NRAMP proteins (Natural Resistance Associated MacroPhage Protein) are H+-stimulated divalent cation transporters. Mammalian NRAMP2/DCT1 mediates intestinal uptake of Fe2+ and Mn2+ and distribution of these cations within cellular compartments. NRAMP1 is expressed in macrophages and is the long known Ity/Lsh/Bcg locus in the mouse that provides resistance to diverse pathogens such as S. typhimurium, Leishmania donovania and Mycobacterial species. Polymorphisms in human NRAMP1 render the bearer more susceptible to M. tuberculosis infection. NRAMP proteins have been characterized primarily in eukaryotic cells; however, many Gram+ and Gram-bacterial species have close homologs (40% sequence identity), suggesting conservation of function. E. coli and S. typhimurium carry a single NRAMP gene which we have cloned and characterized. Our data indicate that a) the bacterial NRAMP protein is a highly selective H+- stimulated Mn2+ transport system (mntH) whose expression is regulated by peroxide and divalent cation. b) MntH is highly induced upon S. typhimurium invasion of NRAMP1+ but not NRAMP1-macrophages, c) virulence of mntH is attenuated in NRAMP1+ mice but not NRAMP1- mice, d) the putative iron transporter SitABCD is a physiologically relevant Mn2+ transporter and 3) mutation of both mntH and sitABCD renders S. typhimurium avirulent. If abrogation of Mn2+ uptake results in loss of virulence, than Mn2+ itself is needed at some point in pathogenesis.
In Aim 1, we will ask where, when and why Mn2+ is important for pathogenesis. Where and when will be determined by investigation of the effect of mutations in mntH (and sitABCD) on invasion, survival and proliferation of S. typhimurium in NRAMP1+ and NRAMP1- macrophages and also epithelial cell lines. Virulence will be determined in congenic NRAMP1+ and NRAMP1- BALB/c mice. Why will be investigated by examining a subset of the very few Mn2+-dependent enzymes: superoxide dismustase (sodA), protein phosphatases 1 and 2 (prpA/B), and phoshoglyceromutatse (gpmM). The effects of mutations at these loci will be measured on invasion of cultured cells and virulence in mice. We will also investigate regulation of mntH expression by Fur, OxyR and a new DtxR homolog MntR.
In Aim 2, we will determine the topology of MntH, further examine its transport properties using 54Mn2+ as tracer and also electrophysiologically after expression in Xenopus oocytes, and investigate the role of conserved and charged intramembrane residues in cation flux by site-directed mutagenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061748-04
Application #
6840847
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Chin, Jean
Project Start
2002-01-11
Project End
2006-12-31
Budget Start
2005-01-01
Budget End
2006-12-31
Support Year
4
Fiscal Year
2005
Total Cost
$271,958
Indirect Cost
Name
Case Western Reserve University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Ikeda, Jack S; Janakiraman, Anuradha; Kehres, David G et al. (2005) Transcriptional regulation of sitABCD of Salmonella enterica serovar Typhimurium by MntR and Fur. J Bacteriol 187:912-22
Agranoff, Daniel; Collins, Lauren; Kehres, David et al. (2005) The Nramp orthologue of Cryptococcus neoformans is a pH-dependent transporter of manganese, iron, cobalt and nickel. Biochem J 385:225-32
Zaharik, Michelle L; Cullen, Vivian Li; Fung, Angela M et al. (2004) The Salmonella enterica serovar typhimurium divalent cation transport systems MntH and SitABCD are essential for virulence in an Nramp1G169 murine typhoid model. Infect Immun 72:5522-5
Hazlett, Karsten R O; Rusnak, Frank; Kehres, David G et al. (2003) The Treponema pallidum tro operon encodes a multiple metal transporter, a zinc-dependent transcriptional repressor, and a semi-autonomously expressed phosphoglycerate mutase. J Biol Chem 278:20687-94
Kehres, David G; Janakiraman, Anu; Slauch, James M et al. (2002) Regulation of Salmonella enterica serovar Typhimurium mntH transcription by H(2)O(2), Fe(2+), and Mn(2+). J Bacteriol 184:3151-8
Kehres, David G; Janakiraman, Anuradha; Slauch, James M et al. (2002) SitABCD is the alkaline Mn(2+) transporter of Salmonella enterica serovar Typhimurium. J Bacteriol 184:3159-66