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Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form (PCF) of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the 2 proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. Moreover, depletion of the mitochondrial proteasome results in increased levels of MaRF11. Thus, we have discovered a protein complex comprising TbPam18, TbPam16, and MaRF11, that controls maxicircle replication. We propose a working model in which the matrix protein MaRF11 functions downstream of the 2 integral inner membrane proteins TbPam18 and TbPam16. Moreover, we suggest that the levels of MaRF11 are controlled by the mitochondrial proteasome.

The Trypanosoma cruzi genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a 'mosaic' structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of T. cruzi and may be involved in parasite-host interactions and/or in diverting the immune response.High-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the T. cruzi seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in in vitro and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection.In spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from T. cruzi MASPs during human infection.

The flagellar pocket constitutes an active and strategic site in the body of trypanosomatids (i.e. parasitic protozoa that cause important human and/or livestock diseases), which participates in several important processes such as cell polarity, morphogenesis and replication. Most importantly, the flagellar pocket is the unique site of surface protein export and nutrient uptake in trypanosomatids, and thus constitutes a key portal for the interaction with the host. In this work, we identified and characterized a novel Trypanosoma cruzi protein, termed TCLP 1, that accumulates at the flagellar pocket area of parasite replicative forms, as revealed by biochemical, immuno-cytochemistry and electron microscopy techniques. Different in silico analyses revealed that TCLP 1 is the founding member of a family of chimeric molecules restricted to trypanosomatids bearing, in addition to eukaryotic ubiquitin-like and protein-protein interacting domains, a motif displaying significant structural homology to bacterial multi-cargo chaperones involved in the secretion of virulence factors. Using the fidelity of an homologous expression system we confirmed TCLP 1 sub-cellular distribution and showed that TCLP 1-over-expressing parasites display impaired survival and accelerated progression to late stationary phase under starvation conditions. The reduced endocytic capacity of TCLP 1-over-expressors likely underlies (at least in part) this growth phenotype. TCLP 1 is involved in the uptake of extracellular macromolecules required for nutrition and hence in T. cruzi growth. Due to the bacterial origin, sub-cellular distribution and putative function(s), we propose TCLP 1 and related orthologs in trypanosomatids as appealing therapeutic targets for intervention against these health-threatening parasites.

BackgroundTcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector.Methodology and principal findingsHere, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy.Conclusion and significanceTogether, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.