Leishmania major virulence attenuation in vitro: An old conundrum revisited in the omics era

BACKGROUND: 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, albeit reduced, 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, this line, as well avirulent parasites passaged five times through the sand fly vector Phlebotomus duboscqi developed very efficiently in sand flies. MATERIALS AND METHODS: 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 minicircle repertoire. RESULTS: While genetic mutations contributed little to differences in host survival, changes in the gene copy number were correlated with alterations to avirulence. Survival capacity strongly correlated with gene expression patterns. Avirulent parasites showed increased abundance of ribosomal and translation-related transcripts compared with lines capable of persistent survival, suggesting selective pressure to restrict translational capacity in hosts. 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 and sand fly passage. CONCLUSIONS: Our results indicate that at the DNA and mRNA levels, L. major survival in the 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.