Evolution and diagnostic potential of major surface proteases from trypanosoma Vivax, T.Brucei Brucei and T.Congolense

Abstract

Tsetse-transmitted trypanosomiasis is a disease unique to Africa, affecting both humans and animals. This disease occurs in about 10 million km2 in 37 sub-Saharan countries corresponding approximately to one-third of Africa's total land area, and threatens an estimated 50 million people and 48 million cattle. The estimated annual losses in cattle production alone are in the range of 1.0-1.2 billion dollars. The diagnosis of trypanosomiasis is notoriously difficult as there are no specific clinical signs and the intermittent and usually low parasitaemia make detection of the difficult. The trypanosomes are infamous for their ability to evade the immune responses by periodically switching their major variant surface glycoprotein (VSG) in a phenomenon called antigenic variation and are likely to be only one of several mechanisms enabling these organisms to thrive in the face of the immune defences. The complexity of the trypanosome parasite's antigenic repertoire has made development of a vaccine based on the VSG coat unlikely hence focus is on identifying invariant surface trypanosome components such as the Major Surface Proteases (MSP) as potential targets for interrupting infection or infection-mediated disease. The MSP are a group Zinc metalloproteases belonging to the M8 metzincin family. MSP-like genes have been identified in the bloodstream-stage of African trypanosomes whose genome contains gene families encoding homologues of the MSP. It is a protein found on the cell surface that contributes to the ability of Leishmania to foil the mammalian immune system. However, very little is known about the MSP from T. congolense, T. b. brucei and T. vivax. The objective of this study was to clone, express and evaluate the evolution of the MSP variants with the aim of determining their possible use as diagnostic targets for African Animal Trypanosomiasis (AAT). We describe the identification and amplification of T. congolense, T. b. brucei and T. vivax MSP using an integrated approach of cloning, expression and phylogenetic analyses with the objective of evaluating and determining their possible use as diagnostic targets for AAT. Samples of T. congolense, T. brucei and T. vivax were obtained from diverse geographical locations throughout East Africa and sequenced for the MSP genes. The MSP genes of T. congolense, T. brucei and T. vivax, the causative agents of AAT, were successfully amplified, cloned and expressed in E. coli cells. The amplification of MSP by PCR yielded stable non-fragmenting products of approximately 1600bp on an agarose gel. The result of a 4-20% SDS–PAGE showed a bigger and broad band of the induced protein, suggesting full induction of the polypeptide. Separation of the proteins based on size on SDS-PAGE gel revealed induced products of approximately 45kDa in size from both the T. congolense and T. b. brucei samples. A protein signature search of the MSP sequences in this study showed characteristics of a typical metallopeptidase such as the peptidase domains and the Zinc finger domains, a catalytic Zinc atom and an active-site helix containing the two histidines of the Zinc proteinase sequence motif HEXXH. Evolutionary studies of these MSP variants showed regions of strong similarity amongst all the three species under study suggesting that they are conserved. Distance and maximum likelihood methods yielded phylogenetic trees of similar topology with indications that geographical placement has no effect on the phylogeny of the MSP. These conserved domains may form useful target sites for the development of a tool for detecting T. congolense, T. brucei and T. vivax.