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 5^th 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.