Lipophosphoglycan expression and virulence in ricin-resistant variants of Leishmania major

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats joined by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the M_r 63 000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies.

All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.     

Interaction of Leishmania mexicana promastigotes with the plasminogen–plasmin system

The binding of human plasminogen and plasmin to the promastigote form of Leishmania mexicana was investigated. L. mexicana was capable to bind both molecules, the binding being inhibited by -aminocaproic acid. Scatchard plot analysis revealed a dissociation constant (K_d) value of 2.4±0.8 μM and 0.9±0.1×10⁴ binding sites per cell for plasminogen and a K_d value of 1.2±0.4 μM and 1.6±0.2×10⁵ binding sites per cell for plasmin. C-terminal lysine residues are involved in plasminogen binding to cells, since carboxypeptidase B treatment reduced this binding by 34%. Ligand blotting analysis showed a group of proteins, with molecular masses between 105 and 115 kDa, capable to interact with plasminogen. Zymogram analysis showed that the protease activity acquired by L. mexicana, due to the interaction with either plasminogen or plasmin, comprises an important fraction of the total protease activity at pH 7.7. Plasminogen activation by tissue-type plasminogen activator (t-PA) was enhanced by the presence of L. mexicana promastigotes. These results raise the question whether the interaction of L. mexicana with components of the fibrinolytic system is involved in the virulence of the parasite.     

Leishmania donovani engages in regulatory interference by targeting macrophage protein tyrosine phosphatase SHP-1

Protozoan parasites of the genus leishmania are obligate intracellular parasites of monocytes and macrophages. These pathogens have evolved to invade the mammalian immune system and typically survive for long periods of time. Leishmania have developed a variety of remarkable strategies to prevent their elimination by both innate and acquired immune effector mechanisms. One particular strategy of interest involves manipulation of host cell regulatory pathways so as to prevent macrophage activation required for efficient microbicidal activity. These interference mechanisms are the main focus of this review. Several lines of evidence have been developed to show that the Src homology-2 domain containing tyrosine phosphatase-1 (SHP-1) becomes activated in leishmania-infected cells and that this contributes to disease pathogenesis. Recent studies aimed at understanding the mechanism responsible for the change in activation state of SHP-1 led to the identification of leishmania EF-1α as an SHP-1 binding protein and SHP-1 activator. This was a surprising finding given that this ubiquitous and highly conserved protein plays an essential role in protein translation in both prokaryotic and eukaryotic cells. The role of leishmania EF-1α as an SHP-1 activator and its contribution to pathogenesis are reviewed with particular attention to the properties that distinguish it from host EF-1α.     

Characterization of the Local and Systemic Immune Responses in Patients with Cutaneous Leishmaniasis Due toLeishmania major

In this study skin biopsies and peripheral blood samples were obtained from patients with cutaneous leishmaniasis caused by Leishmania major. Samples were obtained at diagnosis and during healing when the lesions had regressed to half the original size. At diagnosis most of the cells expressed HLA-DR. The numbers of CD8+ cells in the lesions were higher at diagnosis than during healing. By contrast, a lower percentage of PBMC expressed CD8+ cells at diagnosis probably due to sequestration in the lesion. In the lesion, in situ staining for IFN-gamma, IL-10, and IL-4 showed marked variation between all patients in the number of positive cells for a particular cytokine. The proliferative response of PBMC to leishmanial antigens and IFN-gamma production tended to increase during healing. Cytokine patterns in the PBMC in response to Leishmania antigen was more specific than in the lesion and correlated better with the clinical manifestations. Possible reasons for this are discussed.     

Modulation of the swelling-activated amino acid channel of Leishmania major promastigotes by protein kinases

