Confirmatory Screen for Inhibitors of Bacterial Capsule Biogenesis (2)
Primary Assay Rationale and Summary: Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million UTIs occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. more ..
BioActive Compounds: 58
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. Patrick Seed, Duke University, Durham, NC
Award: 1 R03 MH090791-01
Primary Assay Rationale and Summary: Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million UTIs occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. Antimicrobial resistance among UPEC is on the rise, driving efforts to elucidate vulnerable targets in the molecular pathogenesis of infection. New insights into the roles of K capsules in UPEC virulence during UTI make capsules an attractive target.
During UTI, UPEC lives in intracellular and extracellular locales. UPEC adheres to the apical bladder epithelium and then invades this layer of cells. Within the bladder epithelium, UPEC typically replicates in a biofilm-like state called intracellular bacterial communities (IBC). After maturation of IBCs, UPEC disperses away from the IBC and exits the infected cells. Extracellular UPEC must then re-adhere, initiating the invasion and intracellular replication phases again. Past studies have revealed bacteria encased in the IBC within a complex matrix of fibrous protein assemblies and polysaccharides. Prior studies have also shown that disruption of the IBC pathway aborts experimental UTI, highlighting the importance of this intracellular lifecycle. A detailed study of urine samples from women with acute UTI demonstrated IBC in shed bladder epithelial cell, showing that the pathway is conserved in humans. Investigations have shown that K capsule contributes to multiple aspects of pathogenesis, including IBC formation.
Uropathogenic Escherichia coli (UPEC) produces 80% of community-acquired urinary tract infections (UTI). UPEC is also a leading cause of nosocomial UTI, the most prevalent hospital acquired infection. Dissemination of UPEC from the lower urinary tract is associated with morbidity and mortality through infection of the kidneys, bloodstream, and central nervous system. In recent years, the treatment of outpatient and inpatient UTI has been severely compromised by the rising incidence of antibiotic-resistant UPEC.
Investigators have found that encapsulation is an important UPEC virulence factor. The K1 capsule type is closely associated with pathogenic isolates; not only is it the leading type in UTI, but it also accounts for much of the extra-urinary tract complications. Animals studies of E. coli K1 sepsis demonstrated that injection of K1 capsule degrading enzyme abrogates infection. However, the enzyme treatment is immunogenic; accordingly, chemical inhibition may prove to be a superior approach.
Of the different K types, the Group 2 and Group 3 capsules are most prevalent among UPEC isolates, with K1 and K5 being leading types. Although the capsules have different compositions, they are synthesized, assembled, and exported by functionally homologous factors, leading us to hypothesize that we can develop small molecular inhibitors of K-type encapsulation that target the most medically important K types. Furthermore, the medically important infectious agents Campylobacter jejuni, Hemophilus influenzae, Neisseria meningitides, and Salmonella typhimurium among others, use these homologues in the biogenesis of their capsules.
Confirmatory Assay Protocol: This confirmatory cell-based assay provided an alternative measurement of inhibitory activity on phage-induced lysis. It measures reduction in bacterial capsule formation using an absorbance readout at A600 instead of the alamar blue reagent. Confirmatory assays were performed in 96 well microplate plate format. Overnight starting cultures were grown at 37 degrees C, shaking at 250 RPM in LB. 65 ml of overnight UTI89 culture was inoculated into 6.45 liters of LB (starting OD600 ~0.03) and was sufficient for screening 300 plates at one concentration.
Bacterial cultures of E. coli K1 strain UTI89 (cystitis isolate) were prepared at the screening center immediately prior to use. Overnight starting cultures of UTI89 were grown at 37 degrees C, and diluted 1:100 into LB. Compounds were added to plates in a concentration dependent manner in the range of 100-0.39 microM (10 microL), followed by addition of 90 microL of bacterial culture. Each concentration was tested in duplicate. 1% DMSO (final well concentration) was included. The plates were sealed and shaken vigorously for 1.5 hr. An initial OD600 reading at the time of infection was measured to identify compounds that cause growth retardation or bacterial killing in the absence of phage. Next, K1F phage (5 microL) was added to all of the test wells. The plates were resealed and shaken vigorously at 37 degrees C, and measurements of OD600 for phage-mediated lysis were taken after 2 hr. Each compound was tested in the range of 50 - 0.39 microM for efficacy, and 5 compounds were tested per plate. Control wells contained UTI89 (wt K1) with 1% DMSO as a simulated positive control; and media plus vehicle as a negative control. The plates were read at ambient temperature from the bottom for absorbance at A600 in an Envision plate reader (Perkin Elmer) and the degree of phage-mediated lysis was determined based on the absorbance.
Outcome: Compounds that showed >30% inhibition of phage lysis were considered Active. IC50 values were calculated only for active compounds using a 4 parameter Levenburg-Marquardt algorithm, with the maximum and minimum locked at 0 and 100 respectively.
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In this confirmatory (dose response) screen, active compounds were scored on a scale of 41-80 based on the
IC50 result compounds that did not show activity were given the score 0.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)