PIPOLI DA FONSECA, J. | Search bioRxiv

bioRxiv2026

Initiation codon context governs translation-coupled mRNA decay and coordinated expression in the human parasite Leishmania

In the absence of canonical, promoter-based transcriptional regulation, Leishmania has evolved alternative regulatory mechanisms for adaptive gene expression, including post-transcriptional control via differential mRNA turnover. While this mechanism is recognized as critical in Leishmania, fundamental aspects of transcript stability in these parasites remain to be elucidated, such as the role of translation initiation-mediated mRNA decay. We addressed this important gap by investigating the role of the initiation codon context (Kozak sequence) in gene expression in L. donovani. Mapping Kozak sequences across the trypanosomatid genomes revealed important differences in nucleotide preference across the genus and sub-genus levels, suggesting important cis-regulatory function. Within a single species, only a small subset of possible Kozak sequences is associated with several start codons, further supporting their role in expression control. Transgenic L. donovani lines expressing EGFP under the control of distinct Kozak variants indeed demonstrated that the nucleotide context of the start codon directly modulates both protein expression and mRNA stability, which was associated with increased recruitment of mRNA to heavy polysomes. Parasite exposure to the translation inhibitor cycloheximide restored EGFP expression driven by a weak Kozak sequence, revealing a direct link between mRNA stability and Kozak-mediated translatability. RNA-seq analysis of parasites arrested for transcription or translation elongation revealed transcripts enriched for the GO terms RNA modification and pseudouridine synthesis as key targets for translation-dependent mRNA turnover. The segregation of these transcripts into functional clusters with distinct Kozak profiles further suggests that Kozak sequence composition defines Kozak-governed regulons in Leishmania. Within this regulatory framework, the -3 nucleotide is identified as the key positional determinant driving differential transcript abundance. Our work uncovers a key role for translation initiation-coupled mRNA decay in Leishmania gene expression regulation adding a previously underappreciated layer of post-transcriptional regulation in parasite adaptation.

genomics↗

bioRxiv2026

Genetic introgression and transcriptomic plasticity are associated with enhanced Leishmania infantum pathogenicity causing human cutaneous leishmaniasis in Tunisia

The protozoan parasite Leishmania infantum exhibits significant genetic variability among isolates, influencing disease manifestation and treatment response. Although L. infantum is classically described as the causative agent of Visceral Leishmaniasis (VL) - often associated with immune deficiency, cases of Cutaneous Leishmaniasis (CL) caused by this species in immunocompetent individuals have been reported in different countries. To investigate the molecular basis of this unusual shift in tissue tropism and pathogenicity, we applied comparative genomic and transcriptomic approaches on two canine isolates (CanL) and two human isolates associated with Cutaneous Leishmaniasis (CL) in Tunisia. While the CanL isolates showed close genetic similarity to the L. infantum reference strain (JPCM5), the CL isolates formed a separate, highly divergent cluster based on SNP localization and frequency, differing not only from JPCM5 but also from each other. Utilizing the metagenomics sequence classification tool Kraken, we revealed a complex hybrid nature of the CL isolates, showing introgression from L. donovani and L. tropica, suggesting that hybridization has played a key role in generating novel phenotypic traits. Integration of RNA-seq and DNA-seq data demonstrated that only a minority of gene expression variation within and in-between the CanL or CL groups reflected gene dosage effects due to copy number variation, while the majority of expression differences were independent of gene dosage, implying post-transcriptional regulatory mechanisms contributing to parasite adaptation. In conclusion, our study identifies hybridization, genome instability, and transcriptomic adaptation as interconnected drivers of the L. infantum evolutionary potential. These mechanisms can collectively enhance parasite fitness gain, potentially explaining the emergence of cutaneous disease forms in a species traditionally linked to visceral infection. Author SummaryThis research reveals that hybridization between distinct Leishmania parasite species could be a key mechanism driving the evolution of new disease forms. By demonstrating that cutaneous leishmaniasis (CL) cases are caused by hybrid L. infantum parasites whose genomes show introgression with DNA from L. donovani and L. tropica, this study reveals a molecular mechanism potentially linked to the emergence of tegumentary disease from a species traditionally known to cause visceral infection. These findings contribute to our understanding of Leishmania evolution, the emergence of atypical forms of leishmaniasis linked to hybridization, and the impact of genome instability and transcriptomic adaptation as potent forces for generating phenotypic diversity and enhancing parasite fitness.

genomics↗