Recombination-driven generation of the largest pathogen repository of antigen variants in the protozoan Trypanosoma cruzi

Abstract

BackgroundThe protozoan parasite Trypanosoma cruzi, causative agent of Chagas disease, depends upon a cell surface-expressed trans-sialidase (ts) to avoid activation of complement-mediated lysis and to enhance intracellular invasion. However these functions alone fail to account for the size of this gene family in T. cruzi, especially considering that most of these genes encode proteins lacking ts enzyme activity. Previous whole genome sequencing of the CL Brener clone of T. cruzi identified ~1400 ts variants, but left many partially assembled sequences unannotated.ResultsIn the current study we reevaluated the trans-sialidase-like sequences in this reference strain, identifying an additional 1779 full-length and partial ts genes with their important features annotated, and confirming the expression of previously annotated “pseudogenes” and newly annotated ts family members. Multiple EM for Motif Elicitation (MEME) analysis allowed us to generate a model T. cruzi ts (TcTS) based upon the most conserved motif patterns and demonstrated that a common motif order is highly conserved among ts family members. Using a newly developed pipeline for the analysis of recombination within large gene families, we further demonstrate that TcTS family members are undergoing frequent recombination, generating new variants from the thousands of functional and non-functional ts gene segments but retaining the overall structure of the core TcTS family members.ConclusionsThe number and variety as well as high recombination frequency of TcTS family members supports strong evolutionary pressure, probably exerted by immune selection, for continued variation in ts sequences in T. cruzi, and thus for a unique immune evasion mechanism for the large ts gene family.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3037-z) contains supplementary material, which is available to authorized users.

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins

“…The nearly 100% editing efficiency achieved using RNPs with selected sgRNAs may not be only about transfection efficiency, as it substantially exceeds that reported in mammalian cells using SpCas9-RNP complexes ( 9 ). We hypothesize that this high efficiency may also reflect the well-documented plasticity of the kinetoplastid genomes, wherein recombination is frequent and widespread ( 16 ) and is further facilitated by the targeted cuts introduced by Cas9.…”

“…Our group has focused on the development and application of techniques—including the CRISPR-Cas9 system—for functional analyses of genes in the protozoan parasite Trypanosoma cruzi , the cause of human Chagas disease ( 5 , 11 ). Due to extensive genotypic and phenotypic variation within the species ( 12 , 13 ), its complex genome, with a large number of very-high-copy-number multigene families ( 14 – 16 ), and the absence of tools for the facile manipulation of genes (e.g., unlike the related African trypanosome species, T. cruzi lacks functional RNA interference [RNAi] machinery), T. cruzi is considered one of the least studied and most poorly understood disease-causing pathogens.…”

Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole

“…Using gene ontology annotations, it was possible to link mutations to functional categories. The majority occurred in hypervariable regions, including the large and highly diverse surface antigen gene families, such as the mucins and trans-sialidases, which constitute much of the parasite genome 23 . In functional categories or metabolic pathways that have been tentatively associated with drug-resistance 8 , 14 , 16 , 24 – 27 , there were a range of SNP frequencies ranging from 0.47/kb in genes linked to electron transport to 0.11/kb in ABC transporter genes (Fig.…”

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

“…In Trypanosoma brucei, HR factors have been clearly implicated in the directed recombination of Variant Surface Glycoprotein (VSG) genes during host immune evasion by antigenic variation (21), as well as in maintenance of the massive subtelomeric VSG gene archive (22,23). In T. cruzi, HR has been suggested to be a driver of variability in multigene families (24,25) and in cell hybridisation (26). Finally, in Leishmania, HR related factors have been implicated in mediating the formation or maintenance of episomes, which appear to form stochastically, arise genome-wide and have been implicated in acquisition of drug resistance (27)(28)(29)(30)(31)(32).…”