THE TBMCP1 METALLOCARBOXYPEPTIDASE IN PROCYCLIC TRYPOMASTIGOTES OF TRYPANOSOMA BRUCEI: NULL MUTANTS AND RNAi KNOCK-DOWN CELL LINES

OPPENHEIMER, FLORENCIA MAIA; BOUVIER, LEÓN ALBERTO; CAZZULO, JUAN JOSÉ; NIEMIROWICZ, GABRIELA TERESA

Buenos Aires

Encuentro; XXVII Reunión Anual de la Sociedad Argentina de Protozología; 2015

Sociedad Argentina de Protozología

Metallocarboxypeptidases (MCP) of the M32 family, while broadly distributed among prokaryotic organisms, can only be found in a few eukaryotes including trypanosomatids. Among these organisms are Trypanosoma brucei and T. cruzi, the causative agents of Sleeping Sickness and Chagas disease, respectively. The genomes of these parasites encode M32 MCPs, TcMCP1 and TbMCP1, which share 72% identity. Both orthologues have a cytosolic localization and are expressed in all parasite cultured forms. Although the structure and biochemistry of these enzymes is well known, no function for them has been reported to date. To study the physiological role of TbMCP1 in procyclic trypomastigotes we employed two different strategies. The first strategy included the generation of a double knockout parasite cell line in which both TbMCP1 alleles were replaced with selectable markers. After each selection step correct gene disruption was verified by PCR with primers on both sides of the antibiotic resistance gene. These cell lines were assayed for TbMCP1 expression by Western blot. Although TbMCP1 expression was reduced and completely absent in single and double null mutants respectively, no significant growth differences could be detected among them. With a similar methodology, parasites with TbMCP1 endogenously tagged at the N- or C-terminal with 3xFLAG and HA were obtained. TbMCP1 RNA interference was monitored by Western blot with antibodies against the epitope tags. The knockdown construct was based on pLEW100v5 vector and involved the stem-loop strategy for double stranded RNA generation. In contrast to the knock out parasites, upon induction of interference the cells showed increased duplication times. This suggests that knockout parasites might have developed metabolic compensatory pathways. Phenotype analysis through microscopic methodologies is currently being undertaken to get a better insight of the observed phenomena.