Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors

Abstract

Antibiotic-resistant bacteria are an emerging threat to global public health. New classes of antibiotics and tools for antimicrobial discovery are urgently needed. Type III secretion systems (T3SS), which are required by dozens of Gramnegative bacteria for virulence but largely absent from nonpathogenic bacteria, are promising virulence blocker targets. The ability of mammalian cells to recognize the presence of a functional T3SS and trigger NF-B activation provides a rapid and sensitive method for identifying chemical inhibitors of T3SS activity. In this study, we generated a HEK293 stable cell line expressing green fluorescent protein (GFP) driven by a promoter containing NF-B enhancer elements to serve as a readout of T3SS function. We identified a family of synthetic cyclic peptide-peptoid hybrid molecules (peptomers) that exhibited dose-dependent inhibition of T3SS effector secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa without affecting bacterial growth or motility. Among these inhibitors, EpD-3=N, EpD-1,2N, EpD-1,3=N, EpD-1,2,3=N, and EpD-1,2,4=N exhibited strong inhibitory effects on translocation of the Yersinia YopM effector protein into mammalian cells (Ͼ40% translocation inhibition at 7.5 M) and showed no toxicity to mammalian cells at 240 M. In addition, EpD-3=N and EpD-1,2,4=N reduced the rounding of HeLa cells caused by the activity of Yersinia effector proteins that target the actin cytoskeleton. In summary, we have discovered a family of novel cyclic peptomers that inhibit the injectisome T3SS but not the flagellar T3SS.

Developing Cyclic Peptomers as Broad-Spectrum Gram negative Bacterial Type III Secretion System Inhibitors

“…Secretion of protein substrates through the injectisome T3SS, the flagellar system, and the Tat secretion system require the proton motive force (34)(35)(36). Although cyclic peptomers inhibited secretion from the injectisome T3SS, they did not inhibit the Tat system and only weakly inhibited flagellar substrate secretion, suggesting that the proton motive force is unaffected, as we previously suggested (14), and that the cyclic peptomers do not inhibit bacterial secretion in general. Table S2.…”

“…Previously we identified a group of cyclic peptomers that inhibited secretion of substrates from Y. pseudotuberculosis and P. aeruginosa T3SSs, but did not inhibit bacterial growth, motility, or HeLa cell metabolism (14). The results suggested a potential for development of these cyclic peptomers as pathogen-specific virulence blockers.…”

“…We previously showed that cyclic peptomers inhibited the Ysc T3SS family found in P. aeruginosa and Yersinia ( Fig. 1-2) (14). In order to test whether cyclic peptomers are active against other T3SS families, we evaluated the effect of cyclic peptomers on the Inv-Mxi-Spa T3SS in Y. enterocolitica and Salmonella enterica serovar Typhimurium.…”

“…Interestingly, the Chlamydia T3SS belongs to its own T3SS family (7,8). Here we identify a derivative of a synthetic cyclic peptomer family of T3SS inhibitors (14) that can inhibit the T3SS machinery of three evolutionarily distinct T3SS families used by five different bacterial species to cause human disease, including Chlamydia trachomatis.…”

Fitting Pieces into the Puzzle of Pseudomonas aeruginosa Type III Secretion System Gene Expression

“…Other inhibitors of the T3SS include the anticancer drug cisplatin (197), salicyclidene acylhydrazide INP0341 (198), (Ϫ)-hopeaphenol (199), a selection of synthetic cyclic peptomers (200), and the small-molecule inhibitor fluorothiazinon (197,201). Many of these compounds protect eukaryotic cells from T3SS-induced cytotoxicity (198,199,201).…”