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. 2022 Feb 28;12(1):3266.
doi: 10.1038/s41598-022-07412-z.

Absence of DEATH kinesin is fatal for Leishmania mexicana amastigotes

Suad Gazi Jaafer Husaine Al Kufi  1   2 , Josiah Emmerson  1 , Heidi Rosenqvist  1 , Catarina Mateus Moreira Garcia  1 , Diana Onodelia Rios-Szwed  1   3 , Martin Wiese  4
Affiliations

Absence of DEATH kinesin is fatal for Leishmania mexicana amastigotes

Suad Gazi Jaafer Husaine Al Kufi et al. Sci Rep. .

Abstract

Kinesins are motor proteins present in organisms from protists to mammals playing important roles in cell division, intracellular organisation and flagellum formation and maintenance. Leishmania mexicana is a protozoan parasite of the order Kinetoplastida causing human cutaneous leishmaniasis. Kinetoplastida genome sequence analyses revealed a large number of kinesins showing sequence and structure homology to eukaryotic kinesins. Here, we investigate the L. mexicana kinesin LmxKIN29 (LmxM.29.0350), also called DEATH kinesin. The activated MAP kinase LmxMPK3, a kinase affecting flagellum length in Leishmania, is able to phosphorylate recombinant full length LmxKIN29 at serine 554. Insect promastigote LmxKIN29 Leishmania null mutants showed no obvious phenotype. However, in mouse infection experiments, the null mutants were unable to cause the disease, whereas LmxKIN29 add-backs and single allele knockouts caused footpad lesions. Localisation using promastigotes expressing GFP-tagged LmxKIN29 revealed that the kinesin is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells the GFP-fusion protein was found at the anterior and posterior ends of the cells indicating a role in cytokinesis. The inability to cause lesions in infected animals and the amino acid sequence divergence from mammalian kinesins suggests that LmxKIN29 is a potential drug target against leishmaniasis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Features of LmxKIN29. (A) amino acid sequence of LmxKIN29 with putative MAP kinase phosphorylation sites underlined (serine 333, threonine 440, serine 548, serine 551, and serine 554), the motor domain (R20-I355) is shown in grey (pfam00225), the DEATH motif (D279–H283) is bold and underlined. R20, R22, P23 (↓), G100-F108 (*, Walker A motif GxxxxGKT/S), the switch-1 motif (NxxSSR)(.) and D261 of the switch-2 motif (DxxGxE)(:) are involved in ATP-binding; R308, K311, and R314 are residues of the microtubule-binding site (#); I355 in bold is involved in neck linker binding; K368 marks the start of the coiled-coil dimerisation domain (^). (B) 3D model of LmxKIN29 using 1goj in SWISS-MODEL and displayed by Jmol FirstGlance 3.0 (http://firstglance.jmol.org) showing the localisation of the neck linker, of the ATP-binding and microtubule-binding sites, and of serine 333. (C) coiled-coil prediction for LmxKIN29 (left) and its T. brucei orthologue (right) using COILS version 2.2 and a window of 21 amino acids. (D) partial amino acid sequence alignment of LmxKIN29 from L. mexicana with the amino acid sequences of various kinetoplastid kinesins. Lmex, LmxM.29.0350 Leishmania mexicana MHOM/GT/2001/U1103; Lama, LAMA_000589400 Leishmania amazonensis MHOM/BR/71973/M2269; Lmaj, LmjF.30.0350 Leishmania major strain Friedlin; Linf, LINF_300008500-T1 Leishmania infantum JPCM5; Ldon, LdBPK_300350.1.1 Leishmania donovani BPK282A1; Laet, LAEL147_000550000 Leishmania aethiopica L147; Ltro, LTRL590_300009000 Leishmania tropica L590; Cfas, CFAC1_260021700 Crithidia fasciculata strain Cf-Cl. The asterisk (*) indicates conserved amino acid residues in all orthologues (alignment by Clustal Omega modified).
