The Problem of Mg2+ Transport: Mg2+ is the most charge dense of the biologically active cations. The hydrated cation is much larger and the atomic ion significantly smaller than any other biological cation. Such properties are germane because the initial interaction of a cation transport system is with the hydrated cation whereas the system actually transports a largely ionic (mostly unhydrated) cation. Mg2+ presents as a hydrated cation whose volume is 350 times the volume of the ionic cation. These inherent chemical properties strongly suggest that the transmembrane flux of Mg2+ presents a unique problem to the cell. Our current data strongly support this hypothesis and further suggest a major role for Mg2+ in the regulation of microbial pathogenesis.
The CorA System of Salmonella typhimurium: The corA gene product mediates Mg2+ influx; its expression is constitutive. Our phylogenetic studies demonstrate that CorA is universally distributed in all branches of the Eubacteria and Archaea and therefore forms the primary Mg2+ uptake system for two of the three kingdoms of life, making it the most abundant Mg2+ transporter on Earth. CorA is a 37 kDa protein of 316 amino acids with no homology to any other transport system or protein. It has a large periplasmic N-terminal domain of 240 amino acids followed by 3 membrane spanning segments containing no charged amino acids, leaving a short C-terminus in the cytosol. Selective inhibitors of the CorA system have been developed. Extensive mutagenesis studies have identified the amino acids that Mg2+ interacts with during membrane passage. Uniquely for a divalent cation, Mg2+ transport by CorA does not require negatively charged amino acids within the membrane indicating that transport does not involve electrostatic interactions. CorA appears to function as a pentamer. Several other CorA’s have been cloned from other microbes including the Archeon Methanococcus jannaschii. This Archaeal CorA functions normally when expressed in S. typhimurium and has indistinguishable transport properties compared to the CorA of S. typhimurium. CorA also appears to be a virulence factor. Mutation of corA markedly attenuates S. typhimurium virulence and alters the transcriptional regulation of a subset of known virulence factors. corA mutants are defective for invasion but survive and proliferate normally within macrophages.
The mgtC Virulence Factor of S. typhimurium: mgtC encodes a small membrane protein of 22.5 kDa and is the first gene of a two gene operon with mgtB. Expression of mgtC is regulated by extracellular Mg2+ through the PhoPQ signal transduction system. Strains lacking mgtC appear to be attenuated for mouse virulence after intraperitoneal injection although invasion and survival within macrophages is unimpaired for at least the first several hours after invasion. MgtC is not homologous to any known transporter or other protein and its function is not currently known. Our preliminary electrophysiological data from expression of mgtC in Xenopus oocytes suggests that cellular cation homeostasis is altered but the precise mechanism is not yet understood. Expression of mgtC is unusual. In medium with a low [Mg2+], the mgtC-mgtB operon is turned on several hundred fold. However, only the MgtB protein, encoded by the second gene of the operon, can be detected by Western blot for the first 12-18 hours. Only after long exposure to low [Mg2+] is the cell able to express a detectable amount of MgtC protein. Initial analysis of the promoter indicates a high degree of complexity.
The MgtA and MgtB Systems of Salmonella typhimurium: MgtA and MgtB, while distinct genes and transporters, are siblings and are quite unusual members of the P-type ATPase family. Both are far more homologous to eukaryotic than other prokaryotic P-type ATPases. Moreover, P-type ATPases generally pump cations against their electrochemical gradients, but MgtA and MgtB pump Mg2+ into the cell, with the extremely strong inwardly directed Mg2+ electrochemical gradient. Neither transporter is normally expressed under laboratory growth conditions. However, when S. typhimurium is grown without added exogenous Mg2+, transcription of mgtA and mgtB increases enormously, well over 1,000-fold. Our work showed that the extracellular signal for this transcriptional response is Mg2+ itself, and the Mg2+-responsive signal transduction system was subsequently identified and characterized by Eduardo Groisman and colleagues as the phoP/phoQ two component signal transduction system involved in bacterial virulence. Both MgtA and MgtB are expressed upon Salmonella invasion of macrophage and epithelial cells, implicating Mg2+ and Mg2+ transport systems in microbial pathogenesis.
