Wednesday 2 April 2014

INHIBITORS OF CHOLERA TOXIN (POTENTIAL ALTERNATIVE TO ANTIBIOTICS)

Cholera is an acute infection characterized by vomiting, profuse watery diarrhoea and life-threatening dehydration. These symptoms are induced by cholera toxin (CT) secreted by virulent Vibrio cholerae present in the human gut. CT binds and enters intestinal epithelial cells where it increases cyclic AMP production, which leads to alterations in the sodium and chloride ion uptake and resultant water secretion leading to–diarrhoea-and-dehydration.
CT is a major virulence factor of V. cholerae and, its virulence cascade is regulated by master virulence regulator ToxT. The cascade of transcription of operons of subunits of CT is detailed in the introduction and is shown in the figure. Cell density, anaerobiosis, temperature, pH, osmolarity, bile and amino acids initiate this complex cascade of transcriptions that finally produces CT.
The treatment of cholera includes oral rehydration therapy and antibiotics. But these treatment options are not sufficient in many cases and cholera remains a major public health concern, mainly in the developing world. Finding alternative treatments is an area of active research. One strategy is to inhibit the cascade that produces CT. For example previous work developed ToxT inhibitor called virstatin. Such drugs are called antivirulence drugs that target virulence of the pathogen and give opportunity to immune system of the host to clear the infection. Such drugs have many advantages over antibiotics, which are described in the introduction of this paper. Apart from that, these small antivirulence molecules can act as molecular probes in understanding the biology behind virulence. In this study, high-throughput screening (by using green fluorescent protein) was carried out to identify small molecules that can inhibit expression of toxT.
The authors identified three antivirulence compounds: toxtazin A, B and B’ (structural analogue of B) that inhibited toxT virulence cascade. Optimal concentrations at which these compounds work most effectively were also determined. Inhibition of virulence cascade by these three compounds was tested positive for both of the epidemic biotypes of V. cholerae and under the potential conditions which can induce production of CT. All three compounds target the transcription of toxT.
Host colonisation by V. cholerae depends on the expression of toxin-coregulated pilus which is a component of virulence cascade of V. cholerae. In order to apply these lab findings (in vitro results), effects of these compounds on the host colonisation were tested in vivo by administering the compounds to an infant mouse model 3 hours after injecting it with the 106 bacteria. It was found that toxtazin B decreased V. cholerae colonisation in the infant mouse; however, this was not true for toxtazin A.  Further molecular analyses revealed that toxtazin A and toxtazin B/B’ have different targets and mechanisms of action for inhibiting V. cholerae toxT transcription. Toxtazin B specifically targets tcpP transcription, and in turn toxT transcription. Authors hypothesized that Toxtazin A induces a nonpermissive physiological state in the cell which feeds back to shut off toxT transcription.

In conclusion, this study found two novel classes of small molecules (toxtazin A & B/B’) that are potent inhibitor of toxT virulence cascade and hence in turn decrease the production of CT; furthermore, toxtazin B/B’ also reduced host colonisation by V. cholerae. Antibiotics, in general, target various cellular processes; for example, they inhibit cellular transcription, translation and DNA replication in general, which are vital functions of the host cells as well; thus, having the problem of side effects. But use of such small molecules that specifically target virulence appears to be ideal future alternatives of antibiotics. Moreover, they do not affect the gut microbiota of the host. And also, considering the problem of development of antibiotic resistance in many pathogens and the fear of development of antibiotic-induced multidrug resistant superbugs, this discovery is really significant (this is in relation to comment of Dean and my reply on his comment, on my blog-post published on this blog on 5th February 2014). This paper also gives you an idea of how complex a cellular signal transduction pathway can be that subsequently leads to a transcription of one protein-inducing-the-transcription of the next-and so on in a molecular cascade that ultimately produces a substance (e.g. cholera toxin) in response to certain environmental stimuli, which I find really interesting about biology, in general.


Anthouard, R., & DiRita, V. J. (2013). Small-Molecule Inhibitors of toxT Expression in Vibrio cholerae. mBio, 4(4), e00403-13.

3 comments:

  1. These small inhibitors seem like the ideal cholera treatment! I wonder if their colonisation inhibiting effects are able to clear the gut of Vibrio cholerae more quickly. Inducing diarrhoea using cholera toxin benefits the pathogen because it releases many cells into the environment, where they can infect a new host. I could not find a paper on this, but maybe cholera toxin production is also part of the signal cascade leading to gut wall detachment. So inhibiting toxT expression could possibly cause the pathogen to persist in the gut longer, requiring a long course of this treatment. This is just my speculation.

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    1. Hi Dean,
      thanks for the comment.
      It is very difficult to understand what point you want make here. I am getting confused by what you have written.

      As I have perceived this paper, these small inhibitors inhibit the expression of ToxT which is a master regulator of virulence cascade which eventually produces cholera toxin. This inhibition of toxt is at transcription level and was shown by both classes toxtazin A & toxtazin B/B'.
      Colonisation in the gut is related to toxin coregulated pilus expression which is also the part of virulence cascade. In vivo studies on mouse model showed that this gut colonisation was reduced by only toxtazin B and not by toxtazin A.
      I guess gut colonisation would be the first step that V. cholerae would do after entering the human body. And, after successful colonisation of intestine, it would start expressing other genes of the virulence cascade that eventually produce cholera toxin (or may be before but cholera toxin would really show its effect after bacterial colonisation in the intestine), alter the ion uptake-biochemistry of intestinal epithelial cells. This results in secretion of water into the lumen of intestine from blood (rather than absorption of water from intestinal lumen into the blood). This build-up of water in the intestinal lumen would lead to diarrhoea. And I guess V. cholerae would be continuously multiplying during this in the intestine and, releasing its cells in the watery stool.

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    2. I was reading over our lecture of symbiosis between bobtail squid and Vibrio fisheri and the quorum sensing used by the bacterium in the colonisation of the light organ of the squid and in producing light.

      I was wondering if quorum sensing is also involved in the gut colonisation and cholera toxin production by Vibrio cholerae.
      And, I actually found a paper by Zhu et al. 2002 published in PNAS (in vivo study) that showed involvement of quorum sensing regulators in virulence genes of V. cholerae which include toxin-coregulated pilus necessary for gut colonisation and cholera toxin.
      citation: Zhu, J., Miller, M. B., Vance, R. E., Dziejman, M., Bassler, B. L., & Mekalanos, J. J. (2002). Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proceedings of the National Academy of Sciences, 99(5), 3129-3134.



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