Limits...
Small-molecule inhibitors of toxT expression in Vibrio cholerae.

Anthouard R, DiRita VJ - MBio (2013)

Bottom Line: All three compounds reduce production of both CT and the toxin-coregulated pilus (TCP), an important colonization factor.Treatment with toxtazin B results in a 100-fold reduction in colonization in an infant mouse model of infection, though toxtazin A did not reduce colonization at the concentrations tested.These results add to the growing body of literature indicating that small-molecule inhibitors of virulence genes could be developed to treat infections, as alternatives to antibiotics become increasingly needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan, Medical School, Ann Arbor, Michigan, USA.

ABSTRACT

Unlabelled: Vibrio cholerae, a Gram-negative bacterium, infects humans and causes cholera, a severe disease characterized by vomiting and diarrhea. These symptoms are primarily caused by cholera toxin (CT), whose production by V. cholerae is tightly regulated by the virulence cascade. In this study, we designed and carried out a high-throughput chemical genetic screen to identify inhibitors of the virulence cascade. We identified three compounds, which we named toxtazin A and toxtazin B and B', representing two novel classes of toxT transcription inhibitors. All three compounds reduce production of both CT and the toxin-coregulated pilus (TCP), an important colonization factor. We present evidence that toxtazin A works at the level of the toxT promoter and that toxtazins B and B' work at the level of the tcpP promoter. Treatment with toxtazin B results in a 100-fold reduction in colonization in an infant mouse model of infection, though toxtazin A did not reduce colonization at the concentrations tested. These results add to the growing body of literature indicating that small-molecule inhibitors of virulence genes could be developed to treat infections, as alternatives to antibiotics become increasingly needed.

Importance: V. cholerae caused more than 580,000 infections worldwide in 2011 alone (WHO, Wkly. Epidemiol. Rec. 87:289-304, 2012). Cholera is treated with an oral rehydration therapy consisting of water, glucose, and electrolytes. However, as V. cholerae is transmitted via contaminated water, treatment can be difficult for communities whose water source is contaminated. In this study, we address the need for new therapeutic approaches by targeting the production of the main virulence factor, cholera toxin (CT). The high-throughput screen presented here led to the identification of two novel classes of inhibitors of the virulence cascade in V. cholerae, toxtazin A and toxtazins B and B'. We demonstrate that (i) small-molecule inhibitors of virulence gene production can be identified in a high-throughput screen, (ii) targeting virulence gene production is an effective therapeutic strategy, and (iii) small-molecule inhibitors can uncover unknown layers of gene regulation, even in well-studied regulatory cascades.

Show MeSH

Related in: MedlinePlus

Identification of inhibitors of the virulence cascade in V. cholerae. (A) The wild-type reporter strain NB39 has a high GFP/OD600 ratio when grown overnight under toxin-inducing conditions, while the isogenic ΔtoxR strain NB39 does not. (B) Funnel figure illustrating the triage process used in the screen. (C) Structures of the toxtazins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3735192&req=5

fig1: Identification of inhibitors of the virulence cascade in V. cholerae. (A) The wild-type reporter strain NB39 has a high GFP/OD600 ratio when grown overnight under toxin-inducing conditions, while the isogenic ΔtoxR strain NB39 does not. (B) Funnel figure illustrating the triage process used in the screen. (C) Structures of the toxtazins.

Mentions: To identify small-molecule inhibitors of toxT transcription that do not affect general growth, a reporter strain was constructed consisting of wild-type V. cholerae harboring a plasmid on which the toxT promoter drives the expression of green fluorescent protein (GFP). Culturing the toxT::gfp reporter strain NB39 under toxin-inducing conditions resulted in high numbers of fluorescence intensity units in a wild-type background and relatively low numbers of fluorescence intensity units in the isogenic ΔtoxR strain NB40 (Fig. 1A). Molecules that decrease GFP expression in a wild-type cell could inhibit any event in the virulence cascade prior to toxT transcription.


