Leishmania donovaniRequires Functional Cdc42 and Rac1 To Prevent Phagosomal Maturation

The protozoan parasite Leishmania donovani, the causative agent of visceral leishmaniasis, is transmitted to humans through bites by infected sand flies. Flagellated Leishmania promastigotes are phagocytosed by host macrophages, where they survive inside phagosomes (9, 27). Lipophosphoglycan (LPG), which is the major surface glycoconjugate of promastigotes, is essential for intracellular survival (10, 15, 23, 30). Phagosomes containing L. donovani mutants that lack the repeating sugars of LPG (lpg2−/− knockout [KO]) proceed through the phagolysosomal pathway, and the parasites are killed (10). The mechanism by which LPG arrests phagosomal maturation in macrophages is partially clarified and includes accumulation of F-actin around newly formed phagosomes (17). The effects of LPG on actin are in part related to inhibition of protein kinase Cα (PKCα), an enzyme implicated in F-actin depolymerization at the phagosomal membrane (2, 16, 17, 23, 25). Results from our group indicate that LPG from L. donovani interacts with GM1-enriched lipid microdomains (lipid rafts, detergent-resistant membranes) in the macrophage plasma membrane and that active PKCα colocalizes to a lesser degree with GM1 in these cells (unpublished data). Moreover, it was recently shown that LPG causes a disorganization of the phagosomal membrane, preventing normal assembly of GM1-enriched microdomains (8). In line with this, we found that disrupting membrane microdomains by cholesterol depletion, thereby mimicking the effect of LPG, raised the levels of periphagosomal F-actin around LPG-deficient lpg2−/− KO mutants. However, the levels of periphagosomal F-actin in cholesterol-depleted cells did not reach the levels observed around wild-type (WT) promastigotes (unpublished data). The present study investigates the involvement of the Rho family members Cdc42 and Rac1 in the generation of the F-actin shell formed around phagosomes containing L. donovani promastigotes. The Rho family of small GTPases, including Rho, Rac, and Cdc42, has emerged as main regulators of the cytoskeleton (11, 14). GTP-bound Rho GTPases drive cellular processes such as cytoskeletal reorganization, migration, and proliferation (7, 14, 28). Importantly, Rho GTPases play a profound role in phagocytosis of opsonized prey (6, 22, 26, 29); however, their role in nonopsonic phagocytosis is poorly understood. Dominant negative (N17) and constitutively active (V12) forms of Cdc42 and Rac1, expressed as fusion proteins with the human immunodeficiency virus-derived cell permeable peptide TAT, were used to study the effects of these proteins on periphagosomal F-actin and phagosomal maturation in RAW264.7 macrophages. We found that simultaneous introduction of N17Cdc42 and N17Rac1 prevented the accumulation of F-actin around phagosomes with WT L. donovani. Conversely, introduction of V12Cdc42 and V12Rac1 restored the accumulation of periphagosomal F-actin around phagosomes with LPG-defective promastigotes, resulting in inhibition of phagosomal maturation. Interestingly, introduction of N17Cdc42 and N17Rac1 into the cells, which reduced the levels of periphagosomal F-actin around WT L. donovani, did not promote maturation of these phagosomes. This result points toward a direct role for Cdc42 and Rac1 in phagosomal maturation. In conclusion, our results show that L. donovani requires functional Cdc42 and Rac1 to build up a coat of F-actin around its phagosome to prevent phagosomal maturation.