Leishmania promastigotes respond to hypotonic challenges by a mechanism of regulatory volume decrease (RVD), whereby anionic amino acid channels (HAAC) are hypotonically-activated and intracellular amino acids are released from the cells. Irrespective of the experimental conditions, restoration of isotonicity triggered an immediate blockage of the amino acid release. Both the speed and amplitude of the response depended on the hypotonic stimulus and on the operation of intracellular signaling mechanisms. The initial (5 s) hypotonic-induced release of amino acids (r_i) and the steady state levels of amino acids attained (5 min) or amplitude (A), were markedly affected by modulators of protein kinase C: phorbol 12-myristate 13-acetate, 1-oleoyl-2-acetylglycerol and phorbol 12,13-diacetate whereas staurosporine and the related analog, bis-indolylmaleimide I (GF-109203.X) inhibited the RVD response. Agonists of cAMP-dependent protein kinase A such as forskolin or (8-(4-chlorophenylthio))-adenosine-3′,5′cyclic-monophosphate enhanced the speed of the response but had little effect on its amplitude. Neither 4-phorbol 12,13-didecanoate,1,9-dideoxyforskolin nor genistein, tamoxifen or thapsigargin had any apparent effect on either parameter tested. The most striking stimulation of hypotonic-induced amino acid release was exerted by arachidonic acid or by its non-metabolizable analog, 5,8,11,14-eicosatetraynoic acid (ETYA). These agents caused a major increase in the initial rate of amino acid release as well as a higher amplitude of the response, both of which were markedly inhibited by an anion channel blocker. The present studies indicate not only that hypotonicity is an obligatory and dominant component in HAAC activation, but implicate specific second messengers in the modulation of the RVD response. The modes of activation or attenuation of HAAC activity apparently differ for PKC and PKA modulators as well as for arachidonic acid. The involvement of Ca²⁺ in HAAC was studied in hypotonic challenged cells which were treated with intracellular Ca²⁺-chelators or Ca²⁺-free medium. These cells showed a lag in AA release and a modest inhibition of the amplitude. The inhibition of HAAC was markedly increased when cells were treated with the ionophore A23187 in Ca²⁺-free media. The HAAC activity was accompanied by a significant increase in internal Ca²⁺ when performed in Ca²⁺-containing medium (from 88±9 to 179±22 nM) but by no significant change when measured in Ca²⁺-free medium. These studies indicate that although Ca²⁺ might be involved in the early activation phase of HAAC, it is either not absolutely required or its action might be associated with localized events.     

Uptake and turnover of glucose in Leishmania donovani

Glucose uptake;and metabolism by Leishmania donovani promastigotes was studied using -[¹⁴C]glucose in combination with the silicone oil centrifugation technique on organisms preadapted to different growth rates and glucose availability in the chemostat. The uptake step was differentiated from the subsequent metabolism by separation in time rather than by using non-metabolisable analogues. The uptake of glucose was measured as a function of time and;or the external glucose concentration on cells grown at high or low growth rate with glucose either as growth rate-limiting substrate, or present in excess.Glucose uptake as a function of its external concentration could be described as consisting of two components (1) a rapid equilibration owing to facilitated diffusion, followed by (2) a much slower uptake that involves an enzymatic component. This slower accumulation of label could be explained as the conversion of glucose into metabolites and a storage carbohydrate. Uptake experiments in the presence of inhibitors indicated that the conversion of glucose was an energy dependent process. These experiments indicate that the active uptake of glucose by L donovani, as reported by others does not occur across the plasma membrane and should be reinterpreted as the intracellular conversion of glucose into metabolites and storage carbohydrate.     

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5–30 times the IC₅₀ (30 μg mg ml⁻¹) of parental cells. The vinblastine-resistant parasites were also resistant to promycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug effiux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdrl, and demonstrate that this gene is amplified on an extrachromosomal circle of 35–40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr 1 and 83% identity with the L. donovani 1mdr 1 gene. The lemdr 1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.     

Tn5 mutagenesis of the Salmonella typhimurium 100 kb plasmid: definition of new virulence regions

In this study, the 100 kb plasmid of Salmonella typhimurium, which is known to contribute to the pathogenicity of the organism, was tagged with the transposon Tn5 to define regions of the plasmid contributing to overall virulence. Eleven randomly selected vir::Tn5 plasmids carried by the plasmid-free S. typhimurium strain WS1321 were physically mapped and then examined in mice for subcutaneous LD₅₀ value, ability to induce splenomegaly, and ability to grow to high numbers in the spleens of infected mice. Nine strains were found to be virulence-attenuated and showed varied levels of growth in the spleens of subcutaneously infected BALB/c mice. Eight of these nine strains carried Tn5 insertions which lie outside the previously defined virulence region. These studies corroborate the findings of other investigators as well as defining novel regions of the 100 kb virulence plasmid involved in the pathogenicity of this organism.     

Identification of an essential virulence region on Salmonella plasmids

An 8 kilobase pair (kbp) fragment from the Salmonella dublin 2229 plasmid is sufficient to restore virulence for mice to cured strains of both S. dublin and S. typhimurium but only when cloned on a low copy vector. Two regions previously shown to be associated with virulence lie outside the fragment and complementation results suggest that one of these mutated regions affects expression from the 8 kbp fragment. It therefore appears that there are control elements both within and adjacent to the essential virulence fragment.     

Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells

Monoclonal antibodies raised against Haemophilus influenzae and Neisseria gonorrhoeae were used to investigate similarities or differences in the lipopolysaccharide antigens of pathogenic and commensal strains of several Gram-negative bacteria indigenous to mucosal surfaces of humans. In immunoblotting experiments, 20 of 36 monoclonal antibodies showed cross-reactions between species of Neisseria and Haemophilus. The common epitopes were present on N. gonorrhoeae, N. meningitidis, N. lactamica, H. influenzae including biogroup aegyptius, and occasionally H. parainfluenzae. No other commensal Neisseria or Gram-negative organisms tested reacted with the monoclonal antibodies with one exception; a single strain of pathogenic Escherichia coli was recognised by a N. gonorrhoeae-specific monoclonal antibody. One monoclonal antibody, raised against N. gonorrhoeae lipopolysaccharide, reacted with N. gonorrhoeae (32 of 59 strains), N. meningitidis (9 of 26 strains), H. influenzae (6 of 16 strains). An epitope expressed by H. influenzae and implicated in its virulence was also present on 14 of 59 strains of N. gonorrhoeae and was shown to comprise a digalactoside structure, α-galactosyl-1,4-β-galactose (Galα1, 4Galβ), also found on human cells.     

Leishmania dihydroxyacetonephosphate acyltransferase LmDAT is important for ether lipid biosynthesis but not for the integrity of detergent resistant membranes

Glycerolipid biosynthesis in Leishmania initiates with the acylation of glycerol-3-phosphate by a single glycerol-3-phosphate acyltransferase, LmGAT, or of dihydroxyacetonephosphate by a dihydroxyacetonephosphate acyltransferase, LmDAT. We previously reported that acylation of the precursor dihydroxyacetonephosphate rather than glycerol-3-phosphate is the physiologically relevant pathway for Leishmania parasites. We demonstrated that LmDAT is important for normal growth, survival during the stationary phase, and for virulence. Here, we assessed the role of LmDAT in glycerolipid metabolism and metacyclogenesis. LmDAT was found to be implicated in the biosynthesis of ether glycerolipids, including the ether lipid derived virulence factor lipophosphoglycan and glycosylphosphatidylinositol-anchored proteins. The null mutant produced longer lipophosphoglycan molecules that were not released in the medium, and augmented levels of glycosylphosphatidylinositol-anchored proteins. In addition, the integrity of detergent resistant membranes was not affected by the absence of the LmDAT gene. Further, our genetic analyses strongly suggest that LmDAT was synthetic lethal with the glycerol-3-phosphate acyltransferase encoding gene LmGAT, implying that Leishmania expresses only two acyltransferases that initiate the biosynthesis of its cellular glycerolipids. Last, despite the fact that LmDAT is important for virulence the null mutant still exhibited the typical characteristics of metacyclics.     

A high capacity Alphavirus heterologous gene delivery system

A novel replication competent Sindbis virus based gene delivery vector has been developed for the introduction of genetic cargo into cell lines in vitro and potentially, animal models in vivo. This delivery system expands the previous uses of Sindbis virus as a gene delivery system in that no replicons are required and the resulting cargo containing virus particles are infectious. The heterologous vector is based on a morphological mutant in C, Ser180/Gly183 which produces larger than the normal size T = 4 virus particles of 70 nm in size. This mutant produced particles up to 205 nm in size equal to a triangulation number of 36. It was postulated that because the Ser180/Gly183 mutant was capable of assembling such large particles, that increasing the size of the RNA genome incorporated into this mutant capsid protein would favor the assembly of larger than T = 4 wild type sized virions. The first generation prototype larger vehicle, described here, carries a ~ 18 kb cDNA insert, however it is conceivable that RNA as large as 32 kb could be transcribed and packaged. The large variant produces a high virus titer of ~ 10⁹ pfu/ml from either mammalian or insect cells in culture. Multiple passages of the virus show no loss of the inserted genetic material.     

Heteroduplex analysis of Salmonella virulence plasmids and their prevalence in isolates of defined sources

Strains of the Salmonella serovars S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis harbour large plasmids which are required for extraintestinal colonization after oral infection of mice. Electron microscopic heteroduplex analysis showed that these virulence plasmids share large regions of homology. Nine hundred and eighty-six isolates of different origins were analysed for the presence of these plasmids by using a cloned fragment of a S. choleraesuis virulence plasmid as a gene probe. Virulence plasmids were detected in nearly 100% of strains isolated from animal organs or human blood. Frequencies of detection ranged from 48 to 87% in strains of faecal, food or environmental origin. These results suggest that Salmonella virulence plasmids are required for systemic infections in humans and livestock.