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Summary: Parasites have been engineered to express fluorescent reporter proteins, yet the impact of red fluorescent proteins on Leishmania infections remains largely unknown. We analysed the infection outcome of Leishmania mexicana parasites engineered for the constitutive expression of mKate protein and evaluated their immunogenicity in BALB/c mice. Infection of BALB/c mice with mKate transfected L. mexicana (LmexmKate) parasites caused enlarged lesion sizes, leading to ulceration, and containing more parasites, as compared to LmexWT. The mKate protein showed immunogenic properties inducing antibody production against the mKate protein, as well as enhancing antibody production against the parasite. The augmented lesion sizes and ulcers, together with the more elevated antibody production, were related to an enhanced number of TNF‐α and IL‐1β producing cells in the infected tissues. We conclude that mKate red fluorescent protein is an immunogenic protein, capable of modifying disease evolution of L. mexicana. [ABSTRACT FROM AUTHOR]

The 17-subunit RNA polymerase III (RNAP III) synthesizes essential untranslated RNAs such as tRNAs and 5S rRNA. In yeast and vertebrates, subunit C82 forms a stable subcomplex with C34 and C31 that is necessary for promoter-specific transcription initiation. Little is known about RNAP III transcription in trypanosomatid parasites. To narrow this knowledge gap, we characterized the C82 subunit in Trypanosoma brucei and Leishmania major . Bioinformatic analyses showed that the 4 distinctive extended winged-helix (eWH) domains and the coiled-coil motif are present in C82 in these microorganisms. Nevertheless, C82 in trypanosomatids presents certain unique traits, including an exclusive loop within the eWH1 domain. We found that C82 localizes to the nucleus and binds to RNAP III-dependent genes in the insect stages of both parasites. Knock-down of C82 by RNA interference significantly reduced the levels of tRNAs and 5S rRNA and led to the death of procyclic forms of T. brucei . Tandem affinity purifications with both parasites allowed the identification of several C82-interacting partners, including C34 and some genus-specific putative regulators of transcription. However, the orthologue of C31 was not found in trypanosomatids. Interestingly, our data suggest a strong association of C82 with TFIIIC subunits in T. brucei , but not in L. major .

The Trypanosomatid family includes flagellated parasites that cause fatal human diseases. Remarkably, protein-coding genes in these organisms are positioned in long tandem arrays that are transcribed polycistronically. However, the knowledge about regulation of transcription initiation and termination in trypanosomatids is scarce. The importance of epigenetic regulation in these processes has become evident in the last years, as distinctive histone modifications and histone variants have been found in transcription initiation and termination regions. Moreover, multiple chromatin-related proteins have been identified and characterized in trypanosomatids, including histone-modifying enzymes, effector complexes, chromatin-remodelling enzymes and histone chaperones. Notably, base J, a modified thymine residue present in the nuclear DNA of trypanosomatids, has been implicated in transcriptional regulation. Here we review the current knowledge on epigenetic control of transcription by all three RNA polymerases in this group of early-diverged eukaryotes.

Almost nothing is known about the sequences involved in transcription initiation of protein-coding genes in the parasite Leishmania. We describe here the transcriptional analysis of chromosome 1 (chr1) from Leishmania major Friedlin (LmjF) which encodes the first 29 genes on one DNA strand, and the remaining 50 on the opposite strand. Strand-specific nuclear run-on assays showed that a low level of nonspecific transcription probably takes place over the entire chromosome, but an approximately 10-fold higher level of coding strand-specific RNA polymerase II (Pol II)-mediated transcription initiates within the strand-switch region. 5' RACE studies localized the initiation sites to a <100 bp region. Transfection studies support the presence of a bidirectional promoter within the strand-switch region, but suggest that other factors are also involved in Pol II transcription. Thus, while in most eukaryotes each gene possesses its own promoter, a single region seems to drive the expression of the entire chr1 in LmjF.