Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jun 24;16(6):e0010510.
doi: 10.1371/journal.pntd.0010510. eCollection 2022 Jun.

Leishmania guyanensis M4147 as a new LRV1-bearing model parasite: Phosphatidate phosphatase 2-like protein controls cell cycle progression and intracellular lipid content

Alexandra Zakharova  1 , Amanda T S Albanaz  1 , Fred R Opperdoes  2 , Ingrid Škodová-Sveráková  1   3   4 , Diana Zagirova  1 , Andreu Saura  1 , Lˇubomíra Chmelová  1 , Evgeny S Gerasimov  1 , Tereza Leštinová  5 , Tomáš Bečvář  5 , Jovana Sádlová  5 , Petr Volf  5 , Julius Lukeš  4   6 , Anton Horváth  3 , Anzhelika Butenko  1   4   6 , Vyacheslav Yurchenko  1
Affiliations

Leishmania guyanensis M4147 as a new LRV1-bearing model parasite: Phosphatidate phosphatase 2-like protein controls cell cycle progression and intracellular lipid content

Alexandra Zakharova et al. PLoS Negl Trop Dis. .

Abstract

Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genus Leishmania. Leishmania (Viannia) guyanensis is one of the most common causative agents of the American tegumentary leishmaniasis. It has previously been shown that L. guyanensis strains that carry the endosymbiotic Leishmania RNA virus 1 (LRV1) cause more severe form of the disease in a mouse model than those that do not. The presence of the virus was implicated into the parasite's replication and spreading. In this respect, studying the molecular mechanisms of cellular control of viral infection is of great medical importance. Here, we report ~30.5 Mb high-quality genome assembly of the LRV1-positive L. guyanensis M4147. This strain was turned into a model by establishing the CRISPR-Cas9 system and ablating the gene encoding phosphatidate phosphatase 2-like (PAP2L) protein. The orthologue of this gene is conspicuously absent from the genome of an unusual member of the family Trypanosomatidae, Vickermania ingenoplastis, a species with mostly bi-flagellated cells. Our analysis of the PAP2L-null L. guyanensis showed an increase in the number of cells strikingly resembling the bi-flagellated V. ingenoplastis, likely as a result of the disruption of the cell cycle, significant accumulation of phosphatidic acid, and increased virulence compared to the wild type cells.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Maximum-likelihood phylogenetic tree of 155 PAP2 and PAP2L sequences of 50 kinetoplastid species.
Only bootstrap supports over 75% are shown (red dots indicate maximal supports). The analyzed PAP2L of L. guyanensis is colored in orange. The tree is divided into three main clades colored in orange (clade containing the PAP2L investigated here and L. major Friedlin LmjF.18.0430), green (clade containing LmjF.18.0440 and other 59 PAP2-related sequences, labeled PAP2-1), and blue (clade containing LmjF.19.1350 and other 53 PAP-related sequences, labeled PAP2-2). Numbers of sequences within collapsed clades are shown in brackets. Weblogos of the PAP2 superfamily (PF01569) phosphatase domain are shown on the right for the PAP2L (top), PAP2-1 (middle), and PAP2-2 (bottom) groups. Median sequence length for the three major clades is also shown. Residues of amino acids are colored in accordance with their chemical properties: positive charged side chains are in green; negative charged side chains are in pink; polar uncharged side chains are in red; non-polar hydrophobic side chains are in orange; aromatic hydrophobic side chains are in purple; and special cases are in blue. Residues involved in catalysis are underlined. See S1 Table for additional information.
Fig 2
Fig 2. Genetic ablation of pap2l using CRISPR-Cas9 approach in L. guyanensis.
(A) Schematic representation of the wild type (WT), recombined (KO), and reintroduced (AB) pap2l alleles. Expected sizes of DNA fragments after SalI digestion, positions of the annealed probes for Southern blotting and primers for diagnostic PCR (arrowheads) are indicated. (B) Diagnostic PCR for the WT, KD and KO (left) and AB (right) pap2l alleles in L. guyanensis. NC, negative control. (C) Southern blotting analysis of the SalI-digested total genomic DNA of the WT, Cas9/T7, KD, and KO L. guyanensis. Membranes were probed with 3′UTR and Neo probes. A non-specific band in anti-3′UTR analysis is marked by the asterisk. (D) Quantitative RT-PCR analysis of pap2l expression in the WT, KD, KO, and AB L. guyanensis. Data were normalized to the expression of kmp11 and presented as means and standard deviations of three independent biological replicates. (E) Western blotting confirmation of the LgPAP2L-HA expression in the AB line. Probing with anti-tubulin antibody served as a loading control.
