Original Article | Published:

A small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones suppresses Salmonella infection in vivo

The Journal of Antibiotics volume 69, pages 422427 (2016) | Download Citation

Abstract

Therapeutic strategies that target bacterial virulence have received considerable attention. The type III secretion system (T3SS) is important for bacterial virulence and represents an attractive therapeutic target. A novel compound with a predicted T3SS inhibitory activity named CL-55 (N-(2,4-difluorophenyl)-4-(3-ethoxy-4-hydroxybenzyl)-5-oxo-5,6-dihydro-4H-[1,3,4]-thiadiazine-2-carboxamide) was previously characterized by low toxicity, high levels of solubility, stability and specific efficiency toward Chlamydia trachomatis in vitro and in vivo. In this study, we describe the action of CL-55 on Salmonella enterica serovar Typhimurium. We found that CL-55 does not affect Salmonella growth in vitro but suppresses Salmonella infection in vivo. The i.p. injection of CL-55 at a dose of 10 mg kg−1 for 4 days significantly (500-fold) decreased the numbers of Salmonella in the spleen and peritoneal lavages and increased the survival rates in susceptible (BALB/c, I/St) and resistant (A/Sn) mice. Twelve days of therapy led to complete eradication of Salmonella in mice. Moreover, no pathogen was found 4–6 weeks post treatment. CL-55 was not carcinogenic or mutagenic, did not increase the level of chromosomal aberrations in bone marrow cells and had low toxicity in mice, rats and rabbits. Pharmacokinetic studies have shown that CL-55 rapidly disappears from systemic blood circulation and is distributed in the organs. Our data demonstrates that CL-55 affects S. enterica serovar Typhimurium in vivo and could be used as a substance in the design of antibacterial inhibitors for pharmaceutical intervention of bacterial virulence for infection.

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References

  1. 1.

    Pathobiology of salmonella, intestinal microbiota, and the host innate immune response. Front. Immunol. 26, 252 (2014).

  2. 2.

    et al. Type 3 secretion system island encoded proteins required for colonization by non-O1/non-O139 serogroup V. cholerae. Infect. Immun. 83, 2862–2869 (2015).

  3. 3.

    Mechanism of action of EPEC type III effector molecules. Int. J. Med. Microbiol. 291, 469–477 (2002).

  4. 4.

    , & Shigella: a model of virulence regulation in vivo. Gut Microbes 3, 104–120 (2012).

  5. 5.

    & Interrelationship between type three secretion system and metabolism in pathogenic bacteria. Front. Cell Infect. Microbiol. 27, 150 (2014).

  6. 6.

    , , & The role of the type-three secretion system of the gram-negative bacteria in regulation of chronic infections. Mol. Gen. Mikrobiol. Virusol. 3, 3–13 (2012a).

  7. 7.

    , & Quantitative assessment of cytosolic Salmonella in epithelial cells. PLoS ONE 9, e84681 (2014).

  8. 8.

    & Targeting the type III secretion system to treat bacterial infections. Expert. Opin. Ther. Targets 18, 137–152 (2014).

  9. 9.

    et al. Structural characterisation of Tpx from Yersinia pseudotuberculosis reveals insights into the binding of salicylidene acylhydrazide compounds. PLoS One 7, e32217 (2012).

  10. 10.

    et al. Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence-blocking compounds. J. Biol. Chem. 286, 29922–29931 (2011).

  11. 11.

    et al. Candidate vaginal microbicides with activity against Chlamydia trachomatis and Neisseria gonorrhoeae. Int. J. Antimicrob. Agents 36, 145–150 (2010).

  12. 12.

    et al. Pre-clinical pharmacokinetics and anti-chlamydial activity of salicylidene acylhydrazide inhibitors of bacterial type III secretion. J. Antibiot. 65, 397–404 (2012).

  13. 13.

    et al. Salicylidene acylhydrazide-mediated inhibition of type III secretion system-1 in Salmonella enterica serovar Typhimurium is associated with iron restriction and can be reversed by free iron. FEMS Microbiol. Lett. 302, 114–122 (2010).

  14. 14.

    , , & Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria. J. Intern. Med. 264, 17–29 (2008).

  15. 15.

    et al. Salicylidene acylhydrazides that affect type III protein secretion in Salmonella enterica serovar Typhimurium. Antimicrob. Agents Chemother. 51, 2867–2876 (2007).

  16. 16.

    , & Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton. J. Bacteriol. 191, 563–570 (2009).

  17. 17.

    et al. Inhibition of type III secretion in Salmonella enterica serovar Typhimurium by small-molecule inhibitors. Antimicrob. Agents Chemother. 51, 2631–263 (2007).

  18. 18.

    et al. Small molecule inhibitors of LcrF, a Yersinia pseudotuberculosis transcription factor, attenuate virulence and limit infection in a murine pneumonia model. Infect. Immun. 78, 4683–4690 (2010).

  19. 19.

    , , , & Protection of mice from a Chlamydia trachomatis vaginal infection using a salicylidene acylhydrazide, a potential microbicide. J. Infect. Dis. 204, 1313–1320 (2011).

  20. 20.

    et al. A small-molecule inhibitor of the bacterial type III secretion system protects against in vivo infection with Citrobacter rodentium. J. Antibiot. 64, 197–203 (2011).

  21. 21.

    et al. Development of chlamydial type III secretion system inhibitors for suppression of acute and chronic forms of chlamydial infection. Acta Naturae 4, 87–97 (2012b).

  22. 22.

    et al. Small molecule inhibitor of type three secretion suppresses acute and chronic Chlamydia trachomatis infection in a novel urogenital Chlamydia model. Biomed. Res. Int. 2015, 484853 (2015).

  23. 23.

    et al. Mycobacterium tuberculosis-susceptible I/St mice develop severe disease following infection with taxonomically distant bacteria, Salmonella enterica and Chlamydia pneumoniae. Clin. Exp. Immunol. 146, 93–100 (2006).

  24. 24.

    et al. Comet assay as a tool to screen for mouse models with inherited radiation sensitivity. Mamm. Genome 11, 552–554 (2000).

  25. 25.

    et al. Mammalian in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow cells. Mutat. Res. 189, 157–165 (1987).

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Acknowledgements

This work was supported by grants from the Russian Ministry of Science and Education and the Russian Ministry of Industry.

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Affiliations

  1. Gamaleya Research Center for Epidemiology and Microbiology, Moscow, Russia

    • Ludmila N Nesterenko
    • , Nailya A Zigangirova
    • , Egor S Zayakin
    • , Sergey I Luyksaar
    • , Natalie V Kobets
    • , Denis V Balunets
    • , Ludmila A Shabalina
    • , Tatiana N Bolshakova
    • , Olga Y Dobrynina
    •  & Alexander L Gintsburg

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The authors declare no conflict of interest.

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Correspondence to Natalie V Kobets.

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DOI

https://doi.org/10.1038/ja.2015.131

Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)