develop a disease known as cryptococcosis, a
condition associated with pulmonary infections that may result in lethal
meningoencephalitis. This disease is often accompanied by aneuploidies of single or
multiple chromosomes in a subpopulation of the infecting cells, leading to the idea that
missegregation of chromosomes during infection may be a way for this organism to deal
with stresses and acquiring antifungal resistance. Because this species is rapidly
developing resistance to many of the prevalent antifungal treatments, a need exists for
more potent therapies with fewer side effects. The molecular biology concerning cellular
reproduction and division in C. neoformans is poorly understood for such an important
fungal pathogen. The research performed so far has produced information about such
structures as the centromeric DNA of their chromosomes and the kinetochore protein
superstructure that links the spindle microtubules to the centromeres. Understanding how
chromosome segregation occurs in C. neoformans may bring about better therapies: if it
can be shown that homologous proteins perform the same actions in Cryptococcus as they do in other organisms then these proteins may potentially become targets for future
studies to determine the mechanism linking the presence of antifungal chemicals to
aneuploidy.
The kinetochore protein complex is essential for segregation of chromosomes.
Previous experiments have described a protein essential to the structure and function of
the yeast kinetochore. The necessity of this protein for kinetochore function was first
determined using an Schizosaccharomyces pombe strain carrying a temperature-sensitive
mutation in the Mis12 gene that causes the protein to malfunction above a certain
temperature. It was found that when grown above the restrictive temperature cells were
unable to complete mitotic segregation, with the dividing nucleus stuck in the neck
between budding cells. A later study described the budding yeast homolog MTW1
(Mis12-like protein) in Saccharomyces cerevisiae using a similar temperature-sensitive
mutant and yielded similar results. This protein is part of a subcomplex of kinetochore
proteins called the MIND complex. One goal of this project is to characterize the MTW1
homolog in C. neoformans in order to determine whether it maintains the functional
importance to kinetochore structure exhibited by the previously described homologs.
This has been addressed through the construction of similar temperature-sensitive
mutants and by regulating the gene expression through regulatable CTR4 promoter.
These strains were created through the use of overlap PCR and biolistic transformation.
Molecular and phenotypic characterizations have been performed on these strains and
include growth and morphological analyses. Results have confirmed the presence of the
desired mutations and have shown some inhibitory effect on growth under restrictive conditions. RNA interference was performed in an effort to demonstrate the necessity of various kinetochore proteins. Initial results indicate that MTW1, along with the outer kinetochore gene DAD2, are not essential whereas several other MIND complex genes (NNF1, DSN1) and inner (CSE4, MIF2) and outer (DAD1) kinetochore genes were shown
to be necessary for viability of the yeast cells. These results are in accordance with those
obtained through the use of strains with conditional mutations in MTW1. These results
imply that while CnMTW1 probably acts in the same capacity as that of its ascomycetous
homologs and is necessary for optimal kinetochore function, it is not essential for
chromosome segregation
