Evidence that a naturally occurring single nucleotide polymorphism in the RagC gene of Leishmania donovani contributes to reduced virulence

Abstract

Leishmaniasis is a widespread neglected tropical disease transmitted by infected sand flies resulting in either benign cutaneous infection or fatal visceral disease. Leishmania donovani is the principal species responsible for visceral leishmaniasis, yet an atypical L. donovani has become attenuated in several countries including Sri Lanka and causes cutaneous leishmaniasis. Previous studies have identified 91 genes altered in the atypical cutaneous L. donovani compared to typical visceral disease associated L. donovani including mutations in the RagC and Raptor genes that are part of the eukaryotic conserved TOR pathway and its upstream sensing pathway. In the present study, we investigate whether the RagC R231C mutation present in atypical cutaneous L. donovani introduced into the virulent L. donovani 1S2D chromosome by CRISPR gene editing could affect virulence for survival in visceral organs. Through bioinformatic analysis, we further investigated the presence of sensing pathway components upstream of TOR in L. donovani including RagC complexing proteins, RagA and Raptor. L. donovani 1S2D edited to express mutant RagC R231C were viable in promastigote but had reduced visceral parasitemia in infected BALB/c mice. The RagC R231C mutant retained the ability to interact with RagA and gene knockout experiments revealed that although the RagA gene was essential, the RagC gene was not essential under promastigote culture conditions but was essential for survival in the liver of experimentally infected mice. These results provide evidence that the TOR associated sensing pathway plays a prominent role in L. donovani visceral disease and the RagC R231C mutation contributed to the atypical pathology of cutaneous L. donovani in Sri Lanka.

TORC1 is an essential regulator of nutrient-dependent differentiation inLeishmania

“…Leishmania TORC1 composition, function and regulation is unknown. Bioinformatic analysis by us and others 7,11,32 identified Leishmania homologues for the key TOR1 complex members TOR, RPTOR1 and LST8 (also found in TORC2) but not for DEPTOR and PRAS40, which associate with these TORC1 members in vertebrates 4,33 . In T. brucei two distinct TOR1 and TOR2 complexes were identified with TORC1 containing TOR1 and RPTOR, and TORC2 containing TOR2 and RICTOR.…”

Reconstitution of Mycobacterium marinum Nonhomologous DNA End Joining Pathway in Leishmania

“…Although Mm NHEJ mediated repairs can account for a quarter of the total DSB repair events in Mm Ku and Ligase D expressing Leishmania cells, further optimization of Mm NHEJ is needed to improve its DSB repair efficiency for CRISPR gene targeting without using a donor DNA in Leishmania . Depending on whether a heterologous protein is stable, harmless, or toxic, its expression level could vary greatly with time in Leishmania ( 62 ). It appears the proper expression level and ratio of Mm Ku and Ligase D are necessary for making efficient Mm NHEJ repairs since overexpression of Mm Ku alone may inhibit DSB repair ( 20 , 34 , 35 , 63 ).…”

Gene Annotation and Transcriptome Delineation on a De Novo Genome Assembly for the Reference Leishmania major Friedlin Strain

“…Although two of the SNPs represent silent changes, the other SNP and the InDel would be suggesting a possible co-existence of two proteins, differing each other in two amino acids ( Figure 8 , panel B). If this change in the sequence has a functional role that merits being analyzed, in view of a recent article in which a single amino-acid substitution in the L. donovani RagC protein was found to dramatically affect the virulence of this parasite [ 67 ]. The gene LMJFC_070017800 was annotated as coding for a putative nucleolar RNA-binding protein, but no additional studies on this protein have been reported to date.…”