ABSTRACT
Persistent urinary tract infections (UTIs) in hospitalized patients constitute an important medical problem. It is estimated that 75% of nosocomial UTIs are associated with urinary tract catheters with P. aeruginosa being a species that forms biofilms on these catheters. These infections are highly resistant to standard-of-care antibiotics, and the effects of the host immune defenses, which allows for development of persistent infections. With antibiotics losing their efficacy, new treatment options against resilient infections, such as catheter-associated urinary tract infections (CAUTIs), are critically needed. Central to our anti-biofilm approach is the manipulation of the c-di-GMP signaling pathway in P. aeruginosa to switch bacteria from the protective biofilm to the unprotected planktonic mode of life. We recently identified a compound (H6-335-P1), that stimulates the c-di-GMP degrading activity of the P. aeruginosa BifA protein which plummets the intracellular c-di-GMP content and induces dispersal of P. aeruginosa biofilm bacteria into the planktonic state. In the present study, we formulated H6-335-P1 as a hydrochloride salt (Disperazol), which is water-soluble and facilitates delivery via injection or oral administration. Disperazol can work as a monotherapy, but we observed a 100-fold improvement in efficacy when treating murine P. aeruginosa CAUTIs with a Disperazol/ciprofloxacin combination. Biologically active Disperazol reached the bladder 30 min after oral administration. Our study provides proof of concept that Disperazol can be used in combination with a relevant antibiotic for effective treatment of CAUTIs.
ABSTRACT
The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were re-synthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing, revealing a strong inhibitor of QS-regulated genes, and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump, thereby inhibiting the autogenerated positive feedback and its enhanced signal-molecule synthesis.
Subject(s)
Pseudomonas aeruginosa , Quorum Sensing , Pseudomonas aeruginosa/genetics , Quorum Sensing/genetics , Virulence Factors/genetics , Bacterial Proteins/geneticsABSTRACT
This paper presents the work performed to transition a lab-scale synthesis (1 g) to a large-scale (400 g) synthesis of the 3-5-diamino-1H-Pyrazole Disperazol, a new pharmaceutical for treatment of antibiotic-resistant Pseudomonas aeruginosa biofilm infections. The potentially hazardous diazotisation step in the lab-scale synthesis was transformed to a safe and easy-to-handle flow chemistry step. Additionally, the paper presents an OSHA-recommended safety assessment of active compound E, as performed by Fauske and Associates, LLC, Burr Ridge, IL, USA.
Subject(s)
Pseudomonas aeruginosa , Pyrazoles , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrazoles/chemical synthesis , Pseudomonas aeruginosa/drug effects , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Biofilms/drug effects , Humans , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Risk AssessmentABSTRACT
The crystal violet microtiter plate biofilm assay is often used to compare the amount of biofilm formed by a mutant versus wild-type or a compound-treated biofilm versus the non-treatment control. In many of these studies the amount of biofilm is assessed only at one single time point. However, if the dynamics of biofilm development of the mutant (or compound-treated biofilm) is different than that of the wild-type (or non-treatment control), then biofilm quantification at a single time point may give misleading results. To overcome this shortcoming of the common biofilm quantification technique, we recommend to use a serial dilution-based crystal violet microtiter plate biofilm assay for easy assessment of the dynamics of biofilm development and dispersal. We demonstrate that the dilution-resolved crystal violet assay displays the dynamics of Pseudomonas aeruginosa biofilm development and dispersal as efficient as a time-resolved crystal violet assay. In addition, focusing on mutants of different parts of the c-di-GMP signaling system in P. aeruginosa, we provide an example illustrating the need to assess biofilm dynamics instead of quantifying biofilm biomass at a single time point.