Salicylidene Acylhydrazides and Hydroxyquinolines Act as Inhibitors of Type Three Secretion Systems in Pseudomonas aeruginosa by Distinct Mechanisms

Abstract

Type 3 secretion systems (T3SSs) are major virulence factors in Gramnegative bacteria. Pseudomonas aeruginosa expresses two T3SSs, namely, an injectisome (iT3SS) translocating effector proteins in the host cell cytosol and a flagellum (fT3SS) ensuring bacterial motility. Inhibiting these systems is an appealing therapeutic strategy for acute infections. This study examines the protective effects of the salicylidene acylhydrazide INP0341 and of the hydroxyquinoline INP1750 (previously described as T3SS inhibitors in other species) toward cytotoxic effects of P. aeruginosa in vitro. Both compounds reduced cell necrosis and inflammasome activation induced by reference strains or clinical isolates expressing T3SS toxins or only the translocation apparatus. INP0341 inhibited iT3SS transcriptional activation, including in strains with constitutive iT3SS expression, and reduced the total expression of toxins, suggesting it targets iT3SS gene transcription. INP1750 inhibited toxin secretion and flagellar motility and impaired the activity of the YscN ATPase from Yersinia pseudotuberculosis (homologous to the ATPase present in the basal body of P. aeruginosa iT3SS and fT3SS), suggesting that it rather targets a T3SS core constituent with high homology among iT3SS and fT3SS. This mode of action is similar to that previously described for INP1855, another hydroxyquinoline, against P. aeruginosa. Thus, although acting by different mechanisms, INP0341 and INP1750 appear as useful inhibitors of the virulence of P. aeruginosa. Hydroxyquinolines may have a broader spectrum of activity by the fact they act upon two virulence factors (iT3SS and fT3SS).

Great phenotypic and genetic variation among successive chronic Pseudomonas aeruginosa from a cystic fibrosis patient

“…Elastase activity was determined by the Elastin-Congo-Red assay [ 21 ]. The chloroform-extract method was used for pyocyanin and pyorubin pigments quantification [ 22 ].…”

Inhibition of the type III secretion system by syringaldehyde protects mice from Salmonella enterica serovar Typhimurium

“…Increasing numbers of small molecule compounds have been identified in recent years with the aim of targeting specific key bacterial virulence factors, including those that affect the function of the bacterial type III secretion system. For instance, salicylidene acylhydrazides have a broad spectrum of inhibitory activity against T3SSs in several important Gram‐negative pathogens, including Salmonella spp, Pseudomonas aeruginosa and Chlamydiae . In addition, licoflavonol was shown to exhibit a strong inhibitory effect on the secretion and the transportation of SPI‐1 effector proteins …”

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

“…Recently, whole-cell-based high-throughput screens performed to identify T3SS inhibitors gave several classes of small molecule compounds. Salicylidene acylhydrazides, salicylanilides, sulfonylaminobenzanilides, benzimidazoles, thiazolidinone, and some natural products were shown to be effective against a number of pathogenic bacteria that utilize T3SS, including Yersinia, Chlamydia, Salmonella , enteropathogenic Escherichia coli, Shigella , and Pseudomonas [ 11 , 22 – 25 ].…”

“…These strategies also reduce the risk of selecting resistance, since they only disarm bacteria, allowing the host to employ immune mechanisms to fight the infection [ 8 ]. Different types of T3SS inhibitors are currently reported for Gram-negative bacteria such as therapeutic antibodies against P. aeruginosa PcrV protein [ 9 ], hybrid antibodies against PcrV and Psl [ 10 ], salicylidene acylhydrazides and hydroxyquinolines [ 11 ], and others [ 12 , 13 ]. Recently our laboratory developed a new small molecule inhibitor designated as Fluorothiazinon (FT) that belongs to the class of 2,4-disubstituted-4H-[1,3, 4]-thiadiazine-5-ones.…”