Armando Jardim

Temperature-induced protein secretion by Leishmania mexicana modulates macrophage signalling and function.

Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25–26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.

Down regulation of KMP-11 in Leishmania infantum axenic antimony resistant amastigotes as revealed by a proteomic screen.

The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials, but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory promastigote isolates, but the mechanism leading to drug resistance in amastigote isolates is lagging behind. Here we describe a comparative proteomic analysis of a genetically related pair of antimonial-sensitive and -resistant Leishmania infantum axenic amastigote strains. The proteomics screen has highlighted a number of proteins differentially expressed in the resistant parasite. The expression of the protein argininosuccinate synthetase (ARGG) was increased in the drug resistant mutant while a decrease in the expression of the kinetoplastid membrane protein (KMP-11) correlated with the drug resistance phenotype. This proteomic screen highlighted several novel proteins that are putatively involved in resistance to antimonials.

Identification, expression and immunolocalization of cathepsin B3, a stage-specific antigen expressed by juvenile Fasciola gigantica.

To identify antigens that could potentially be developed as vaccines against Fasciola gigantica, somatic antigens were analyzed by immunoprecipitation using pooled sera from rats infected with F. gigantica metacercariae. A prominent antigen of the newly excysted juveniles (NEJ), cathepsin B3 protease (FgCatB3), was identified by N-terminal sequencing and PCR screening of a cDNA library. Recombinant FgCatB3 (rFgCatB3) was expressed in Pichia pastoris, and shown to catalyse the digestion of gelatin, the fluorometric substrate Z-Phe-Arg-AMC and native fibronectin, suggesting that this enzyme may be involved in digesting host connective tissues during the flukes penetration and migration in the host. Rabbit polyclonal sera against rFgCatB3 was produced and used to determine the distribution of the native cathepsin B3 protease in various developmental stages of F. gigantica. By Western blotting, cathepsin B3 was detected in the whole body (WB) extract of metacercariae and NEJ but not in 4-week-old juveniles or adult parasites which confirmed the stage-specific characteristics of cathepsin B3. Immunolocalization of cathepsin B3 protease in each parasite stage showed that high levels of FgCatB3 were present in the caecal epithelium of the metacercariae and NEJ. The differential distribution of FgCatB3 in the different life cycle stages suggests that this protease is functionally important for the juvenile stage of F. gigantica.

Peroxisomal targeting protein 14 (PEX14) from Leishmania donovani. Molecular, biochemical, and immunocytochemical characterization.

Pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other nutritional pathways in glycosomes, unique subcellular organelles related to the peroxisomes of mammals and yeasts. Most glycosomal proteins are targeted to the glycosomes by a COOH-terminal tripeptide signal similar to the peroxisomal targeting signal-1 (PTS-1). It has been proposed that PTS-1 forms a complex with the PEX5 receptor protein which then docks to the glycosomal membrane through interactions with the membrane associated PEX14 protein. To analyze the role of PEX14 in glycosomal protein import, the gene encoding the L. donovani PEX14 (LdPEX14) was isolated and shown to encode a 464 amino acid protein that exhibited very limited sequence homology with peroxisomal PEX14 proteins. In vitro binding experiments with purified recombinant LdPEX14 and LdPEX5 confirmed that LdPEX14-LdPEX5 interacted with a K(d) of 2.75 microM. When LdPEX5 was preloaded with a PTS-1 peptide, the affinity of the LdPEX14-LdPEX5 interaction affinity increased. Furthermore, binding experiments with truncated forms of LdPEX5 and LdPEX14 showed that the interaction domains localized to the amino terminal region of both proteins. Finally, confocal microscopy, subcellular fractionation, and differential extraction experiments indicated that LdPEX14 is a soluble protein that associates tightly with the glycosomal membrane and further support the role of LdPEX14 in forming a docking complex involved in glycosome biogenesis.

