Insights on a putative aminoacyl-tRNA-protein transferase of Leishmania major

Abstract

The N-end rule pathway leads to regulated proteolysis as an adaptive response to external stress and is ubiquitous from bacteria to mammals. In this study, we investigated a gene coding for a putative core enzyme of this post-translational regulatory pathway in Leishmania major, which may be crucial during cytodifferentiation and the environment adaptive responses of the parasite. Leucyl, phenylalanyl-tRNA protein transferase and arginyl-tRNA protein transferase are key components of this pathway in E. coli and eukaryotes, respectively. They catalyze the specific conjugation of leucine, phenylalanine or arginine to proteins containing exposed N-terminal amino acid residues, which are recognized by the machinery for the targeted proteolysis. Here, we characterized a conserved hypothetical protein coded by the LmjF.21.0725 gene in L. major. In silico analysis suggests that the LmjF.21.0725 protein is highly conserved among species of Leishmania and might belong to the Acyl CoA-N-acyltransferases (NAT) superfamily of proteins. Immunofluorescence cell imaging indicates that the cytosolic localization of the studied protein and the endogenous levels of the protein in promastigotes are barely detectable by western blotting assay. The knockout of the two alleles of LmjF.21.0725 by homologous recombination was only possible in the heterozygous transfectant expressing LmjF.21.0725 as a transgene from a plasmid. Moreover, the kinetics of loss of the plasmid in the absence of drug pressure suggests that maintenance of the gene is essential for promastigote survival. Here, evidence is provided that this putative aminoacyl tRNA-protein transferase is essential for parasite survival. The enzyme activity and corresponding post-translational regulatory pathway are yet to be investigated.

Fig 1. Amino-acid sequence alignment of LmjF.21.0725 homologs derived from various Leishmania species.

Fig 2

(A) PANTHER classification system categorizing LmjF.21.0725 as L/F transferase; InterProScan results showing the signature motif acyl-CoA-N-acyltransferase (NAT) and the predicted amino acid sequence of LmjF.21.0725. (B) Three-dimensional structure of LmjF21.0725 predicted by the Phyre2 server. (C) Template c2cxA (L/F transferase from E. coli) (D) Superimposed modeled structure of LmjF21.0725 with template c2cxA (E) Amino-acid sequence alignment between LmjF21.0725 and L/F transferase from E. coli.

Fig 3

(A) Phylogenetic tree based on sequences of 23 selected homologues of LmjF21.0725, constructed by the UPGMA algorithm. (B) Pairwise sequence alignment between arg-tRNA protein transferase, e.g., Aat of P. falciparum and Lmj.21.0725. (C) Multiple sequence alignment of putative L/F transferase from various species of Plasmodium.

Fig 4. Analysis of the LmjF.21.0725 locus anatomy in wild-type and transfectants of L. major.

(A) In silico physical map of HYG replacement of the endogenous allele is depicted in the cartoon and includes SalI digestion sites (above); expected fragment size of SalI digested DNA is indicated below (interrupted line) and HYG probe fragment is depicted as coarse line above diagram. Southern blotting membrane hybridized with HYG probe is shown below. (B) The in silico physical map of PAC replacement of endogenous allele is depicted in cartoon and includes SalI digestion sites (above); expected fragment size of SalI digested DNA is indicated below (interrupted line) and PAC probe fragment is depicted as coarse line above diagram. The Southern blotting membrane hybridized with PAC probe is shown below. (C) The in silico physical map of endogenous LmjF.21.0725 allele is depicted in cartoon and includes SalI digestion sites (above); the expected 2 fragments of SalI digested DNA are indicated below (interrupted line), and the LmjF.21.0725 probe fragment is depicted as the coarse line above the diagram. The southern blotting membrane hybridized with the LmjF.21.0725 probe is shown below. (D) Map of pxNeoGFP-LmjF21.0725 used for overexpression of LmjF.21.0725, and SalI digestion predicted fragment sizes are indicated below. Leishmania wild-type (WT) and transfectants are indicated in each blot as follows: +/+ = WT; +/PAC = heterozygous line with a PAC cassette replacing one endogenous allele; +/PAC/(+): heterozygous transfectant bearing PAC and plasmid expressing LmjF21.0725 (pxNeoGFP-LmjF21.0725); PAC/HYG/(+) = double replacement transfectant with both selectable markers and plasmid expressing LmjF21.0725 (pxNeoGFP-LmjF.21.0725, addback transfectant). Marker: Lambda DNA-HindIII digested.

