Genetic regulation of leishmanial and mycobacterial infections: the Lsh / Ity / Bcg gene story continues

Abstract

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 activity. This relies on the TNF-α-dependent sustained expression of the inducible nitric oxide synthase (iNOS) gene responsible for the generation of large amounts of toxic nitric oxide (NO). The Lsh / Ity / Bcg gene has many pleiotropic effects, including differential expression of the early response gene KC following stimulation of macrophages with bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM). The major signal transduction pathway involved in KC induction requires the generation of low levels of NO via constitutive nitric oxide synthase (cNOS) activity, leading to activation of guanylate cyclase 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 the later production of NO as the final effector mechanism for kill. The recently cloned candidate for Lsh / Ity / Bcg, designated Nramp for Natural resistance associated macrophage protein, encodes a polytopic integral membrane protein that has structural features common to prokaryotic and eukaryotic transporters and includes a conserved binding-protein-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 role for tyrosine kinases in regulating Nramp function. This is also supported by the demonstration that ligation of β 1 integrins, which signal via tyrosine kinases, by plating of macrophages onto extracellular matrix proteins is sufficient to mediate differential TNF-α release by macrophages from congenic Lsh resistant and susceptible mice. Transfection studies with the resistant allele demonstrate that Nramp plays a role, either directly or as an additional pleiotropic effect, in interferon-γ/LPS upregulated L-arginine transport across the macrophage membrane, thus providing the substrate required to generate NO for both signal transduction and antimicrobial activity. Nramp also shows 55–58% sequence similarity with the yeast genes SMF1 and SMF2, which influence protein import into mitochondria. A high degree of conservation over the region of Nramp which contains the susceptible Nramp mutation indicates a possible common function at the level of protein translocation across membranes of intracellular compartments. Analysis of human NRAMP has identified a novel 3 × 9 nucleotide repeat in the putative SH3 binding domain, with a rare second allele bearing a 2 × 9 nucleotide repeat occurring at low frequency in the Brazilian population. Studies in progress will attempt to determine the function of human NRAMP, and hence to identify its role in parallel activation pathways in man. This is of particular interest in the light of studies demonstrating ( 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-γ-inducible JAK tyrosine kinases are candidates for two other major genes, Scl1 and Scl2, identified and mapped in mice for their role in controlling different leishmanial resistance phenotypes. 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 strategies for disease control.