Integrity of the Actin Cytoskeleton of Host Macrophages is Essential for Leishmania donovani Infection

Abstract

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.

Statin-induced chronic cholesterol depletion inhibits Leishmania donovani infection: Relevance of optimum host membrane cholesterol

“…In support of these results, Rodríguez et al showed that plasma membrane cholesterol is necessary for the entry of Leishmania chagasi into host macrophages [23]. In addition, we have recently shown that destabilization of the actin cytoskeleton of macrophages results in reduction in the entry of Leishmania donovani into host cells, due to a possible cross-talk between membrane cholesterol and the actin cytoskeleton [24]. This observation is relevant in light of the emerging relationship between membrane cholesterol and the actin cytoskeleton [25-28; P. Sarkar, G.A.…”

“…Viability of macrophages upon treatment with lovastatin or MβCD-cholesterol complex was assessed using MTT assay as described earlier [24,37].…”

“…Murine macrophage cell line J774A.1 (American Type Culture Collection) was cultured as described previously [24]. Briefly, cells were maintained in IMDM medium supplemented with 2.4 g/l sodium bicarbonate, 10% heat-inactivated FCS, and antibiotic antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B) solution in a humidified atmosphere with 5% CO 2 at 37°C.…”

“…Labeling of L. donovani promastigotes with FITC was carried out as described previously [24]. Parasites in the mid log phase were incubated in 0.1% FITC in buffer A (50 mM carbonate buffer, pH 8.0) at 22°C for 1 h. Parasites were thoroughly washed in PBS and suspended in IMDM medium containing 2% heat-inactivated FCS.…”

GPCRs: Lipid-Dependent Membrane Receptors That Act as Drug Targets

“…Membrane cholesterol is often found to be distributed in a nonrandom fashion in domains (or pools) in biological and model membranes [50][51][52][53]. These domains are envisaged to be crucial since various cellular processes such as membrane sorting and trafficking [54], signal transduction [55], and the entry of pathogens [56][57][58] have been attributed to these types of domains. As mentioned earlier, the role of cholesterol in the function and organization of membrane proteins and receptors constitutes an emerging and exciting area of research [36,[41][42][43][44].…”