Protozoan parasites of the genus Leishmania undergo a complex life cycle involving transmission by biting sand flies and replication within mammalian macrophage phagolysosomes. A major component of the Leishmania surface coat is the glycosylphosphatidylinositol (GPI)-anchored polysaccharide called lipophosphoglycan (LPG). LPG has been proposed to play many roles in the infectious cycle, including protection against complement and oxidants, serving as the major ligand for macrophage adhesion, and as a key factor mitigating host responses by deactivation of macrophage signaling pathways. However, all structural domains of LPG are shared by other major surface or secretory products, providing a biochemical redundancy that compromises the ability of in vitro tests to establish whether LPG itself is a virulence factor. To study truly lpg ؊ parasites, we generated Leishmania major lacking the gene LPG1 [encoding a putative galactofuranosyl (Galf) transferase] by targeted gene disruption. The lpg1 ؊ parasites lacked LPG but contained normal levels of related glycoconjugates and GPIanchored proteins. Infections of susceptible mice and macrophages in vitro showed that these lpg ؊ Leishmania were highly attenuated. Significantly and in contrast to previous LPG mutants, reintroduction of LPG1 into the lpg ؊ parasites restored virulence. Thus, genetic approaches allow dissection of the roles of this complex family of interrelated parasite virulence factors, and definitively establish the role of LPG itself as a parasite virulence factor. Because the lpg1 ؊ mutant continue to synthesize bulk GPI-anchored Galf-containing glycolipids other than LPG, a second pathway distinct from the Golgi-associated LPG synthetic compartment must exist.trypanosomatid parasites ͉ macrophages ͉ glycoconjugates ͉ galactofuranose

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.

The abundant cell surface glycolipid lipophosphoglycan (LPG) was implicated in many steps of the Leishmania infectious cycle by biochemical tests. The presence of other abundant surface or secreted glycoconjugates sharing LPG domains, however, has led to uncertainty about the relative contribution of LPG in vivo. Here we used an Leishmania major lpg1 ؊ mutant, which lacks LPG alone and shows attenuated virulence, to dissect the role of LPG in the establishment of macrophage infections in vivo. lpg1 ؊ was highly susceptible to human complement, had lost the ability to inhibit phagolysosomal fusion transiently, and was oxidant sensitive. Studies of mouse mutants defective in relevant defense mechanisms confirmed the role of LPG in oxidant resistance but called into question the importance of transient inhibition of phagolysosomal fusion for Leishmania macrophage survival. Moreover, the limited lytic activity of mouse complement appears to be an ineffective pathogen defense mechanism in vitro and in vivo, unlike human hosts. In contrast, lpg1 ؊ parasites bound C3b and resisted low pH and proteases normally, entered macrophages efficiently and silently, and continued to inhibit host-signaling pathways. These studies illustrate the value of mechanistic approaches focusing on both parasite and host defense pathways in dissecting the specific biological roles of complex virulence factors such as LPG.phosphoglycans ͉ trypanosomatid protozoan parasite ͉ oxidant resistance ͉ inhibition of macrophage activation ͉ adhesin

Protozoan parasites of the genus Leishmania have the remarkable ability to avoid destruction in the hostile environments they encounter throughout their life cycle. The molecular details of how these pathogens persevere with impunity under harsh conditions are beginning to be understood. The fact that Leishmania parasites have adapted to not only survive, but to proliferate probably is due to the protection conferred by specialized molecules on the parasite's cell surface. One such macromolecule is a novel glycoconjugate called lipophosphoglycan. This heterogeneous, lipid-containing polysaccharide is the major surface molecule of the parasite and has been implicated in a surprisingly large number of functions that may contribute the the parasite's pathogenesis. This review emphasizes the structural aspects of lipophosphoglycan and its possible functions and biosynthesis.