Authors

V Yurchenko¹; ES Gerasimov³; P Volf²; SL Zimmer⁴;  ¹ University of Ostrava, Czechia;  ² Charles University, Czechia;  ³ Moscow State University, Moscow, Russian Federation;  ⁴ University of Minnesota Medical School, Duluth campus, United States

Discussion

It has been known for decades that long-term cultivation of Leishmania in vitro frequently leads to a loss of virulence, which is attributed to the selective advantage of avirulent subpopulations that outgrow the virulent ones. In the case of L. major, avirulent parasites retained the ability to develop nearly normally in sand fly vectors, however, they could not induce lesions in BALB/c mice. Residual persistence in the inguinal lymph nodes permitted re-isolation and subsequent additional murine passages of these flagellates. While a parasite line obtained after five consecutive passages of avirulent Leishmania in mice was not fully restored to virulence, passage of avirulent parasites five times through the sand fly vector Phlebotomus duboscqi yielded a cell line that developed very efficiently in sand flies. The primary goal of the current study was to understand the molecular mechanisms responsible for the alterations in host persistence and virulence among these virulent and avirulent L. major lines.
Leishmania major cell lines with differing capacities for host survival were studied using genomic and transcriptomic approaches. Specifically, we focused on genetic mutations, gene copy number variation, differential gene expression, and differences in kinetoplast DNA, including RNA editing and repertoire of minicircles.
While genetic mutations contributed little to differences in host survival, changes in the gene copy number were documented to be involved in alterations to avirulence. Even more so, survival capacity strongly correlated with gene expression patterns. Avirulent parasites showed reduced abundance of ribosomal and translation-related transcripts compared with lines capable of persistent survival, suggesting deliberate restriction of translational capacity. Most interestingly, the relative abundance of kinetoplast DNA minicircle classes, encoding guide RNAs, was altered during culture but reverted to the virulent pattern following mouse passage. 
Our results indicate that L. major survival in insect vector or mammalian host is primarily driven by adaptive regulation of gene expression rather than fixed genetic changes. Modulation of translational capacity and host-specific expression programs appear central to parasite persistence, highlighting flexible cellular strategies that support survival in natural transmission cycles.