Modulation of the Leishmania donovani Peroxin 5 Quaternary Structure by Peroxisomal Targeting Signal 1 Ligands

ABSTRACT The import of proteins containing the peroxisomal targeting signal 1 (PTS1) into the Leishmania glycosome is dependent on the docking of the PTS1-loaded LdPEX5 cytosolic receptor with LdPEX14 on the glycosome surface. Here we show that, in the absence of PTS1, LdPEX5 is a tetramer that is stabilized by two distinct interaction domains; the first is a coiled-coil motif encompassing residues 277 to 310, whereas the second domain is localized to residues 1 to 202. By using microcalorimetry, surface plasmon resonance, and size exclusion chromatography techniques, we show that PTS1 peptide binding to LdPEX5 tetramers promotes their dissociation into dimeric structures, which are stabilized by a coiled-coil interaction. Moreover, we demonstrated that the resulting LdPEX5-PTS1 complex is remarkably stable and exhibits extremely slow dissociation kinetics. However, binding of LdPEX14 to LdPEX5 modulates the LdPEX5-PTS1 affinity as it decreases the thermodynamic dissociation constant for this latter complex by 10-fold. These changes in the oligomeric state of LdPEX5 and in its affinity for PTS1 ligand upon LdPEX14 binding may explain how, under physiological conditions, LdPEX5 can function to deliver and unload its cargo to the protein translocation machinery on the glycosomal membrane.

An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion.

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Leishmania donovani Peroxin 14 Undergoes a Marked Conformational Change following Association with Peroxin 5

The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size >670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiled-coil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (Kd) of ∼74 nm. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.

Interaction of Leishmania PTS2 receptor peroxin 7 with the glycosomal protein import machinery

Leishmania proteins containing a peroxisomal targeting signal sequence 2 (PTS2) are selectively trafficked to the glycosome by associating with the peroxin 7 receptor protein (PEX7). The L. major PEX7 (LmPEX7) encodes a approximately 41 kDa protein that exhibits limited sequence identity with PEX7 homologues from other eukaryotic organisms. Functional characterization of recombinant and native LmPEX7 revealed that this receptor bound the PTS2 protein fructose-1,6-bisphosphate aldolase. Moreover, LmPEX7 also formed a tight association with the Leishmania PEX5, the cytosolic PTS1 receptor, and PEX14, a glycosomal peripheral membrane protein required for protein import into the glycosome. Mapping studies revealed that the Leishmania PEX7 binds to a domain on LdPEX5 encompassing residues 111-148 and to a site on LdPEX14 spanning residues 120-148. Finally, subcellular localization studies revealed that Leishmania PEX7 has a dual distribution within the cytosolic compartment and glycosomal lumen.

Peroxin 5–peroxin 14 association in the protozoan Leishmania donovani involves a novel protein–protein interaction motif

Import of proteins with a PTS1 (peroxisomal targeting signal 1) into the Leishmania glycosomal organelle involves docking of a PTS1-laden LdPEX5 [Leishmania donovani PEX5 (peroxin 5)] receptor to LdPEX14 on the surface of the glycosomal membrane. In higher eukaryotes, the PEX5-PEX14 interaction is mediated by a conserved diaromatic WXXXY/F motif. Site-directed and deletion mutageneses of the three WXXXY/F repeats in LdPEX5 did not abolish the LdPEX5-LdPEX14 association. Analysis of the equilibrium dissociation constant (K(d)) revealed that ldpex5-W53A (Trp53-->Ala), ldpex5-W293A, ldpex5-W176,293A and ldpex5-W53,176,293A mutant receptors were capable of binding LdPEX14 with affinities comparable with wild-type LdPEX5. That the diaromatic motifs were not required for the LdPEX5-LdPEX14 interaction was further verified by deletion analysis that showed that ldpex5 deletion mutants or ldpex5 fragments lacking the WXXXY/F motifs retained LdPEX14 binding activity. Mapping studies of LdPEX5 indicated that the necessary elements required for LdPEX14 association were localized to a region between residues 290 and 323. Finally, mutational analysis of LdPEX14 confirmed that residues 23-63, which encompass the conserved signature sequence AX2FLX7SPX6FLKGKGL/V present in all PEX14 proteins, are essential for LdPEX5 binding.

Adenylosuccinate synthetase and adenylosuccinate lyase deficiencies trigger growth and infectivity deficits in Leishmania donovani.

Background: Purine salvage in Leishmania is an essential nutritional function. Results: Null mutants deficient in either adenylosuccinate synthetase or adenylosuccinate lyase impact growth and infectivity phenotypes of Leishmania donovani. Conclusion: Adenylosuccinate synthetase and adenylosuccinate lyase are central enzymes in purine salvage by L. donovani. Significance: Adenylosuccinate lyase has been validated as a potential drug target in L. donovani. Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent Km of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.