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Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. We have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir-Leishmania interactions, we selected Nelfinavir-resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir-resistant and -sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time-dependent manner. Furthermore, high-resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug-induced intracellular vesicles., (© 2013 John Wiley & Sons Ltd.)

Whereas bacterial pathogens take over the control of their host cell actin cytoskeleton by delivering an array of protein effectors through specialized secretion systems, promastigotes of the protozoan parasite Leishmania donovani rely entirely upon a cell surface glycolipid to achieve this feat. Here, we review recent evidence that L. donovani promastigotes subvert host macrophage actin dynamics during the establishment of infection and we discuss the potential mechanisms involved.

Phagocytosis of Leishmania donovani promastigotes is characterized by an inhibition of phagolysosome biogenesis mediated by the surface glycolipid lipophosphoglycan (LPG). However, the consequences of this inhibition on macrophage function remain to be determined. In this study, we investigated the impact of LPG-mediated phagosome remodelling on the assembly and function of the NADPH oxidase complex. Phagocytosis of both wild-type and LPG-defective L. donovani promastigotes triggered the release of similar levels of superoxide. However, wild-type promastigotes, but not LPG-defective mutants, inhibited generation of superoxide at the phagosome. Confocal microscopy imaging revealed that the membrane component gp91(phox) and the Rho-family GTPase Rac1 were present on phagosomes containing either wild-type or LPG-defective promastigotes. In contrast, the NADPH oxidase cytosolic components p47(phox) and p67(phox) were excluded from phagosomes in a LPG-dependent fashion. This inhibition is not the consequence of a general defect in the initiation of the NADPH oxidase activation process because both wild-type and LPG-defective promastigotes induced p47(phox) phosphorylation and the formation of complexes containing p47(phox) and p67(phox). Thus, by remodelling their intracellular habitat, L. donovani promastigotes prevent the assembly of a functional phagosomal NADPH oxidase complex, thereby evading an important host innate defence mechanism.

Macrophages produce little superoxide during phagocytosis of Leishmania donovani amastigotes. In this study, we characterized molecular events associated with L. donovani amastigotes uptake by mouse macrophages, to further define the mechanisms by which they are internalized without triggering superoxide production. Using transient transfections, we first showed that internalization of L. donovani amastigotes is mediated by the GTPases Rac1 and Arf6, of which Rac1 is recruited and retained on parasite-containing phagosomes. Next, we showed that, whereas internalization of amastigotes induced no superoxide release, co-internalization of serum-opsonized zymozan and amastigotes resulted in superoxide production. Furthermore, in co-internalization experiments, we detected superoxide production in over 95% of phagosomes containing IgG-opsonized SRBC compared to 5% of amastigote-harboring phagosomes. These results suggest that amastigotes evade the ability of macrophages to produce superoxide during phagocytosis. Consistently, we observed that amastigotes induced barely detectable phosphorylation of the NADPH oxidase component p47phox, leading to a defective phagosomal recruitment of p67phox and p47phox. Finally, we showed that amastigotes disrupt phagosomal lipid raft integrity, potentially interfering with NADPH oxidase assembly. Collectively, our results indicate that internalization of L. donovani amastigotes is a Rac1- and Arf6-dependent process that occurs in the absence of significant NADPH oxidase activation.

Leishmania donovani promastigotes inhibit phagosome maturation and induce the accumulation of periphagosomal F-actin during the establishment of infection within macrophages. These events are mediated by the surface glycolipid lipophosphoglycan (LPG), but the underlying mechanisms remain to be elucidated. In this study, we addressed the role of the Rho-family GTPases RhoA, Rac1 and Cdc42 in the uptake of L. donovani promastigotes and in the accumulation of periphagosomal F-actin. Confocal microscopy analyses revealed that association of both Rac1 and RhoA to phagosomes containing L. donovani promastigotes was independent of the presence of LPG. In contrast, Cdc42 and proteins required for F-actin assembly (Arp2/3, WASP, Myosin, alpha-actinin) were retained on phagosomes in a LPG-dependent manner. Expression of the RhoA inhibitor C3-transferase blocked the internalization of complement-opsonized promastigotes, whereas the dominant-negative Rac1N17 blocked the uptake of unopsonized promastigotes. The dominant-negative Cdc42N17 inhibited LPG-mediated phagosomal accumulation of F-actin and retention of Arp2/3 and Myosin. Thus, our data suggest that the effect of LPG on the accumulation of periphagosomal F-actin is the consequence of an abnormal retention or activation of Cdc42 at the phagosome.

Promastigotes of the protozoan parasite Leishmania are inoculated into the mammalian host by an infected sandfly and are phagocytosed by macrophages. There, they differentiate into amastigotes, which replicate in phagolysosomes. A family of glycoconjugates, the phosphoglycans (PGs), plays an important role in the ability of promastigotes to survive the potentially microbicidal consequences of phagocytosis. Lipophosphoglycan (LPG), an abundant promastigote surface glycolipid, has received considerable attention over the past several years. Of interest for this review, lipophosphoglycan confers upon Leishmania donovani promastigotes the ability to inhibit phagolysosome biogenesis. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that prevents L. donovani promastigote-harboring phagosomes from interacting with late endosomes and lysosomes. Thus, similar to several other pathogens, Leishmania promastigotes hijack the host cell's cytoskeleton early during the infection process. Here, we review this phenomenon and discuss the potential underlying mechanisms.