Background: Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania’s ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. Methodology/Principal findings: The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. Conclusions/Significance: L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana’s general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.
Bibliographical noteFunding Information:
Support from the Czech Grant Agency (www.gacr.cz) awards 17-10656S to VY and JV, Moravskoslezsk? kraj research initiative (www.msk.cz) 01211/2016/RRC to VY and AK, ERC CZ (www.msmt.cz) grant LL1601 to JL, and the COST action (www.cost.eu) CM1307 and LD14076 is kindly acknowledged. AI was funded by grant from the University of Ostrava (www.osu.cz) SGS16/PRF/2017. This work was also financially supported by the Ministry of Education, Youth and Sports of the Czech Republic (www.msmt.cz) in the ?National Feasibility Program I?, project LO1208 ?TEWEP?. JH and TL were supported by Charles University (www.cuni.cz) project UNCE 204017/2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank members of our laboratories for stimulating discussions, Tatiana Spitzov? and Jovana S?dlov? for their help with the statistical analysis and morphometry, respectively, and Vladim?ra Najdrov?, Lubo? Voleman and Ale? Benda for their help with confocal immunofluorescence microscopy.
© 2017 Ishemgulova et al.