High efficacy treatment of murine Pseudomonas aeruginosa catheter-associated urinary tract infections using the c-di-GMP modulating anti-biofilm compound Disperazol in combination with ciprofloxacin.

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.

Total Linear Chemical Synthesis of LC3A and LC3B.

Solid-phase peptide synthesis (SPPS) enables the synthesis of chemically modified peptides and proteins. Chemically synthesized ubiquitin(-like) proteins containing a fluorescent tag or reactive warhead have proven to be important tools in elucidating biological processes. Here, we describe the first fully synthetic method for the linear synthesis of two LC3 ubiquitin-like proteins using disaggregating building blocks and heated synthesis. Both LC3A and LC3B were synthesized and equipped with a fluorescent rhodamine tag, followed by folding of the proteins and liquid chromatography-mass spectrometry and SDS-PAGE analysis to prove that the quality of the synthetic material is comparable to expressed material.

Identification of small molecules that interfere with c-di-GMP signaling and induce dispersal of Pseudomonas aeruginosa biofilms.

Microbial biofilms are involved in a number of infections that cannot be cured, as microbes in biofilms resist host immune defenses and antibiotic therapies. With no strict biofilm-antibiotic in the current pipelines, there is an unmet need for drug candidates that enable the current antibiotics to eradicate bacteria in biofilms. We used high-throughput screening to identify chemical compounds that reduce the intracellular c-di-GMP content in Pseudomonas aeruginosa. This led to the identification of a small molecule that efficiently depletes P. aeruginosa for c-di-GMP, inhibits biofilm formation, and disperses established biofilm. A combination of our lead compound with standard of care antibiotics showed improved eradication of an implant-associated infection established in mice. Genetic analyses provided evidence that the anti-biofilm compound stimulates the activity of the c-di-GMP phosphodiesterase BifA in P. aeruginosa. Our work constitutes a proof of concept for c-di-GMP phosphodiesterase-activating drugs administered in combination with antibiotics as a viable treatment strategy for otherwise recalcitrant infections.