Identification and characterization of yeast SNF1 kinase homologs in Leishmania major

Abstract

Background: Sucrose Non Fermenting1 (SNF1) constitutes a family of protein kinases conserved in eukaryotes, plants, and fungi. SNF1 has been known to play a crucial role in stress adaptation and metabolism, enabling organisms to respond to changing environmental conditions. Initially identified in yeast, SNF1 is essential for shifting from the primary carbon source, glucose, to secondary carbon sources like sucrose. Homologs of this protein family were identified in Leishmania major, a protozoan parasite and we aimed to determine their role in this parasite.

Methods: In the present study, we identified the putative homologs of SNF1 kinase in L. major and knock out strains were prepared using the CRISPR-Cas9 knock-out strategy. The developed strains were evaluated for their growth, characteristics, protein expression and ultra structural changes in vitro and virulence in a mouse model.

Results: One of the strain named N2, was found to be completely avirulent and showed limited growth, lack of glycosomes and had a fewer mitochondria with deformed cristae. The N2 strain failed to produce infection in mice when compared to WT mice. Proteome analysis revealed an increase in ribosomal proteins in the N2 strain, highlighting the role of ribosomes in stress adaptation.

Conclusion: The essentiality of this gene for developing infections in mice underscores its potential in the development of future antileishmanial therapies and live attenuated strains.

Keywords: CRISPR-Cas9; Leishmania major; SNF1; ribosomes; stress adaptation.

FIGURE 1

Confirmation of Leishmania major KOs and sequencing of the PCR products. (A) The PCR products of N1, N2, N3 were sequenced, the sequences marked in red correspond to WT DNA and those marked in green correspond to the inserted cassette. (B) Agarose gel electrophoresis of PCR products amplified from different KO strains. N1, N2 and N3 PCR show a single band of about 1800 base pairs which corresponds to the size of inserted cassette. N4 shows two bands, One corresponding to the WT gene fragment and the other to the inserted cassette.

FIGURE 2

Characteristics of WT and KO strains and metacyclogenesis. (A) Growth curve analysis of WT, N1, N2 and N3 strains. Cells were seeded on blood agar slants overlaid with medium containing 10% serum at 1 × 10 ⁵ Cell/mL. Ten independent readings were taken at each time point for each sample. Results are obtained from three independent experiments with 10 readings captured in individual groups. Significant growth difference was found between WT vs N2 (***p < 0.001) and WT vs N3 (***p < 0.001) data is represented in mean ± SD, n = 10. (B) (Two Way Anova). Flowcytometry for WT, N2 and N3 KO strain showing variation in size and shape by increased forward scattered gated plot on the right side. N2 strain had showed increased FSC which described their bulbous shape. (C) Metacyclic parasite population quantification by peanut agglutination. Percentage of metacyclic parasite was significantly reduced in N2 strain when compared with WT (***p < 0.001) and WT vs. N3 (***p < 0.001) (One Way Anova). Data were represented by mean and SD value, n = 10.

FIGURE 3

Transmission Electron Microscopy (TEM) of the WT and N2 strains. The WT micrograph shows numerous glycosomes and mitochondria distributed throughout the cellular cytoplasm. In contrast, the N2 strain electron micrograph reveals promastigotes lacking glycosomes and having fewer mitochondria. Additionally, the mitochondria in the N2 strain exhibit deformed, irregularly shaped cristae compared to those in the WT cells. N = nucleus, M = Mitochondria, G = Glycosomes.

FIGURE 4

Whole proteome analysis of WT and N2 strains. (A) Heat map showing the differentially expressed proteins between the two strains. (B) Volcano plot illustrates the distribution of differentially expressed proteins, with green dots representing proteins with increased expression and red dots representing proteins with decreased expression in the N2 strain. (C) STRING pathway analysis of upregulated proteins in the WT. (D) STING pathway analysis for N2 strain. The clustering of proteins in the STRING pathway indicates that upregulated proteins are part of specific pathways and not randomly distributed, suggesting that the KO affects entire pathways rather than individual proteins at random.

FIGURE 5

Loss of virulence in N2 KO strain and development of infection in mice. (A) Foot pad thickness was measured for 8 weeks, per week post infection. No foot pad swelling was observed in the N2 infected mice. Five mice were taken for each time point and infection was given in both the hind foot pads (n = 10) ***, P < 0.001 (Two Way Anova). (B) Draining lymph node weight was measured at 1 month, (C) at 2 months post infection and (D) at 3 months post infection. Significantly increased lymph node weight was observed in WT mice in all 3 months post infection when compared with other groups (***p < 0.001). N2 strain infected mice. lymph node showed unaltered weight over the period of 1 to 3 months and remained constant when compared with WT mice (***p < 0.001). At 3 months post infection, WT virulent and N3 mice showed increased lymph node weight significantly increased when compared to N2 mice (***p < 0.001). Data are represented by mean ± SD, n = 10 (One Way Anova).