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Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis.

ABSTRACT: Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis, a major cause of mortality and morbidity worldwide. The host plasma membrane serves as the portal of entry for Leishmania to gain access to the cellular interior. Although several host cell membrane receptors have been shown to be involved in the entry of Leishmania donovani into host cells, the endocytic pathway involved in the internalization of the parasite is not known. In this work, we explored the endocytic pathway involved in the entry of Leishmania donovani into host macrophages, utilizing specific inhibitors against two major pathways of internalization, i.e., clathrin- and caveolin-mediated endocytosis. We show that pitstop 2, an inhibitor for clathrin-mediated endocytosis, does not affect the entry of Leishmania donovani promastigotes into host macrophages. Interestingly, a significant reduction in internalization was observed upon treatment with genistein, an inhibitor for caveolin-mediated endocytosis. These results are supported by a similar trend in intracellular amastigote load within host macrophages. These results suggest that Leishmania donovani utilizes caveolin-mediated endocytosis to internalize into host cells. Our results provide novel insight into the mechanism of phagocytosis of Leishmania donovani into host cells and assume relevance in the development of novel therapeutics against leishmanial infection.

SUBMITTER: Kumar GA

PROVIDER: S-EPMC6718660 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis.

Kumar G Aditya GA Karmakar Joyshree J Mandal Chitra C Chattopadhyay Amitabha A

Scientific reports 20190902 1

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis, a major cause of mortality and morbidity worldwide. The host plasma membrane serves as the portal of entry for Leishmania to gain access to the cellular interior. Although several host cell membrane receptors have been shown to be involved in the entry of Leishmania donovani into host cells, the endocytic pathway involved in the internalization of the parasite is not known. In this work, we explored th ...[more]

PMID: 31477757

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Project description:BACKGROUND:Insulin-like growth factor-I receptor (IGF-IR) is a tyrosine kinase receptor (RTK) associated with caveolae, invaginations of the plasma membrane that regulate vesicular transport, endocytosis and intracellular signaling. IGF-IR internalization represents a key mechanism of down-modulation of receptors number on plasma membrane. IGF-IR interacts directly with Caveolin-1 (Cav-1), the most relevant protein of caveolae. Recently it has been demonstrated that the Polymerase I and Transcript Release Factor I (PTRF/Cavin) is required for caveolae biogenesis and function. The role of Cav-1 and PTRF/Cavin in IGF-IR internalization is still to be clarified. METHODOLOGY/PRINCIPAL FINDINGS:We have investigated the interaction of IGF-IR with Cav-1 and PTRF/Cavin in the presence of IGF1in human Hacat cells. We show that IGF-IR internalization triggers Cav-1 and PTRF/Cavin translocation from plasma membrane to cytosol and increases IGF-IR interaction with these proteins. In fact, Cav-1 and PTRF/Cavin co-immunoprecipitate with IGF-IR during receptor internalization. We found a different time course of co-immunoprecipitation between IGF-IR and Cav-1 compared to IGF-IR and PTRF/Cavin. Cav-1 and PTRF/Cavin silencing by siRNA differently affect surface IGF-IR levels following IGF1 treatment: Cav-1 and PTRF/Cavin silencing significantly affect IGF-IR rate of internalization, while PTRF/Cavin silencing also decreases IGF-IR plasma membrane recovery. Since Cav-1 phosphorylation could have a role in IGF-IR internalization, the mutant Cav-1Y14F lacking Tyr14 was transfected. Cav-1Y14F transfected cells showed a reduced internalization of IGF-IR compared with cells expressing wild type Cav-1. Receptor internalization was not impaired by Clathrin silencing. These findings support a critical role of caveolae in IGF-IR intracellular traveling. CONCLUSIONS/SIGNIFICANCE:These data indicate that Caveolae play a role in IGF-IR internalization. Based on these findings, Cav-1 and PTRF/Cavin could represent two relevant and distinct targets to modulate IGF-IR function.

Project description:UnlabelledThe prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani.ImportanceLeishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in L. donovani. Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in Leishmania.

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Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis.

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Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis.

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