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A Leishmania-specific gene upregulated at the amastigote stage is crucial for parasite survival

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Abstract

The morphological and biochemical alterations between the two life stages of Leishmania are governed by stage-regulated expression of several genes. Amastigote-specific genes are believed to have a role in the survival and replication of the parasite in the hostile environment of the mammalian host. Previously, we have reported the upregulated expression of A1 gene (LdA1) at the amastigote stage at RNA level. In the present study, we have further characterized LdA1, in order to understand its role in Leishmania. Sequence homology search revealed that LdA1 is unique to the Leishmania genus. Sequence- and structure-level functional annotations predicted the involvement of LdA1 in a range of biological processes critical for survival of the parasites. Western blot confirmed the upregulated expression of LdA1 at the protein level at the amastigote stage. Overexpression of LdA1 in Leishmania did not affect its growth, phenotype, or infectivity. Attempts to generate null mutants of LdA1 by homologous recombination were not successful. Repeated inability to create null mutants of LdA1 was suggestive of gene essentiality. Mutant parasites with a single allele deletion of LdA1 (LdA1+/−) showed reduction in motility, size, and growth rate at both the life stages in vitro, which was restored following gene add-back by episomal expression of LdA1 in mutant parasites. Although LdA1+/− parasites were able to infect macrophages ex vivo, their capacity to survive inside macrophages was reduced significantly (P < 0.01) beyond 72 h of infection. In conclusion, LdA1 is a Leishmania-specific gene having upregulated expression at the amastigote stage and is important for survival of Leishmania parasite.

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Acknowledgments

We thank the Biomedical Informatics Centre, National Institute of Pathology, for providing the technical assistance with in silico analysis.

Funding

This work was funded by grants from the Department of Biotechnology (DBT), Government of India (Grant no. BT/PR441/MED/15/7/2011). KA1 and KA2 are grateful to the Council for Scientific and Industrial Research (CSIR) and the Department of Science and Technology (DST), India, for fellowships. PS is grateful to DST for JC Bose Fellowship.

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Authors and Affiliations

  1. Molecular Parasitology, National Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi, India

    Kumar Avishek, Dibyabhaba Pradhan & Poonam Salotra

  2. Jamia Hamdard-Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India

    Kavita Ahuja & Angamuthu Selvapandiyan

  3. Division of Emerging and Transfusion Transmitted Diseases, CBER, FDA, Bethesda, MD, USA

    Sreenivas Gannavaram & Hira L. Nakhasi

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  1. Kumar Avishek
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  2. Kavita Ahuja
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  3. Dibyabhaba Pradhan
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  4. Sreenivas Gannavaram
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  5. Angamuthu Selvapandiyan
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  6. Hira L. Nakhasi
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  7. Poonam Salotra
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Contributions

Kumar Avishek (KA1), AS, SG, HLN, and PS conceived of and designed the experiments. KA1 and Kavita Ahuja (KA2) performed the experiments. PS and AS contributed to the reagents/materials/analysis tools. DP, KA1, and PS performed the in silico analysis, and DP contributed in its write up. KA1 and PS wrote the paper. AS, HLN, and SG critically revised the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Poonam Salotra.

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The authors declare that they have no conflict of interest.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Electronic supplementary material

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Supplementary Fig. 1

Phylogenetic analysis showing evolutionary patterns of putative LdA1 protein in Leishmania. Phylogenetic tree was constructed using Ensemblprotists gene tree database showing the different clusters of LdA1. (PNG 78 kb)

High resolution image (TIFF 1020 kb)

Supplementary Fig. 2

Multiple sequence alignment for LdA1 from Leishmania species. (A) Sequence alignment was developed for LdA1 sequence from different species of Leishmania in Clustal W-alignment format. Amino acids are listed in the standard one-letter code, and residues identical to Leishmania LdA1 are indicated by stars. (B) The percentage sequence identity of LdA1 between different species of Leishmania. (PNG 3052 kb)

High resolution image (TIF 4154 kb)

Movie 2

Reduced size, motility and number of LdA1+/− mutant parasites after replacement of LdA1 with neor (movie 1) and hygr (movie 2) as compared to the wild type parasites (movie 3) in log phage culture. Defect in growth and morphology was rescued after episomal expression of LdA1 in LdA1+/− mutant parasites (Movie 4). (MP4 4737 kb)

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Avishek, K., Ahuja, K., Pradhan, D. et al. A Leishmania-specific gene upregulated at the amastigote stage is crucial for parasite survival. Parasitol Res 117, 3215–3228 (2018). https://doi.org/10.1007/s00436-018-6020-6

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