Small-molecule inhibitors of toxT expression in Vibrio cholerae.

Anthouard R, DiRita VJ - MBio (2013)

Identification of inhibitors of the virulence cascade in V. cholerae. (A) The wild-type reporter strain NB39 has a high GFP/OD600 ratio when grown overnight under toxin-inducing conditions, while the isogenic ΔtoxR strain NB39 does not. (B) Funnel figure illustrating the triage process used in the screen. (C) Structures of the toxtazins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3735192&req=5

fig1: Identification of inhibitors of the virulence cascade in V. cholerae. (A) The wild-type reporter strain NB39 has a high GFP/OD600 ratio when grown overnight under toxin-inducing conditions, while the isogenic ΔtoxR strain NB39 does not. (B) Funnel figure illustrating the triage process used in the screen. (C) Structures of the toxtazins.
Mentions: To identify small-molecule inhibitors of toxT transcription that do not affect general growth, a reporter strain was constructed consisting of wild-type V. cholerae harboring a plasmid on which the toxT promoter drives the expression of green fluorescent protein (GFP). Culturing the toxT::gfp reporter strain NB39 under toxin-inducing conditions resulted in high numbers of fluorescence intensity units in a wild-type background and relatively low numbers of fluorescence intensity units in the isogenic ΔtoxR strain NB40 (Fig. 1A). Molecules that decrease GFP expression in a wild-type cell could inhibit any event in the virulence cascade prior to toxT transcription.

Bottom Line: All three compounds reduce production of both CT and the toxin-coregulated pilus (TCP), an important colonization factor.Treatment with toxtazin B results in a 100-fold reduction in colonization in an infant mouse model of infection, though toxtazin A did not reduce colonization at the concentrations tested.These results add to the growing body of literature indicating that small-molecule inhibitors of virulence genes could be developed to treat infections, as alternatives to antibiotics become increasingly needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan, Medical School, Ann Arbor, Michigan, USA.

ABSTRACT

Unlabelled: Vibrio cholerae, a Gram-negative bacterium, infects humans and causes cholera, a severe disease characterized by vomiting and diarrhea. These symptoms are primarily caused by cholera toxin (CT), whose production by V. cholerae is tightly regulated by the virulence cascade. In this study, we designed and carried out a high-throughput chemical genetic screen to identify inhibitors of the virulence cascade. We identified three compounds, which we named toxtazin A and toxtazin B and B', representing two novel classes of toxT transcription inhibitors. All three compounds reduce production of both CT and the toxin-coregulated pilus (TCP), an important colonization factor. We present evidence that toxtazin A works at the level of the toxT promoter and that toxtazins B and B' work at the level of the tcpP promoter. Treatment with toxtazin B results in a 100-fold reduction in colonization in an infant mouse model of infection, though toxtazin A did not reduce colonization at the concentrations tested. These results add to the growing body of literature indicating that small-molecule inhibitors of virulence genes could be developed to treat infections, as alternatives to antibiotics become increasingly needed.

Importance: V. cholerae caused more than 580,000 infections worldwide in 2011 alone (WHO, Wkly. Epidemiol. Rec. 87:289-304, 2012). Cholera is treated with an oral rehydration therapy consisting of water, glucose, and electrolytes. However, as V. cholerae is transmitted via contaminated water, treatment can be difficult for communities whose water source is contaminated. In this study, we address the need for new therapeutic approaches by targeting the production of the main virulence factor, cholera toxin (CT). The high-throughput screen presented here led to the identification of two novel classes of inhibitors of the virulence cascade in V. cholerae, toxtazin A and toxtazins B and B'. We demonstrate that (i) small-molecule inhibitors of virulence gene production can be identified in a high-throughput screen, (ii) targeting virulence gene production is an effective therapeutic strategy, and (iii) small-molecule inhibitors can uncover unknown layers of gene regulation, even in well-studied regulatory cascades.

Show MeSH
Related in: MedlinePlus