Friday, March 18, 2011

vibrio chloerae

Most Americans don't fear the attack of vibrio cholerae anymore, but pandemics of this bacteria have killed millions of people and it continues to strike in communities using contaminated water. The latest well-known outbreak hit Haiti following the devastation of the earthquake. People infected with cholera suffer debilitating bouts of diarrhea and the ensuing dehydration and loss of electrolytes causes the victims to appear blue-black and shriveled. Without rapid treatment, death can occur in just four hours.

Many theories were proposed in attempts to describe the mechanism of attack. In the 18th and 19th centuries, cholera was thought to be caused by "bad air" or the temperature of ingested food and water. It wasn't until 1854, when British physician John Snow mapped the outbreak clusters in London, that people realized the infection was transmitted through water contaminated by human feces.

An overview (source 1):
The culprit at the heart of the destruction is cholera toxin (CT). Secreted by Vibrio cholerae upon entry into the wall of the small intestine, the toxin acts fast in the intestinal lumen. It is divided into A and B subunits. The B unit is the stabilizer; its five identical units form a ring to anchor the complex. It binds to the epithelial cell membranes by attaching to a specific ganglioside, GM1, found in lipid rafts. This connection allows a portion of subunit A, the afflictor, to enter the cytosol. It acts as an ADP-ribosyltransferase, catalyzing the addition of ADP-ribose to Gof a G-protein regulator. This ADP-ribosylation irreversibly activates Gsα, resulting in an overproduction of cAMP. Without the capability to turn off intracellular rush of cAMP, both salt and water accumulate in the cell and the cell dies. Occurring in a massive amount of cells, this process creates the flood of diarrhea characterizing the disease. 

                                                      CT                     AC
                                G  +  NAD    →   ADPR-Gsα    →     cAMP

Interesting points:
  • The A unit has two subunits: A1 separates from the rest of the complex to enter the cell and A2 mediates the vital connection between A and B. A1 and A2 are connected via a disulfide bond.
  • ADP-ribosylation of G is irreversible because it increases the sensitivity of G to its activator GTP, as well as decreasing its GTPase activity, which normally would hydrolyze GTP to GDP and dissociate the G-AC complex - this can induce cAMP production enhancements of several hundred-fold!

 An expansion of A1 function (source 2):
 A1, the catalytic portion of the protein, is divided further into three subdomains, each with their own unique functions. A11 is the largest (132 residues) and functions as the catalyzer. A12 consists of 29 residues and is short and flexible, and A13 (31 residues) is globular and largely hydrophobic, interfacing with A11 and supplying the disulfide bond that links A1 with A2.

This study focused on the unique role of A13, hypothesizing that it facilitated two processes: entry of A1 into the cytosol through ER-associated degradation (ERAD)-mediated transport and interactions with ADP-ribosylation factors (ARFs) that allosterically activate A1. 

Although they could not demonstrate that ERAD plays a role in A1 transport, the concept is interesting. ERAD removes misfolded proteins from the ER to the cytosol for degradation. By disguising itself as a misfolded protein, A13 would trigger the normally beneficial action of ERAD, taking advantage of its technique to eventually destroy the entire cell. It was demonstrated that A13 was not necessary for the translocation of A1 using an ERAD system. They propose that the trigger for ERAD export into the cytosol might not be a specific sequence of residues, but an inherent physical quality of A1 instead.

ARFs  are known to be allosteric activators of A1, the exact mechanism is not yet determined. Here, the researchers demonstrated that A13 is necessary for interaction with one ARF, using examples of A13 mutants that lack toxicity because of poor ARF interactions. Without A13, ARF6 and A1 interaction was reduced to 15% of that of the wild type.

An expansion of subunit B binding effects (source 3):
It is difficult to study the mechanism of cholera toxin's adherence to the membrane in a laboratory setting for a variety of reasons: triggered endocytosis removes CTB from the membrane before it can be studied and CTA contamination can increase intracellular cAMP which can't be differentiated from some of CTB's effects. This study used integrative approaches such as surface functionalization and single cell photometry to overcome these obstacles. CTB binding induces multiple cell processes, including vesicle docking, trafficking, and exocytosis.

They demonstrated that this process is influenced by both cholesterol and calcium signaling. The recognition of cholesterol's role gives more significance to the purpose of lipid rafts When lipid rafts are disrupted, the functions performed by CTB are decreased. The processes of CTB binding increase intracellular concentrations of calcium, probably through the action of the ganglioside, GM1. It is suggested that CTB attachment reduces the inhibition normally acted by gangliosides on enzymes that induce exoctyosis.


 Sources:
1. http://www.ncbi.nlm.nih.gov/pubmed/1480112
2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1418936/
3. http://pubs.rsc.org/en/content/pdf/article/2010/ib/c0ib00006j

Tuesday, March 1, 2011

 purely aesthetic view, using a space-filling model and colouring the five B subunits red and the A subunit green and black in order to imitate a poppy

side cartoon view, colouring each chain separately in order to emphasize the separation of the A and B subunits and the alpha-helix bridge connecting them

 top view of B subunit, highlighting separate secondary structures, with alpha-helices coloured in blue and beta-sheets coloured in purple
includes water molecules associated with the protein

 space-filling model, using colours to display the amino and carboxy terminals of the individual chains, as well as the insertion of the A subunit at the center (in red)

side view using separate colours and models to emphasize the different subunits