Identification and Characterization of Genes that Play a Role in the Trophozoite to Cyst Ttransition of Giardia lamblia

Sinha, Abhishek (2013) Identification and Characterization of Genes that Play a Role in the Trophozoite to Cyst Ttransition of Giardia lamblia. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

The focus of this thesis has been towards understanding the process of protein degradation in Giardia lamblia. Although this organism is of considerable medical importance as it is one of the leading causes of diarrhea worldwide, study of its cellular processes, which are very different from those of its higher eukaryotic hosts, has lagged behind because of several reasons. One of the primary reasons is that the unique biology of Giardia lamblia makes it recalcitrant to genetic manipulations. Two of the major reasons for this lack of genetic approaches are: i) gene knockouts are not an option as this parasite is at the very least tetraploid because it has two diploid nuclei in each cell and ii) lack of an efficient recombination machinery that precludes genomic manipulations (Takumi et al., 2012, Xu et al., 2012). In addition, approaches relying on the use of orthologous sequences from other eukaryotes to search for functionally orthologous Giardia proteins are tenuous as the sequences of most Giardia proteins are highly diverged from other eukaryotic orthologues. Thus any candidate gene identified by the use of BLAST searches must be validated by using functional assay, which is difficult because of the lack of genetic approaches as stated above. To circumvent these issues, the research approach described in this thesis has relied on the use of BLAST searches to identify putative genes that may be involved in a given pathway, which have then been validated by the use of functional complementation of the corresponding yeast S. cerevisiae mutants. Overall, this thesis has relied on sequence analysis to identify target genes or domains in the parasite, then adopted genetic methods in S. cerevisiae to establish their function, and finally used cell biology techniques to validate the results. Experiments were performed to understand two major processes of protein degradation in Giardia i.e. proteasomal protein degradation and lysosomal protein degradation. The studies in this thesis suggests UPS of Giardia can become an important drug target as it is involved in encystation process and hence in the propagation of disease. The subunit structures of proteasome are also different when compared to the host proteasome. Hence selective proteasomal inhibition is likely to be possible. Thus it will be worthwhile to search/design a new set of chemotherapeutic agents specific for the Giardia proteasome. Considering the involvement of proteasome in development of flagella and possible degradation of IFT components and other flagellar proteins, future study should be undertaken to determine if flagellar proteins are degraded via the UPS. Recent studies have shown that IFT proteins are involvement in the trafficking of the T-cell receptor complex to the immune synapse (Finetti et al., 2009). Considering the role of IFT proteins in receptor trafficking, and the possible involvement of the proteasome with the flagella revealed by this study it may be speculated that there may be direct and/or indirect links between the two major degradation processes i.e. proteasomal and lysosomal degradation.

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