Kharon1 Null Mutants of Leishmania mexicana Are Avirulent in Mice and Exhibit a Cytokinesis Defect within Macrophages

Abstract

In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δ kharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δ kharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δ kharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δ kharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

“…Fixed cells were post-fixed in osmium tetroxide, stained with uranyl acetate and embedded in Agar 100 epoxy resin as described in [58]. Sections (90 nm) were cut on an Ultracut 7 microtome (Leica), post stained with lead citrate and imaged in a Tecnai 12 transmission electron microscope (Hillsboro, OR, USA) at 120 kV.…”

Targeting the Cytochrome bc 1 Complex of Leishmania Parasites for Discovery of Novel Drugs

“…All cultures were maintained at 26°C. Axenic amastigotes of L. mexicana were cultivated at 32°C as described previously (3).…”

“…Parasites exist in several life cycle forms, including extracellular promastigotes that live in the gut of the sand fly vector and intracellular amastigotes that reside within the phagolysosomal vesicles of mammalian host macrophages. Promastigotes can be cultured readily in a variety of media and represent the most facile system for in vitro studies, and amastigotes can be grown inside cultured macrophages such as the J774.A1 cell line (2) or axenically at elevated temperatures (32 to 37°C) and at pH 5.6 to 5.7 (3). All of the drugs employed to treat the leishmaniases are suboptimal (4) and suffer from problems such as toxicity, difficulties in delivery, high expense, and development of resistance.…”

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes

“…Macrophage infections were performed by infecting differentiated macrophages from the human acute leukemia monocyte cell line (THP-1) in triplicate samples, as described (57). Briefly, 3 ϫ 10 5 differentiated THP-1 cells were infected with stationary phase L. mexicana (10:1 promastigotes:macrophages ratio) for 4 h. Infected macrophages were then washed exhaustively to remove extracellular promastigotes.…”

“…Transmission electron microscopy samples were prepared and imaged as previously described (57). Serial block face scanning electron microscopy samples were washed in 0.1 M PIPES buffer and then embedded in a mix of 4% low melting point agarose and 4% gelatin from porcine skin.…”