Antigenicity and immunogenicity of phage library-selected peptide mimics of the major surface proteophosphoglycan antigens of Entamoeba histolytica

Entamoeba histolytica is the protozoan parasite responsible for intestinal amoebiasis and amoebic liver abscess, which cause significant morbidity and mortality in many countries of the world. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) represent dominant surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against these components protected SCID mice from amoebic liver abscess, so PPGs might be regarded as vaccine candidates; however, their structure is very complex and only known in part. They are glycosylphosphatidylinositol-linked polypeptides of unknown sequence carrying glycan side-chains linked to serine residues via phosphodiester bonds. In order to identify peptide mimics of the E. histolytica PPG antigens, we screened six different phage-displayed random peptide libraries with the antibody EH5. Various peptide mimics of different length were identified and, in all the peptides, a distinct consensus sequence Gly-Thr-His-Pro-X-Leu could be identified. The phages strongly bound to the antibody, and the natural antigen inhibited binding of the phages to antibody EH5. In addition, several of the phages induced a significant immunoglobulin G response against amoebic antigens in immunized mice.     

Trichomoniasis: evaluation to execution

Trichomoniasis is the most common sexually transmitted disease, caused by a motile flagellate non-invasive parasitic protozoan, Trichomonas vaginalis (T. vaginalis). More than 160 million people worldwide are annually infected by this protozoan. T. vaginalis occupies an extracellular niche in the complex human genito-urinary environment (vagina, cervix, penis, prostate gland, and urethra) to survive, multiply and evade host defenses. T. vaginalis (strain G3) has a ∼160 megabase genome with 60,000 genes, the largest number of genes ever identified in protozoans. The T. vaginalis genome is a highly conserved gene family that encodes a massive proteome with one of the largest coding (expressing ∼4000 genes) capacities in the trophozoite stage, and helps T. vaginalis to adapt and survive in diverse environment. Based on recent developments in the field, we review T. vaginalis structure, patho-mechanisms, parasitic virulence, and advances in diagnosis and therapeutics.     

Adversarial relationship between the leishmania lipophosphoglycan and protein kinase C of host macrophages

The dominant glycoconjugate on the cell surface of all Leishmania promastigotes is an unusual glycoconjugate named lipophosphoglycan (LPG). Its relative abundance, unique structure, and cellular location have implicated LPG as an essential virulence determinant. One feature of LPG resides in its strong inhibitory effect on the activity of protein kinase C (PKC) of host macrophages. This article summarizes the evidence that LPG is inhibitory toward PKC activation in macrophages and discusses the implication of such inhibition on intramacrophage survival of the parasite.     

Enrichment of invariant natural killer T cells in the bone marrow of visceral leishmaniasis patients

Lipid antigens of Leishmania donovani like lipophosphoglycans are shown as a potent ligand for the activation of invariant natural killer T (iNKT) cells. It is reported that activation of iNKT cells augments the disease pathology in experimental visceral leishmaniasis (VL). In this study, we demonstrate the enrichment of iNKT cells in the bone marrow, one of the disease sites among patients with VL.     

Typing of Leishmania lipophosphoglycans by electrospray mass spectrometry.

A method has been developed to identify the repeating phosphosaccharide units of Leishmania lipophosphoglycans using electrospray mass-spectrometry (ES–MS). Cone voltage-induced fragmentation of intact lipophosphoglycan was found to be as effective as analysis of mild acid hydrolysates in identifying the degree of modification of the repeating units of lipophosphoglycans derived from Leishmania mexicana and Leishmania major. This finding was exploited in a ‘rapid-analysis’ method in which a crude organic extract of 2×10⁹ L. major promastigote cells was loaded onto a reverse-phase cartridge for immediate elution into the mass-spectrometer. Using this approach, it was possible to identify the repeating units by total ion scanning and scanning for parents of the m/z 79 (PO₃^–) fragment ion. This approach is suitable for quick-typing of lipophosphoglycan repeats and was shown to detect alterations in repeat side chains caused by: (1) culturing L. major promastigotes in the presence of L-fucose; and (2) in vitro metacyclogenesis of L. major promastigotes. It is anticipated that the method will be applicable to small samples of cultured field isolates or genetically-manipulated strains.     

Analysis of the human T cell responsiveness to purified antigens of Leishmania: lipophosphoglycan (LPG) and glycoprotein 63 (gp 63).