The NRAMP Mn2+ Transport System of S. typhimurium: S. typhimurium possesses a single Mn2+ influx system encoded by the mntH locus. The MntH protein is highly homologous to mammalian NRAMP proteins which cotransport a spectrum of transition metal cations with proton; Fe2+ and Mn2+ are likely the physiological substrates of the mammalian transporters. Bacterial NRAMPs from S. typhimurium, E. coli, Pseudomonas aeruginosa, and Burkholderia cepacia have been cloned. All appear to be extremely highly selective Mn2+ transporters involved in the bacterium’s response to reactive oxygen, especially peroxide. Inactivating polymorphisms in mammalian NRAMPs are very common and certain relatively common polymorphisms in NRAMP1, expressed in macrophages and monocytes, renders the carrier several fold more susceptible to infection by a variety of pathogens, including S. typhimurium. Likewise, the mntH locus of S. typhimurium is also important for virulence. When tested in mice that carry a functional NRAMP1 allele, S. typhimurium mntH mutant strains are markedly attenuated. Interestingly, mammalian and bacterial NRAMPs interact functionally, since mntH has no effect on virulence in mice that do not express a functional NRAMP1 protein.
Pseudomonas aeruginosa Gene Expression in Cystic Fibrosis Analyzed by Microarray Analysis: Pseudomonas aeruginosa is the most common opportunistic pathogen that infects lungs of cystic fibrosis patients. It is exceedingly difficult to control, and the lung scarring and lack of sufficient uptake capacity caused by P. aeruginosa is the most common cause of death in these patients. With the genome of this microbe recently completed, inexpensive gene chips of the entire genome are now available. In concert with the CWRU Cystic Fibrosis Center, we are beginning analysis of the changes in gene expression in P. aeruginosa that occur upon exposure to lung epithelial cells which do or do not express CFTR and after lung infection of congenic mouse strains with or without a function CFTR allele.
Role of Small G-proteins in S. typhimurium Pathogenesis: During invasion of macrophage or epithelial cells, S. typhimurium and many other enteric bacteria gain control of and manipulate the mammalian cell’s actin cytoskeleton. This requires a functional Cdc42 small G-protein in the mammalian cell. We are collaborating with Dr. Amy Wilson to investigate S. typhimurium invasion, survival and proliferation in cell lines carrying wild type, dominant negative, non-prenylated and other forms of Cdc42 to help determine the requirements for and mechanism of the bacterium’s control of the cytoskeleton.
Development of Novel Antibiotics: Under a contract from DARPA (Defense Advanced Research Projects Agency), we are developing novel bivalent antibiotics both for use as countermeasures to biological terrorist attack and for eventual use against civilian infectious disease.
Selected Publications
Kucharski, L.M., W.J. Lubbe and M.E. Maguire (2000) “Cation Hexaammines Are Potent and Selective Inhibitors of the CorA Magnesium Transport System,” J. Biological Chemistry 275:16767-16773. [PubMed]
Kehres, D.G., M.L. Zaharik, B.B. Finlay and M.E. Maguire (2000) The NRAMP Proteins of Salmonella typhimurium and Escherichia coli Are Mn2+ Selective Transporters Involved in the Response to Reactive Oxygen, Molec. Microb 36:1085-1100.[PubMed]
Szegedy, M.A. and M.E. Maguire (1999) "The CorA Magnesium Transport Protein of Salmonella typhimurium: Mutagenesis of Conserved Residues in the Second Membrane Domain,” J. Biological Chemistry 274: 36973-36979. [PubMed]
Moncrief, M.B.C., and M.E. Maguire (1999) “Structure-Function of Microbial Mg2+ Transport Systems,” J. Bio-Inorganic Chemistry 4:523-527. [PubMed]
Smith, R.L. M.A. Szegedy, C. Walker, R.M. Wiet, A. Redpath, M. Kaczmarek, L.M. Kucharski and M.E. Maguire (1998) "The CorA Magnesium Transport Protein of Salmonella typhimurium: Mutagenesis of Conserved Residues in the Third Membrane Domain Identifies a Mg2+ Pore,” J. Biological Chemistry 273:28663-28669. [PubMed]
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