The Bcg/Ity/Lsh locus: genetic transfer of resistance to infections in C57BL/6J mice transgenic for the Nramp1 Gly169 allele.

The murine Bcg/Ity/Lsh locus determines the susceptibilities of inbred strains to infection with unrelated intracellular parasites, such as Mycobacterium bovis, Salmonella typhimurium, and Leishmania donovani. A candidate for Bcg/Ity/Lsh, designated Nramp1, has been recently identified and shown to encode a novel integral membrane protein that is expressed exclusively in professional phagocytes but whose function remains unknown. In inbred strains, the susceptibility to infection is associated with a single glycine-to-aspartic acid substitution at position 169 (G169D) in the predicted TM4 of the protein. To confirm the candidacy of Nramp1 as Bcg/Ity/Lsh and to determine the importance of the G169D mutation on Nramp1 function, we constructed transgenic mice in which the G169 allele of Nramp1 was transferred onto the background of a homozygous D169 allele. These transgenic mice were analyzed for their sensitivity to infections under the control of Bcg/Ity/Lsh. The transgene constructed for these studies contained the entire Nramp1G169 gene together with approximately 5 kb of sequences upstream of the transcription initiation site of this gene. We observed that these sequences were sufficient to direct Nramp1G169 expression in transgenic macrophages, resulting in the appearance of a mature protein of 90 to 100 kDa over a background of Nramp1G169 characterized by the complete absence of the mature Nramp1 polypeptide. The appearance of the Nramp1G169-encoded protein in transgenic macrophages was concomitant with the emergence of resistance to infection by M. bovis BCG, as measured by the extent of bacteria] replication in the spleen, and by S. typhimurium, as measured by survival after an intravenous challenge. The gain of function detected in transgenic Nramp1G169 animals establishes unambiguously that Nramp1 and Bcg/Ity/Lsh are allelic.

Govoni G ，Vidal S ，Gauthier S ，Skamene E ，Malo D ，Gros P ... -  《-》

The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.

In mice, natural resistance or susceptibility to infection with intracellular parasites is determined by a locus or group of loci on chromosome 1, designated Bcg, Lsh, and Ity, which controls early microbial replication in reticuloendothelial organs. We have identified by positional cloning a candidate gene for Bcg, Nramp1, which codes for a novel macrophage-specific membrane transport protein. We have created a mouse mutant bearing a null allele at Nramp1, and we have analyzed the effect of such a mutation on natural resistance to infection. Targeted disruption of Nramp1 has pleiotropic effects on natural resistance to infection with intracellular parasites, as it eliminated resistance to Mycobacterium bovis, Leishmania donovani, and lethal Salmonella typhimurium infection, establishing that Nramp1, Bcg, Lsh, and Ity are the same locus. Comparing the profiles of parasite replication in control and Nramp1-/- mice indicated that the Nramp1Asp169 allele of BcgS inbred strains is a null allele, pointing to a critical role of this residue in the mechanism of action of the protein. Despite their inability to control parasite growth in the early nonimmune phase of the infection, Nramp1-/- mutants can overcome the infection in the late immune phase, suggesting that Nramp1 plays a key role only in the early part of the macrophage-parasite interaction and may function by a cytocidal or cytostatic mechanism distinct from those expressed by activated macrophages.

Vidal S ，Tremblay ML ，Govoni G ，Gauthier S ，Sebastiani G ，Malo D ，Skamene E ，Olivier M ，Jothy S ，Gros P ... -  《-》

Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1 D169) mouse strains.

The mouse Nramp1 gene (Bcg/Ity/Lsh) controls innate defense to infection with intracellular parasites such as Mycobacterium, Salmonella, and Leishmania. Sequence analysis of Nramp1 predicts a hydrophobic, membrane-associated protein expressed exclusively in monocyte/macrophage lineages. A single G169D substitution within the fourth predicted transmembrane domain of Nramp1 is associated with susceptibility to infection (Bcg) in inbred mouse strains. To initiate the biochemical characterization of the Nramp1 protein and to analyze the molecular basis of susceptibility associated with the Nramp1(D169) allele, oligopeptides derived from Nramp1 and two fusion proteins containing the first 54 and the last 35 residues of Nramp1 were used to raise specific anti-Nramp1 antisera. In addition, a c-Myc epitope (EQKLISEEDL) was introduced in-frame at the C terminus of Nramp1 to follow its expression in a yeast heterologous system. Western analysis of crude membrane fractions from yeast demonstrated that Nramp1 is indeed an integral membrane protein (resistant to urea extraction). In resident Bcg(r) (129sv, Nramp1(G169)) macrophages, one of the anti-Nramp1 antisera identified by immunoprecipitation a protein of 90,000 to 100,000 apparent m.w. that was absent in macrophages from knockout mice bearing a null mutation at Nramp1. The Nramp1 protein could be metabolically labeled with orthophosphate and was sensitive to glycosylase treatment, verifying that Nramp1 is an integral membrane phosphoglycoprotein. Parallel analysis of macrophage extracts from Bcg(s) mouse strains (C57BL6/J and BALB/c, Nramp1(D169)) failed to detect mature Nrampl protein in these cells, suggesting that the G169D mutation at Nramp1 prevents proper maturation or membrane integration of the protein, resulting in its rapid degradation.

