title: Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells.
abstract: Mammalian heterotrimeric GTP-binding protein (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the alpha-, beta- or the gamma-subunits of heterotrimeric G proteins. Such antibodies also after the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk-G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian alpha- and beta-G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the alpha-, beta- and gamma-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the alpha- and gamma-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.
authors.affiliation: Department of Microbiology & Immunology, University of Illinois College of Medicine, Chicago 60612, USA.
authors.forename: S
authors.initials: S
authors.lastname: Shankar
authors.forename: V
authors.initials: V
authors.lastname: Kapatral
authors.forename: A M
authors.initials: AM
authors.lastname: Chakrabarty
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003429:Cross Reactions; D004912:Erythrocytes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D015398:Signal Transduction; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D000906:Antibodies; D006160:Guanosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D019204:GTP-Binding Proteins; D014544:Uridine Triphosphate