Protein Interaction Between Basal Body and Kinetoplast DNA Complex Within Trypanosoma brucei

Trypanosoma brucei is responsible for the disease Human African Trypanosomiasis (HAT), also known as African Sleeping Sickness, in humans as well as N’gana in livestock. Our work was done on the sub-species T. brucei brucei, which is non-pathogenic in humans, and thus safe for undergraduate laboratory research. Trypanosoma brucei is part of a diverse group of flagellated protozoans known as the kinetoplastids. Kinetoplastids are named for the concentration of mitochondrial DNA located at the posterior end of the mitochondrion and adjacent to the flagellum, called the kinetoplast DNA (kDNA). The kinetoplast is connected to the basal body (motor) of the flagellum by proteins through both membranes of the mitochondrion. The flagella drive the process of binary fission (cell division), suggesting that the replicated kDNA is separated through these protein interactions. Specific proteins were tested for their participation in the connection between kDNA and basal body. Our genes of interest were inserted into plasmid vectors. The plasmids were then linearized, followed by transfection into trypanosomes. Immunofluorescence was used to identify where these proteins localized within the cell. INTRODUCTION Trypanosoma brucei is responsible for the Human African Trypanosomiasis (HAT), also known as African Sleeping Sickness, in humans as well as N’gana in livestock. This parasite is present in 36 countries of Sub-Saharan Africa, putting approximately 60 million people at risk, and affecting an estimated 300,000 annually (World Health Organization, 2006). T. brucei also affects humans indirectly by decreasing the economic value of the livestock it infects. Infected cattle have decreased milk production and the milk that is produced is of lower quality. Strength and body weight is also decreased which provides less meat and affects their ability to plow fields. Because of the underproduction of cattle, an estimated $4.5 billion is lost each year. Trypanosome transmission to a human or animal host occurs mainly through the insect vector Glossina morsitans, the Tsetse fly. A form of T. brucei resides in the salivary gland of an insect vector. When an infected fly bites a mammalian host, the parasite is transmitted via the fly’s saliva. Once in the host, the trypanosome goes through a series of transformations, evolving into the epidemiological T. brucei (Center for Disease Control, 2008). Initial symptoms include fever, severe headaches and extreme fatigue. The trypanosomes then attack the central nervous system, affecting mental functionality, leading to coma and eventually death. Trypanosoma brucei is part of a diverse group of flagellated protozoans known as the kinetoplastids. Kinetoplastids are named for the concentration of mitochondrial DNA located at the posterior end of the mitochondrion and adjacent to the flagellum, called the kinetoplast DNA (kDNA). The kDNA consists of two types of circular DNA, one being “maxicircles” of about 25 kilobases and the other being “minicircles” of about 1 kilobase. Trypanosomes contain about 50 maxicircles and several thousand minicircles (Downey et al., 2005). Mini and maxi circles are intertwined in one another and must be separated for cell division. The kinetoplast is connected to the basal body (motor) of the flagellum by proteins through both membranes of the mitochondrion. The flagella drive the process of binary fission (cell division), suggesting that the replicated kDNA is separated through these protein interactions. The objective of this study was to further our understanding of the kDNA and basal body complex. We tested specific proteins for their participation in the connection between these two structures. We did so by identifying where these proteins localized within the cell, and allowing for interpretation of protein function by blocking its production using RNA interference and studying the effects on the cell.