Vidal SM ，Pinner E ，Lepage P ，Gauthier S ，Gros P ... -  《JOURNAL OF IMMUNOLOGY》

Natural resistance to infection with intracellular parasites: molecular genetics identifies Nramp1 as the Bcg/Ity/Lsh locus.

Natural resistance to infection with intracellular parasites is controlled in the mouse by the expression of a locus or group of loci on chromosome 1 alternatively named Bcg, Lsh, and Ity. Bcg affects the capacity of mature tissue macrophages to restrict the intracellular proliferation of ingested parasites in the reticuloendothelial organs of the host during the early phase of infection. This review summarizes our molecular genetic approach to the isolation and characterization of the Bcg locus. We have used a positional cloning strategy based on genetic and physical mapping, YAC cloning, and exon trapping to isolate a candidate gene for Bcg, named Nramp1, which codes for a macrophage-specific polytopic protein with 12 predicted transmembrane domains and a consensus transport motif. Sequence analysis of Nramp1 cDNA clones from 27 Bcgs and Bcgr mouse strains reveals that susceptibility to infection (Bcgs) is associated with a single nonconservative Gly to Asp substitution at position 169 within predicted transmembrane domain 4 of the Nramp protein. Cloning experiments and homology search in available databases demonstrated that the Nramp1 gene belongs to a small gene family with several members in vertebrates and in such distantly related species as yeast and plants. Nramp proteins share a remarkable degree of similarity, with strong amino acid sequence conservation in the transmembrane domains, suggesting a common transport function for the Nramp family. Finally, we generated Nramp1-/- gene knockout mice, and analysis of their phenotypic characteristics established that (1) Nramp1 plays a key role in natural defense against infection with intracellular parasites and therefore demonstrated allelism between Nramp1 and Bcg/Ity/Lsh, (2) Nramp1 functions by a novel cytocidal/cytostatic mechanism distinct from those expressed by the activated macrophage, and (3) the Nramp1Asp169 allele of Bcgs inbred strains is a null allele, pointing to a critical role of this residue in Nramp1 function.

Vidal S ，Gros P ，Skamene E 《JOURNAL OF LEUKOCYTE BIOLOGY》

Evidence inconsistent with a role for the Bcg gene (Nramp1) in resistance of mice to infection with virulent Mycobacterium tuberculosis.

The superior resistance of some strains of mice over others to infection with certain intracellular pathogens, including the vaccine strain of Mycobacterium bovis, bacillus Calmette Guerin (BCG), is determined by a gene associated with a small segment of chromosome 1 designated by Ity/Lsh/Bcg locus, referred to here as the Bcg locus. DBA/2 mice containing the dominant resistant allele of the Bcg gene (Bcgr), major histocompatibility complex-compatible BALB/c mice containing the recessive susceptible allele (Bcgs), and congenic C.D2-N20 Bcgr, which are genetically the same as BALB/c mice except for possessing a small piece of DBA/2 chromosome 1 containing the Bcg locus, were used to determine whether the Bcg gene determines resistance to infection with the virulent H37Rv strain of Mycobacterium tuberculosis (Mtb). According to the survival times of Bcgr and Bcgs mice infected via either the intravenous or respiratory route, Bcgr mice proved much less, rather than more, resistant to Mtb infection than Bcgs mice. Shorter survival times of Bcgr mice were associated with an inferior capacity to control Mtb growth in their lungs and to retard the development of Mtb-induced pathology in this organ. Resistance to Mtb infection was a dominant trait in the F1 progeny of Bcgr and Bcgs mice. The results show that resistance to Mtb is not determined by the resistance allele of the Bcg gene nor by the recently isolated candidate Bcg gene Nramp1, located in the Bcg locus.

Medina E ，North RJ 《-》