Vivapain : a cysteine peptidase from Trypanosoma vivax.
African animal trypanosomosis is a devastating disease affecting livestock mainly found in sub-Saharan Africa. This disease is known as nagana and is transmitted by the trypanosome parasite from the tsetse fly vector to a mammalian host. There are three African trypanosomes namely Trypanosoma vivax, T. congolense and T. brucei brucei that are the causative agents responsible for this disease in African cattle. This disease is serious since it not only affects livestock but also has a negative impact on the sub-Saharan African economy. There is, therefore, a great demand for better control methods of the disease and suitable diagnostic methods. Current control measures such as the use of trypanocidal drugs, tsetse fly eradication methods and trypanotolerant cattle have become inadequate. The defence mechanism of the trypanosome to continuously change its surface coat by a process of antigenic variation has made it impossible to produce a suitable vaccine. Therefore, chemotherapy is still one of the key approaches for control of this wasting disease. The long existence of the current trypanocidal drugs has allowed the development of drug resistance. The development of new chemotherapeutic drugs is focused on targeting the pathogenic factors such as parasite cysteine peptidases that contribute to the disease. Vivapain is the main cysteine peptidase of T. vivax and shares high sequence identity with congopain, the main cysteine peptidase of T. congolense, which was previously shown to be a pathogenic factor contributing to trypanosomosis. Vivapain, thus, has potential as a target for chemotherapeutic drug design. Hence, the first part of this study involved the recombinant expression and enzymatic characterisation of vivapain for future production of new synthetic inhibitors for the use in new trypanocidal drugs. The catalytic domain of vivapain (Vp) was recombinantly expressed in the Pichia pastoris yeast expression system and enzymatically characterised. The main finding from this study was that Vp was only able to hydrolyse a substrate if the P2 position was occupied by either a hydrophobic Phe or Leu residue. Vp was also found to be active close to physiological pH and was inhibited by the reversible cysteine peptidases, leupeptin, antipain and chymostatin and the irreversible cysteine peptidases L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E-64), iodoacetic acid (IAA) and iodoacetamide (IAN). A further important aspect of controlling trypanosomosis is the diagnosis of the disease. Clinical, parasitological, molecular and serological techniques have been applied and used to diagnose trypanosomosis. One of the most promising serological techniques has proven to be the enzyme-linked immunosorbent assay (ELISA), more specifically the antibody and antigen detection ELISAs. The main requirement for this technique is a readily available and reproducible antigen such as that produced by recombinant expression. While there are recombinant antigens that are available to be used to detect T. congolense, T. brucei brucei and even T. evansi infections, there are none available to detect T. vivax infections. In the second part of this study, a mutant inactive full length form of vivapain (FLVp) was expressed in a bacterial expression system for the detection of T. vivax infections. Antibodies against this antigen were produced in both chickens and mice. Both the chicken IgY and mice sera were able to detect the recombinant FLVp in western blots. The mice sera were also able to detect native vivapain in a T. vivax lysate, which is very promising for future use of the FLVp antigen and the corresponding antibodies in diagnosis of T. vivax infections in sera of infected animals.