GENETIC-REGULATION OF LEISHMANIAL AND MYCOBACTERIAL INFECTIONS - THE LSH ITY/BCG GENE STORY CONTINUES/

A common basis to genetic regulation of leishmanial and mycobacterial infections is provided by the action of the murine Lsh/Ity/Bcg gene in controlling the priming/activation of macrophages for antimicrobial a ctivity. This relies on the TNF-alpha-dependent sustained expression o f the inducible nitric oxide synthase (iNOS) gene responsible for the generation of large amounts of toxic nitric oxide (NO). The Lsh/Ity/Bc g gene has many pleiotropic effects, including differential expression of the early response gene KC following stimulation of macrophages wi th bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinoman nan (LAM). The major signal transduction pathway involved in KC induct ion requires the generation of low levels of NO via constitutive nitri c oxide synthase (cNOS) activity, leading to activation of guanylate c yclase and the cGMP-dependent kinase pathway. NO therefore appears to provide a common link between the early influence of Lsh in regulating the expression of genes which mediate many pleiotropic effects, and t he later production of NO as the final effector mechanism for kill. Th e recently cloned candidate for Lsh/Ity/Bcg, designated Nramp for Natu ral resistance associated macrophage protein, encodes a polytopic inte gral membrane protein that has structural features common to prokaryot ic and eukaryotic transporters and includes a conserved binding-protei n-dependent transport motif which may be involved in interaction with peripheral ATP-binding subunits. The N-terminal sequence also carries a proline/serine rich putative SH3 binding domain, consistent with a r ole for tyrosine kinases in regulating Nramp function. This is also su pported by the demonstration that ligation of beta(1) integrins, which signal via tyrosine kinases, by plating of macrophages onto extracell ular matrix proteins is sufficient to mediate differential TNF-alpha r elease by macrophages from congenic Lsh resistant and susceptible mice . Transfection studies with the resistant allele demonstrate that Nram p plays a role, either directly or as an additional pleiotropic effect , in interferon-gamma/LPS upregulated L-arginine transport across the macrophage membrane, thus providing the substrate required to generate NO for both signal transduction and antimicrobial activity. Nra,mp al so shows 55-58% sequence similarity with the yeast genes SMF1 and SMF2 , which influence protein import into mitochondria. A high degree of c onservation over the region of Nramp which contains the susceptible Nr amp mutation indicates a possible common function at the level of prot ein translocation across membranes of intracellular compartments. Anal ysis of human NRAMP has identified a novel 3X9 nucleotide repeat in th e putative SH3 binding domain, with a rare second allele bearing a 2X9 nucleotide repeat occurring at low frequency in the Brazilian populat ion. Studies in progress will attempt to determine the function of hum an NRAMP, and hence to identify its role in parallel activation pathwa ys in man. This is of particular interest in the light of studies demo nstrating (i) that NO generated by iNOS is not used for antimicrobial activity in human macrophages, and (ii) that the iNOS gene itself and the interferon-gamma-inducible JAK tyrosine kinases are candidates for two other major genes, Sell and Scl2, identified and mapped in mice f or their role in controlling different leishmanial resistance phenotyp es. Further analysis of genetic regulation of pathways leading to iNOS -mediated NO production may provide the key to understanding why human macrophages do not use this as an antimicrobial pathway, and may also provide the basis for development of novel immunotherapeutic strategi es for disease control.