Small Molecule Inhibitor of Type Three Secretion System Belonging to a Class 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones Improves Survival and Decreases Bacterial Loads in an AirwayPseudomonas aeruginosaInfection in Mice

Abstract

Pseudomonas aeruginosa is a cause of high mortality in burn, immunocompromised, and surgery patients. High incidence of antibiotic resistance in this pathogen makes the existent therapy inefficient. Type three secretion system (T3SS) is a leading virulence system of P. aeruginosa that actively suppresses host resistance and enhances the severity of infection. Innovative therapeutic strategies aiming at inhibition of type three secretion system of P. aeruginosa are highly attractive, as they may reduce the severity of clinical manifestations and improve antibacterial immune responses. They may also represent an attractive therapy for antibiotic-resistant bacteria. Recently our laboratory developed a new small molecule inhibitor belonging to a class 2,4-disubstituted-4H-[1,3, 4]-thiadiazine-5-ones, Fluorothiazinon (FT), that effectively suppressed T3SS in chlamydia and salmonella in vitro and in vivo. In this study, we evaluate the activity of FT towards antibiotic-resistant clinical isolates of P. aeruginosa expressing T3SS effectors ExoU and ExoS in an airway infection model. We found that FT reduced mortality and bacterial loads and decrease lung pathology and systemic inflammation. In addition, we show that FT inhibits the secretion of ExoT and ExoY, reduced bacteria cytotoxicity, and increased bacteria internalization in vitro. Overall, FT shows a strong potential as an antibacterial therapy of antibiotic-resistant P. aeruginosa infection.

Animal Models of Type III Secretion System-Mediated Pathogenesis

“…This section covers selected in vivo techniques to study the T3SS. Mice are the most common rodent used to study T3SS pathogenesis [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]. They are small, inexpensive, and well described in comparison to other models.…”

“…The most common bacterial load assay is organ homogenization. It is performed after the animal is sacrificed [38,43,46]. The animal's infected organs are harvested, cleaned, and homogenized in order to be plated using serial dilution.…”

“…Organ homogenization of lung and spleen samples was used by the Kobets laboratory in a T3SS inhibition assay with P. aeruginosa [46]. They found that treatment with a small molecule T3SS inhibitor led to reduced CFU counts.…”

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).…”

Prevalence of ExoY Activity in Pseudomonas aeruginosa Reference Panel Strains and Impact on Cytotoxicity in Epithelial Cells

“…Moreover, the expression of the type III secretion system (T3SS), which enables direct delivery of virulence factors into human endothelial or epithelial cells, was also shown to change over the course of CF infections (Jain et al, 2004). As strains with a functional T3SS cause higher bacterial burden and mortality in acute respiratory infections (Hauser et al, 2002), the T3SS is considered as a potential therapeutic target (Lee et al, 2007;Aiello et al, 2010;Anantharajah et al, 2016;Sheremet et al, 2018).…”