guanosine-triphosphate and Cholera

Topics: Adenosine Diphosphate; Adenylyl Cyclases; ADP-Ribosylation Factors; Cholera; Cholera Toxin; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Protein Binding

The bacterial secondary messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been implicated in the pathogenesis of Vibrio cholerae, due to its significant role in regulating the virulence, biofilm formation and motility of the host organism. The VC0395_0300 protein from V. cholerae, possessing a GGEEF sequence has been established as a diguanylate cyclase (DGC) capable of catalyzing the conversion of two GTP molecules to form cyclic-di-GMP. This in turn, plays a crucial role in allowing the organism to adopt a dual lifestyle, thriving both in human and aquatic systems. The difficulty in procuring sufficient amounts of homogenous soluble protein for structural assessment of the GGDEF domain in VC0395_0300 and the lack of soluble protein yield, prompted the truncation into smaller constructs (Sebox31 and Sebox32) carrying the GGDEF domain. The truncates retained their diguanylate cyclase activity comparable to the wild type, and were able to form biofilms as well. Fluorescence and circular dichroism spectroscopy measurements revealed that the basic structural elements do not show significant changes in the truncated proteins as compared to the full-length. This has also been confirmed using homology modeling and molecular docking of the wild type and truncates. This led us to conclude that the truncated constructs retain their activity in spite of the deletions in the N terminal region. This is supportive of the fact that DGC activity in GGDEF proteins is predominantly dependent on the presence of the conserved GGD(/E)EF domain and its interaction with GTP.

Topics: Amino Acid Sequence; Bacterial Proteins; Cholera; Cyclic GMP; Escherichia coli Proteins; Guanosine Triphosphate; Humans; Models, Molecular; Phosphorus-Oxygen Lyases; Vibrio cholerae

Feo is the most widely conserved system for ferrous iron transport in prokaryotes, and it is important for virulence in some pathogens. However, its mechanism of iron transport is not fully understood. In this study, we used full-length Vibrio cholerae FeoB (VcFeoB) as a model system to study whether its enzymatic activity is affected by regulatory factors commonly associated with FeoB proteins from other species or with G-proteins that have homology to FeoB. VcFeoB showed a higher rate of hydrolysis of both ATP and GTP than its N-terminal domain alone; likewise, ions such as K+ and Fe2+ did not modulate its nucleotide hydrolysis. We also showed that the three V. cholerae Feo proteins (FeoA, FeoB, and FeoC) work in a 1 : 1 : 1 molar ratio in vivo. Although both FeoA and FeoC are required for Feo-mediated iron transport, neither of these proteins affected the VcFeoB NTPase rate. These results are consistent with an active transport mechanism independent of stimulatory factors and highlight the importance of using full-length FeoB proteins as a reliable proxy to study Feo-mediated iron transport in vitro.

Topics: Adenosine Triphosphate; Bacterial Proteins; Cholera; Guanosine Triphosphate; Humans; Hydrolysis; Iron; Potassium; Vibrio cholerae

In the rod outer segment membranes of the bovine retina at least two members of the small molecular weight guanine nucleotide binding proteins were identified by means of the technique of binding radiolabeled GTP to nitrocellulose Western blots of proteins separated by sodium dodecyl sulphate gel electrophoresis. Such proteins, of 23 and 25 kDa, are able to specifically bind guanine nucleotides after denaturing treatments, and are not labeled by pertussis or cholera toxin-catalyzed ADP-ribosylation. The binding site is specific for GTP.

Topics: Adenosine Diphosphate Ribose; Animals; Antibodies, Monoclonal; Binding Sites; Blotting, Western; Cattle; Cholera; Cross Reactions; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanosine Triphosphate; Molecular Weight; Pertussis Toxin; Photoreceptor Cells; Rod Cell Outer Segment; Transducin; Virulence Factors, Bordetella

The cholera vibrio, unlike most pathogens, neither invades the body's cells nor causes them direct physical damage and thus does not engender inflammation or fever. Rather, the organism attaches itself to the intestinal epithelium, where it secretes a toxin that deranges cellular function. Study of the toxin's structure and mechanism of action is helping to elucidate normal enzymatic processes.

Topics: Adenylyl Cyclases; Bacterial Toxins; Cholera; Cholera Toxin; Cyclic AMP; Dehydration; Epithelium; Escherichia coli; Gangliosides; Guanosine Triphosphate; Humans; Intestinal Mucosa; NAD; Structure-Activity Relationship; Water-Electrolyte Imbalance

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Adult; Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Diarrhea, Infantile; Enterotoxins; Epithelial Cells; Epithelium; Escherichia coli Infections; Fluorides; Guanosine Triphosphate; Guanylate Cyclase; Humans; Infant; Intestinal Mucosa; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes; Prostaglandins; Temperature; Tritium