Fig 5. Analysis of the LmjF.21.0725 locus structure on independent transfectant clones and comparative analysis of their molecular karyotypes.

(A) Southern blot of genomic DNA from five different PAC/HYG/ (+) Leishmania clones digested with SalI and hybridized with an LmjF21.0725 fragment as probe. Arrows pointing to unexpected bands present in all PAC/HYG/ (+) clones (lane 1 to 5) and in +/PAC (lane 6) cell lines, +/+ (lanes 7 and 8) that revealed the expected locus architecture (lane 7- L. major CC1; lane 8—L. major Friedlin). (B) Southern blot of genomic DNA digested with SalI and hybridized with PAC-specific probe; arrows indicate unexpected 8 kb bands in all mutants. (C) Molecular karyotype of L. major strains by pulsed field gel electrophoresis. +/+ = WT; +/PAC = heterozygous line with a PAC cassette replacing one endogenous allele; PAC/HYG/(+) = double replacement transfectant bearing both selectable markers and plasmid expressing LmjF21.0725 (pxNeoGFP-LmjF21.0725, addback transfectant). Marker: Lambda ladder (New England Biolabs).

Fig 6. LmjF.21.0725 is essential for L. major promastigotes.

(A) FACS analysis was used to quantify GFP levels and estimate dynamics of passive loss of pXNeoGFP-LmjF.21.0725 in four independent transfectants in the absence of G418(depicted as numbers 1 to 4, top of the panel). (B) With same culture conditions, western blot analysis indicated differences in LmjF.21.0725 expression levels in transfectant lines, using anti-LmjF.21.0725 antibody. (C) Fluorescent microscopy images showing GFP expression in transfectants (10^th passage in absence of G418). Lines: +/PAC (+) = heterozygous cell line with pXNeoGFP-LmjF21.0725; HYG/PAC(+) = double replacement transfectant bearing both selectable markers and plasmid expressing LmjF.21.0725 (pxNeoGFP-LmjF21.0725, addback transfectant).

Fig 7. Immunolocalization of LmjF.21.0725 in L. major.

(A) Subcellular localization of Lmj21.0725 in L. major strains: (+/+: WT; +/PAC/ (+): heterozygous cell line with addback pxNeoGFP-LmjF21.0725; PAC/HYG/ (+): Null mutant with addback pxNeoGFP-LmjF21.0725; Representative cells were visualized with bright field (B/F), DAPI and anti-LmjF.21.0725 antibody overlays. Nuclei and kinetoplasts were stained with DAPI (blue). (B) Western blot analysis with anti-LmjF.21.0725 antibody confirming expression of ~42 kDa LmjF.21.0725 in L. major promastigotes (+/+: WT; +/PAC/ (+): heterozygous cell line with addback pxNeoGFP-LmjF21.0725; HYG/PAC(+): Null mutant with addback pxNeoGFP-LmjF.21.0725; +/PAC/ (V) L. major carrying Leishmania expression vector pXNeoGFP. (C) Growth curve of L. major WT and mutant promastigotes (+/+: WT; +/PAC/ (+): heterozygous cell line with addback pxNeoGFP-LmjF21.0725; HYG/PAC(+): Null mutant with addback pxNeoGFP-LmjF.21.0725; +/PAC/ (V) L. major carrying Leishmania expression vector pXNeoGFP.