Cloning and expression analysis of two novel paraflagellar rod domain genes found in Trypanosoma cruzi

Abstract

The eukaryotic flagellum is one of the most complex macromolecular structures found in cells, containing more than 250 proteins. One unique structure in the flagella of trypanomastids is the paraflagellar rod (PFR). The PFR constitutes a lattice of cytoskeletal filaments that lies alongside the axoneme in the flagella. This unique and complex structure is critical for cell motility, though little is known about its molecular assembly or its role in the lifecycle of trypanosomatids. These proteins are of particular importance in Trypanosoma cruzi, as purified or recombinant PFR proteins have been demonstrated to be immunogenic, protecting mice from a lethal challenge with the parasite. We have searched the T. cruzi databases and discovered two novel genes containing PFR domains. Both these genes are transcribed in vivo and are significantly larger than the previously described PFR genes identified in T. cruzi (>2 Kb). Real-time PCR was used to examine the relative expression levels of six PFR genes, including the two we describe here, in all three stages of T. cruzi's lifecycle. Database searches have further provided EST and genomic sequence support for the presence of these genes in two other pathogenic trypanosomatids, Trypanosoma brucei and Leishmania spp. One of these genes, designated PFR5 contains a carboxy terminal SH3 domain not previously seen in PFR family genes. We propose that this proline-binding SH3 domain may play an important role in the assembly of the PFR.

Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum

“…As expected, motifs consistent with adenylate kinase signatures were detected for ADKA and ADKB. A Pfam analysis of the data set also identified two IQ calcium-independent calmodulin binding motifs (PF00612) in PFC15, a Beige/BEACH domain (PF02138) in PFC10, as previously reported (43), an SH3 (PF00018) domain in PFR5, and a phosphatidylinositol 3-and 4-kinase (PF00454) and FATC (PF02260) domain in Tb11.01.6300, consistent with the automatic PI3K-related kinase annotation.…”

“…The other major component of the PFR, PFR1 is also reduced, and although it is difficult to resolve these two proteins using twodimensional DIGE, the average log 2 ratios of induced to noninduced samples detected by iTRAQ are Ϫ2.4 (PFR1) and Ϫ2.6 (PFR2), which is consistent with a near stoichiometric loss of these two proteins after RNAi. Four other proteins have previously been given the soubriquet of PFR (or PAR) (39,42,43), and of these PAR1 and PFR5 are both present in our data set.…”

“…PFR5, TbI2, TbI17, PDEB1, and PDEB2 (29,(41)(42)(43)61). Their absence could be due to sampling variations, low protein abundance, masking by other proteins in the case of DIGE, or physical properties that may be refractory to MS identification.…”

The paraflagellar rod of kinetoplastid parasites: From structure to components and function

“…This process drew attention to two proteins that contain domains in common with PFR1 and PFR2 (Clark et al, 2005). These have been named PFR5 and PFR6 and have predicted molecular weights of 87 kDa and 75 kDa, respectively.…”

Expresión diferencial entre estadios de Trypanosoma cruzi I en el aislamiento de un paciente con cardiomiopatía chagásica crónica de zona endémica de Santander, Colombia

“…En el presente estudio se observó expresión diferencial de las proteínas de unión al calcio flagelar (PUFCa) y el componente 3 del bastón paraflagelar (BPF-3) en las formas de tripomastigote en relación con las de amastigote; se obtuvieron resultados similares en los niveles de expresión de BPF-3, utilizando reacción en cadena de la polimerasa (PCR) en tiempo real con la cepa Perú (T. cruzi I) (24). Por su localización en la membrana flagelar, estas proteínas son importantes en la movilidad y, además, cumplen funciones metabólicas, reguladoras, de señalamiento y adherencia (25)(26)(27).…”