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》

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 infection with intracellular parasites: isolation of a candidate for Bcg.

Vidal SM ，Malo D ，Vogan K ，Skamene E ，Gros P ... -  《Cell》

Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites.

The mouse chromosome 1 locus Bcg (Ity, Lsh) controls the capacity of the tissue macrophage to restrict the replication of antigenically unrelated intracellular parasites and therefore determines the natural resistance (BCG-R, dominant) or susceptibility (BCG-S, recessive) of inbred mouse strains to infection with diverse pathogens, including several Mycobacterium species, Salmonella typhimurium, and Leishmania donovani. 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 (Nramp) that encodes a predicted macrophage-specific transport protein. We have analyzed a total of 27 inbred mouse strains of BCG-R and BCG-S phenotypes for the presence of nucleotide sequence variations within the coding portion of Nramp and have carried out haplotype typing of the corresponding chromosome 1 region in these mice, using 11 additional polymorphic markers mapping in the immediate vicinity of Nramp. cDNA cloning and nucleotide sequencing identified 5 nucleotide sequence variations within Nramp in the inbred strains; while 4 of these represented silent sequence polymorphisms, one G to A substitution at nucleotide position 783 resulted in the non-conservative replacement of Gly105 to Asp105 within the second predicted transmembrane domain (TM2) of the Nramp protein. An absolute association of this allelic variation and Bcg phenotype was observed in the 20 BCG-R strains (Gly105) and 7 BCG-S strains (Asp105) tested. Moreover, sequence analysis of the corresponding region of the Nramp gene from distantly related species indicated strong amino acid sequence conservation of TM2, including an invariant glycine at position 105. Haplotype mapping using sequence polymorphism identified within Nramp and additional RFLPs and SSLPs from the region revealed that although the 20 BCG-R strains analyzed showed diverse allelic combinations for these markers, the 7 BCG-S strains tested share a conserved core haplotype of 2.2 Mb overlapping and including Nramp. Taken together, these results suggest that (1) Gly105 is the wildtype form of Nramp and that the nonconservative substitution to Asp105 underlies the BCG-S phenotype, and (2) Bcg8 alleles carry the same Gly105-->Asp105 mutation and are identical by descent.

Malo D ，Vogan K ，Vidal S ，Hu J ，Cellier M ，Schurr E ，Fuks A ，Bumstead N ，Morgan K ，Gros P ... -  《GENOMICS》

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 ... -  《-》