The responsiveness of peripheral blood lymphocytes from 15 cutaneous leishmaniasis patients and five healthy volunteers to purified surface antigens of Leishmania was studied. The following purified antigens were independently used as stimuli for lymphocyte proliferation in vitro: lipophosphoglycan of L. braziliensis, (Lb-LPG), lipophosphoglycan of L. mexicana (Lm-LPG), glycoprotein 63 of L. braziliensis (Lb-gp 63) and glycoprotein 63 of L. mexicana (Lm-gp 63). Among six patients tested with Lb-gp 63 and Lm-gp 63, two borderline responses and two positive responses were observed, respectively, all the others being negative. Lb-LPG and Lm-LPG induced positive responses in all patients tested (11 with Lb-LPG and five with Lm-LPG). A positive dose-response correlation was observed in the responses of the patients to both Lb-LPG and Lm-LPG. Significant differences (P less than 0.01) were found between the two groups (patients and healthy subjects) with regard to the responses to Lb-LPG. The proliferating cells after stimulation with Lb-LPG were shown to belong to the CD4+ T cell subset by flow cytometry. However, the following evidence suggests that the T cell responses were not induced by LPG itself but rather by protein contaminants in the LPG preparations: a highly purified Lb-LPG batch that had undergone additional purification induced positive responses in cells from only three out of six patients and a proteinase K-treated Lb-LPG batch did not stimulate any positive response among the same six patients.     

Inhibition of HIV-1-mediated syncytium formation and virus replication by the lipophosphoglycan from Leishmania donovani is due to an effect on early events in the virus life cycle

Previous findings have indicated that the major surface molecule of Leishmania, lipophosphoglycan (LPG), could abrogate HIV-1-induced syncytium formation and virus replication. In the present work, we were interested in characterizing this inhibitory process. Data from a new luciferase-based semiquantitative assay for syncytium formation, relying on the coincubation of a T-cell line containing an HIV-1 LTR-driven luciferase construct with a cell line chronically infected with HIV-1, confirmed that LPG was indeed a strong inhibitor of HIV-1-dependent syncytium formation and that this inhibition was dose-dependent. As determined by flow cytometric analyses, this inhibition was not apparently due to downregulation of CD4, CXCR4 or LFA-1, three distinct surface glycoproteins known to be important in HIV-1 mediated syncytium formation. Furthermore, LPG did not seem to affect signal transduction pathways in T cells as judged by measurement of HIV-1 LTR-driven reporter gene activity upon treatment with different stimuli. However, pretreatment of either of the cell lines used in the assay with LPG led to a significant decrease of virus-mediated syncytium formation, which was further accentuated when both cell lines were pretreated. LPG inhibition of HIV-1 replication was next assessed. When measuring either infection with luciferase-encoding recombinant HIV-1 particles or multinucleated giant cell formation following an acute virus infection, we again observed that LPG was efficient at blocking HIV-1 replication. Specific assays probing different steps of viral entry demonstrated that attachment was not hindered by LPG but that viral entry was modulated, suggesting that LPG targets a postbinding step. Hence, incorporation of LPG into a target cell membrane could influence its fluidity and diminish both the virus-cell and cell-to-cell fusion processes initiated by HIV-1.     

Parasite antigens recognized by patients with cutaneous leishmaniasis.

Humoral and cell-mediated responses to crude and purified parasite antigens were examined in patients with active cutaneous leishmaniasis caused by Leishmania major. The patients had serum antibody titres against parasite lysates ranging from 1/500 to 1/10,000 and recognized multiple components by Western blotting with molecular weights between 5000 and greater than 200,000. Several components, particularly at 5 and 50 kD, were recognized by most of the patients. The lymphoproliferative responses to two pure antigens, promastigote surface protease and lipophosphoglycan, both considered potential candidates for the development of a human vaccine were measured. No response to promastigote surface protease was observed even at 10 micrograms/ml; however, weak proliferation to lipophosphoglycan was consistently present. T cell blots examining peripheral blood lymphocyte proliferation, showed antigen-specific responses to a 72-82 kD component in promastigote lysates.     

Typing of Leishmania lipophosphoglycans by electrospray mass spectrometry

A method has been developed to identify the repeating phosphosaccharide units of Leishmania lipophosphoglycans using electrospray mass-spectrometry (ES–MS). Cone voltage-induced fragmentation of intact lipophosphoglycan was found to be as effective as analysis of mild acid hydrolysates in identifying the degree of modification of the repeating units of lipophosphoglycans derived from Leishmania mexicana and Leishmania major. This finding was exploited in a ‘rapid-analysis’ method in which a crude organic extract of 2×10⁹ L. major promastigote cells was loaded onto a reverse-phase cartridge for immediate elution into the mass-spectrometer. Using this approach, it was possible to identify the repeating units by total ion scanning and scanning for parents of the m/z 79 (PO₃^–) fragment ion. This approach is suitable for quick-typing of lipophosphoglycan repeats and was shown to detect alterations in repeat side chains caused by: (1) culturing L. major promastigotes in the presence of L-fucose; and (2) in vitro metacyclogenesis of L. major promastigotes. It is anticipated that the method will be applicable to small samples of cultured field isolates or genetically-manipulated strains.