Figure 2
Figure 2
Determination of the LmxMPK3 LmxKIN29 phosphorylation site. (A) radiometric kinase assay of activated His-LmxMPK3 with different GST-LmxKIN29 proteins resolved on 14% SDS-PAGE and Coomassie-stained; (B) autoradiograph after 24 h of exposure. Lane 1, His-LmxMPK3 + GST-LmxKIN29; lane 2, His-LmxMPK3 + GST-LmxKIN29S551A; lane 3, His-LmxMPK3 + GST-LmxKIN29A2; lane 4, His-LmxMPK3 + GST-LmxKIN29S554A; M, marker in kDa.
Figure 3
Figure 3
PCR to confirm deletion of LmxKIN29. Lanes 1, 4, 7, 10, and 13, detection of LmxKIN29 by an 860 bp PCR fragment; lanes 2, 5, 8, 11 and 14, detection of correct integration of BSD by a 937 bp PCR fragment; lanes 3, 6, 9, 12, and 15, detection of correct integration of PHLEO by a 766 bp PCR fragment. Marker in kb.
Figure 4
Figure 4
Morphological analysis of promastigotes of LmxKIN29 deletion mutants in L. mexicana. (A) cell body length in µm; (B) cell body width in µm; (C) flagellum length in µm. Boxplot of distribution of measurements. Different shades of grey from left to right indicate results for wild type (WT) L. mexicana, single allele knockout with BSD (BSD), single allele knockout with PHLEO (PHLEO), two null mutants with BSD and PHLEO, A3 and D11. × , means. Stars indicate significant differences (p** < 0.01, p*** < 0.001) (Student’s t-Test). Measurements of 189 random cells were taken using Image J.
Figure 5
Figure 5
LmxKIN29 genomic add-back. (A) genomic situation in ΔLmxKIN29-/- null mutant. (B) LmxKIN29 add-back construct; (C) add-back replacing PHLEO; (D) add-back replacing BSD; (E) PCR to prove correct replacement. Lanes 1 and 2, null mutant A3; lanes 3 and 4, LmxKIN29 add-back A3E2; lanes 5 and 6, LmxKIN29 add-back D11H2; lanes 1, 3, and 5, primer pair to amplify a 1481 bp DNA fragment showing correct integration of 3’-end of PACLmxKIN29 construct; lanes 2, 4, and 6 primer pair to amplify 1308 bp fragment indicating correct integration of 5’-end of PACLmxKIN29; all lanes including lanes for the null mutants show amplification of either one or two additional unspecific bands between 400–800 bp, which serve as internal controls. M, DNA marker in kb.
Figure 6
Figure 6
Footpad infection of female Balb/c mice with wild type, single-allele knockout, double-allele knockout, and add-back clones. WT, L. mexicana wild type; Phleo, ΔLmxKIN29+/- single-allele knockout; A3 and D11, independet ΔLmxKIN29-/- double allele knockout clones; A3E2, add-back clone for A3; D11H2, add-back clone for D11.
Figure 7
Figure 7
Fluorescence microscopy of LmxKIN29 in live promastigotes. (A, B) L. mexicana promastigotes carrying pTHKin29GFP clone A3C12 (GFP fused to C-terminus); (C, D) L. mexicana promastigotes carrying pTHGFPKin29, D11C2 (GFP fused to N-terminus). Bar, 10 μm.
Figure 8
Figure 8
Expression of GFP-tagged LmxKIN29 in L. mexicana promastigotes. (A) 14% Coomassie-stained SDS-PAGE; (B) immunoblot using an anti-GFP antibody. Lanes 1 and 2, pTHGFPLmxKIN29 A3C10 and D11C2, respectively (N-terminal GFP); lane 3, L. mexicana expressing GFP only; lanes 4 and 5, pTHKin29GFP A3C12 and D11A1, respectively (C-terminal GFP). Marker in kDa.
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