Gerald F. Späth

The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts

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.

Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes.

Hepatocyte growth factor induces proliferation, motility and differentiation of epithelial cells through the tyrosine kinase receptor encoded by the MET proto‐oncogene. The cytoplasmic portion of Met (referred to as cyto‐Met) is activated but only weakly transforming. In order to determine the effect of activated Met on hepatocytes, we have targeted truncated Met expression to the liver by incorporating the cDNA into a vector carrying the entire human a‐1‐antitrypsin transcriptional unit. Transgenic expression in the liver of truncated human Met, containing the regulatory and the catalytic cytoplasmic domains, renders hepatocytes constitutively resistant to apoptosis and reproducibly permits immortalization. The emerging stable cell lines are not transformed and maintain a highly differentiated phenotype judged by the retention of epithelial cell polarity and the expression of hepatocyte‐enriched transcription factors as well as hepatic products.

Is lipophosphoglycan a virulence factor? A surprising diversity between Leishmania species

Lipophosphoglycan is a prominent member of the phosphoglycan-containing surface glycoconjugates of Leishmania. Genetic tests enable confirmation of its role in parasite virulence and permit discrimination between the roles of lipophosphoglycan and related glycoconjugates. When two different lipophosphoglycan biosynthetic genes from Leishmania major were knocked out, there was a clear loss of virulence in several steps of the infectious cycle but, with Leishmania mexicana, no effect on virulence was found. This points to an unexpected diversity in the reliance of Leishmania species on virulence factors, a finding underscored by recent studies showing great diversity in the host response to Leishmania species.

Vaccination with Phosphoglycan-Deficient Leishmania major Protects Highly Susceptible Mice from Virulent Challenge without Inducing a Strong Th1 Response

Long-term immunity to Leishmania may require the continued presence of parasites, but previous attempts to create attenuated parasites that persist without causing disease have had limited success. Since Leishmania major mutants that lack lipophosphoglycan and other secreted phosphoglycans, termed lpg2−, persist indefinitely in infected mice without inducing any disease, we tested their ability to provide protection to virulent L. major challenge. In response to leishmanial Ag stimulation, cells from lpg2−-infected mice produced minimal levels of IL-4 and IL-10, as well as very low levels of IFN-γ. Nevertheless, when BALB/c mice infected with lpg2− parasites were challenged with virulent L. major they were protected from disease. Thus, these findings report on attenuated parasites that may be used to induce long-term protection against leishmaniasis and indicate that the immunity induced can be maintained in the absence of a strong Th1 response.

Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major.

ABSTRACT Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.

Inflammation Recapitulates the Ontogeny of Lymphoid Stromal Cells

Stromal cells in lymphoid tissues regulate lymphocyte recruitment and survival through the expression of specific chemokines and cytokines. During inflammation, the same signals recruit lymphocytes to the site of injury; however, the “lymphoid” stromal (LS) cells producing these signals remain poorly characterized. We find that mouse inflammatory lesions and tumors develop gp38+ LS cells, in recapitulation of the development of LS cells early during the ontogeny of lymphoid organs and the intestine, and express a set of genes that promotes the development of lymphocyte-permissive tissues. These gp38+ LS cells are induced by a robust pathway that requires myeloid cells but not known Toll- or NOD-like receptors, the inflammasome, or adaptive immunity. Parabiosis and inducible genetic cell fate mapping experiments indicate that local precursors, presumably resident fibroblasts rather that circulating precursors, massively proliferate and give rise to LS cells during inflammation. Our results show that LS cells are both programmed during ontogeny and reinduced during inflammation.

Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1−/− null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3′ UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3′ UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3′ UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

Phosphoproteomic analysis of Leishmania donovani pro- and amastigote stages.

Following transmission to the vertebrate host, the protozoan parasite Leishmania donovani differentiates into the pathogenic amastigote stage that is adapted for intracellular survival. This developmental transition is induced by environmental factors including elevated temperature and acidic pH and is likely transduced by signaling cascades involving protein kinases and their downstream phosphoprotein substrates. These signaling networks are highly adapted to the specific nutritional and physiological requirements of the organism and thus studying Leishmania phosphorylation may allow important insight into the parasite‐specific biology. We used a gel‐based approach to investigate qualitative and quantitative changes of the phosphoproteome of the major L. donovani life cycle stages. Phosphoproteins were purified by immobilized metal affinity chromatography (IMAC), separated by IEF and SDS‐PAGE using pH 4–7 IPG immobiline strips, revealed by fluorescent multiplex staining, and identified by MALDI‐MS and MS/MS. Our analysis allowed us to establish a first repertoire of the Leishmania phosphoproteome and to identify phosphoproteins implicated in stress‐ and heat shock response, RNA/protein turnover, metabolism, and signaling.

Identification of a Compensatory Mutant (lpg2−REV) of Leishmania major Able To Survive as Amastigotes within Macrophages without LPG2-Dependent Glycoconjugates and Its Significance to Virulence and Immunization Strategies

ABSTRACT Different Leishmania species rely to different extents on abundant glycoconjugates, such as lipophosphoglycan (LPG) and related molecules, in mammalian infections. Previously, we showed that Leishmania major deletion mutants lacking the Golgi GDP-mannose transporter LPG2, which is required for assembly of the dominant phosphoglycan (PG) repeats of LPG, were unable to survive in macrophages. These lpg2− mutants, however, retained the ability to generate asymptomatic, persistent infections in mice. In contrast, Ilg and colleagues showed that Leishmania mexicana LPG2 mutants retained virulence for mice. Here we identified a partial revertant population of the L. major lpg2− mutants (designated lpg2−REV) that had regained the ability to replicate in macrophages and induce disease pathology through a compensatory change. Like the lpg2 parent, the lpg2−REV revertant was unable to synthesize LPG2-dependent PGs in the promastigote stage and thus remained highly attenuated in the ability to induce infection. However, after considerable delay lpg2−REV revertant-infected mice exhibited lesions, and amastigotes isolated from these lesions were able to replicate within macrophages despite the fact that they were unable to synthesize PGs. Thus, in some respects, the lpg2−REV amastigotes resemble L. mexicana amastigotes. Future studies of the gene(s) responsible may shed light on the mechanisms employed by L. major to survive in the absence of LPG2-dependent glycoconjugates and may also improve the potential of the lpg2−L. major line to serve as a live parasite vaccine by overcoming its tendency to revert toward virulence.