Turtle, Lance; (2001) Chemical and immunological characterisation of glycophospholipid and phospho-oligosaccharide from mycobacteria. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Following ligand binding by a wide variety of cytokines and growth factors, a glycosylphosphatidyl inositol phospholipase D (GPI-PLD) cleaves free membrane glycosylphosphatidyl inositol (GPI) to liberate water-soluble inositol phosphoglycan (IPG) second messengers. IPG are released outside cells and mediate many of the immediate metabolic effects of insulin. Many other cytokines and growth factors use IPG to signal, including IL-2, NGF, IGF-1 and ACTH. The composite structure of IPG molecules is known and they consist of hexose, hexosamine, inositol, phosphate and divalent cations. The development of clinical tuberculosis in a susceptible host appears to involve a highly complex interaction between the infecting organism and the host immune system, with much or indeed all the tissue damage characteristic of the disease being immune mediated. Many Mycobacterium tuberculosis colonised individuals do not develop active disease, and there are demonstrable immunological differences between the asymptomatic immune carrier state and the state of active disease in both humans and animal models. Mycobacterium tuberculosis contains PLD activity and contains phosphatidyl inositol linked glycans that could theoretically give rise to IPG like structures. Such glycolipids include lipoarabinomannan (LAM), lipomannan (LM) and phosphoinositol mannoside (PIM). Previously it has been shown in our laboratory and that of our collaborators that two strains of the pathogen M. tuberculosis and a non-pathogen, M. vaccae, contain IPG- like biological activity. This mycobacteria derived material has been provisionally named phospho-oligosaccharide (POS). The aim of this study was to examine the hypothesis that mycobacteria may contain a homologous system to the mammalian GPI/IPG signalling mechanism. Using an established protocol for the purification of mammalian GPI a glycophospholipid (GPL) was isolated from M. tuberculosis and M. vaccae that showed similar characteristics to mammalian GPI. This GPL was a substrate for (glycosyl) phosphatidylinositol phospholipase C (GPI-PLC), contained phosphate, sugar residues and did not contain amino groups. GPL was biologically active in a cell free pyruvate dehydrogenase (PDH) phosphatase activation assay and induced inflammatory mediator release from monocyte/macrophage cells. GPL is distinct from LAM, LM and PIM because it was sensitive to GPI-PLC and incorporated radioactive galactose. POS fractions were isolated from mycobacteria that were biologically active in PDH phosphatase, lipogenesis, cell proliferation and nitric oxide production assays, contained carbohydrate, phosphate (except for one fraction) and amino groups. On the basis of amino group analysis, which revealed that POS contained amino groups and GPL did not, POS is not a cleavage product of mycobacterial GPL, as is the case with GPI and IPG in mammalian cells. However, there were interesting similarities in biological activity between GPL and POS. Serological studies were undertaken to characterise mycobacterial POS both structurally and immunologically. Although POS exhibit biological activity in systems where mammalian IPG are also active, there are clearly structural differences between POS and IPG because some antibodies that bind IPG do not bind POS. POS are immunogenic: administration of complete Freund's adjuvant induced an anti-POS antibody response in rabbits (but not in mice) and sera from tuberculosis patients contained significantly higher levels of anti-POS antibody than healthy controls. POS is proliferogenic for human peripheral blood mononuclear cells in vitro, in particular, POS activates human B cells in vitro causing a greater increase in B cell CD25 expression than T cell CD25 expression. Thus the material described has some interesting immunological properties, although its role in the pathogenesis of tuberculosis remains to be determined. The growth factors IL-2 and insulin have both been shown to signal using IPG cleaved from GPI, it is therefore intriguing that mycobacteria appear to contain a homologue of an immunologically relevant mammalian second messenger.

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