Fig 3
Fig 3. Morphometric analysis of L. guyanensis with ablated LgPAP2L in vitro for days 3, 5, and 7 of cultivation.
A, anterior end of the cell; K, kinetoplast; N, nucleus. Boxes and error bars indicate the median along with the upper and lower quartiles, and 97.5 percentiles, respectively. Asterisks show significant differences (*–P ≤ 0.05; **–P ≤ 0.01; ***–P ≤ 0.001), no other differences were statistically significant.
Fig 4
Fig 4. Growth curve, analysis of viral load, and bi-flagellated state of L. guyanensis with ablated LgPAP2L in vitro.
A) Growth curves of the WT, KO, and AB L. guyanensis. (B) RT-qPCR analysis of the LRV1 capsid RNA level in the WT, KO, and AB L. guyanensis after 5 days of cultivation. Data are presented as normalized fold expression over kmp11 and reported as the mean and standard deviation of three replicates. Two-tailed Student’s t-test was used for statistical analysis; ns–not significant. (C) The proportion of mono- and bi-flagellated cells in the WT, KO, and AB L. guyanensis after 5 days of cultivation. Asterisks indicate significant differences (**–P ≤ 0.01; ****–P ≤ 0.0001).
Fig 5
Fig 5. Leishmania guyanensis infection in Lutzomyia longipalpis.
Intensity and localization of infection are presented in panels (A) and (B), respectively. Numbers of dissected females are indicated above the bars in A). AMG, abdominal midgut; TMG, thoracic midgut; SV, stomodeal valve. (C) Percent of mono- and bi-flagellated L. guyanensis cells 7- and 9-days p.i. in Lutzomyia longipalpis gut. *–P ≤ 0.05; ns–not significant.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Maslov DA, Opperdoes FR, Kostygov AY, Hashimi H, Lukeš J, Yurchenko V Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution. Parasitology. 2019;146: 1–27. doi: 10.1017/S0031182018000951 - DOI - PubMed
    1. Lukeš J, Butenko A, Hashimi H, Maslov DA, Votýpka J, Yurchenko V Trypanosomatids are much more than just trypanosomes: clues from the expanded family tree. Trends Parasitol. 2018;34: 466–480. doi: 10.1016/j.pt.2018.03.002 - DOI - PubMed
    1. Butenko A, Hammond M, Field MC, Ginger ML, Yurchenko V, Lukeš J Reductionist pathways for parasitism in euglenozoans? Expanded datasets provide new insights. Trends Parasitol. 2021;37: 100–116. doi: 10.1016/j.pt.2020.10.001 - DOI - PubMed
    1. Lukeš J, Skalický T, Týč J, Votýpka J, Yurchenko V Evolution of parasitism in kinetoplastid flagellates. Mol Biochem Parasitol. 2014;195: 115–122. doi: 10.1016/j.molbiopara.2014.05.007 - DOI - PubMed
    1. Stuart K, Brun R, Croft S, Fairlamb A, Gurtler RE, McKerrow J, et al.. Kinetoplastids: related protozoan pathogens, different diseases. J Clin Invest. 2008;118: 1301–1310. doi: 10.1172/JCI33945 - DOI - PMC - PubMed

Publication types

Supplementary concepts

Grants and funding

This work was supported by the Grant Agency of Czech Republic (20-07186S for investigating bi-flagellated phenotype and lipid content to JL and VY, and 20-22689S to VY for establishing a system for genetic manipulations in L. guyanensis) and the European Regional Development Funds (CZ.02.1.01/0.0/0.0/16_019/0000759) to VY, JS, PV, and JL, grant SGS/PřF/2022 from the University of Ostrava to AZ and ATSA, Moravskoslezský kraj research initiative (RRC/10/2019 to AZ and RRC/02/2020 to ATSA) and the Ministry of Education, Youth and Sports of the Czech Republic in the frame of the Operational Program “Research, Development and Education” (CZ.02.2.69/0.0/0.0/19_073/0016939) to AZ, Scientific Grant Agency of the Slovak Ministry of Education and the Academy of Sciences (VEGA 1/0553/21) and Slovak Research and Development Agency Contracts (APVV-20-0129) to AH. FRO was supported by a grant from the de Duve Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.