High-throughput identification of chemical inhibitors of E. coli Group 2 capsule biogenesis as anti-virulence agents

Abstract

Rising antibiotic resistance among Escherichia coli, the leading cause of urinary tract infections (UTIs), has placed a new focus on molecular pathogenesis studies, aiming to identify new therapeutic targets. Anti-virulence agents are attractive as chemotherapeutics to attenuate an organism during disease but not necessarily during benign commensalism, thus decreasing the stress on beneficial microbial communities and lessening the emergence of resistance. We and others have demonstrated that the K antigen capsule of E. coli is a preeminent virulence determinant during UTI and more invasive diseases. Components of assembly and export are highly conserved among the major K antigen capsular types associated with UTI-causing E. coli and are distinct from the capsule biogenesis machinery of many commensal E. coli, making these attractive therapeutic targets. We conducted a screen for anti-capsular small molecules and identified an agent designated "C7" that blocks the production of K1 and K5 capsules, unrelated polysaccharide types among the Group 2-3 capsules. Herein lies proof-of-concept that this screen may be implemented with larger chemical libraries to identify second-generation small-molecule inhibitors of capsule biogenesis. These inhibitors will lead to a better understanding of capsule biogenesis and may represent a new class of therapeutics.

Figure 1. Group 2 capsules and overview of screening strategy to identify capsule biogenesis inhibitors.

(A) Organization of the genomic region encoding for major K capsule synthesis and assembly products. The Group 2 capsules are encoded from similar loci. The SYNTHESIS gene products derive the primary monosaccharides necessary for capsule and render the monosaccharides competent for polymerization. The ASSEMBLY and EXPORT products co-assemble to polymerize the capsule and transport it out of the bacterium where it envelops the cell. As examples, K1 and K5 capsules differ in Region II gene number and identity. Interconnecting lines indicate degree of amino acid identity between key structural components of capsule biogenesis encoded from Region I and Region III. Thick, thin, and doted lines indicate >97%, >94%, and >70% identity, respectively (further details in Table S1). (B) Diagram of high-throughput screen for capsule inhibitors. Wells of a 96-well plate were seeded with a dilution of UPEC K1 strain UTI89. Compounds from the DTP small molecule library were placed in appropriate wells at a final concentration 100 µM. After a short incubation, the K1 capsule specific K1F phage was added to these wells. Growth was monitored and compounds that did not produce an initial growth inhibition but did inhibit phage lysis, suggesting altered capsule biosynthesis or phage inhibition, were further tested. Secondary assays (indicated by shaded block) were conducted using the engineered K-12:K1 strain EV36. T7 phage, inhibited by T7 capsule but genetically similar to K1F phage, was used to infect this strain. Compounds that sensitized cells to T7 phage were classified as having capsule-specific effect(s) and selected for further analysis. Finally, the K-12 strain MG1655, which is readily lysed by T7 phage, was used in a final assay to determine which compounds inhibited phage lysis.

Figure 2. C7 inhibition of K1 and K5 capsule dependent phage lysis.

(A) C7 inhibits K1 capsule-dependent phage lysis of UPEC. UPEC K1 strain UTI89 was grown in the presence of 1% DMSO or indicated concentrations of C7 for 1 hr and K1F phage was added where indicated. Relative OD₆₀₀ change after 4 hrs is shown. Increasing C7 concentrations inhibit lysis of UPEC K1. P value: <0.0001 No C7 (0 µM) compared to each of C7 treatments >12.5 µM. (B) C7 sensitizes K12:K1 strain to T7 phage. The K12 strain EV36 producing K1 capsule was infected with T7 phage (T7φ), which is inhibited by the presence of K1 capsule. Addition of 100 µM C7 sensitized EV36 to T7 phage (p = <0.0001). (C) C7 inhibits K5 capsule-dependent phage lysis. UPEC K5 strain DS17 was incubated with (K5φ) or without phage and relative change in OD₆₀₀ was recorded. Addition of 100 µM C7 prevented lysis by K5 capsule specific phage (p<0.0001).

Figure 3. C7 treated cells produce less orcinol-reactive carbohydrates.

Cells were (A) sonicated or (B) treated with mild acid to release cell surface polysaccharides. Material was then extracted and polysaccharides were detected using the orcinol reagent. C7 treated cells had levels of orcinol-reactive carbohydrates comparable to those of a capsule synthesis (K1 SYNTHESIS) mutant for both whole cell sonicates as well as released surface material. This suggests that capsule levels are reduced by C7 treatment, and the compound does not simply release capsule from the bacterial surface. Absorbance readings were expressed as the percent reactivity of the wild-type UTI89 K1 strain. K1 vs. SYNTHESIS mutant or C7 treated, p<0.01; SYNTHESIS mutant vs. C7 treated p>0.05 by Tukey's t-test for both (A) and (B).

Figure 4. C7 increases C3 binding to UPEC K1.

UTI89 was incubated with human serum and C3 binding was quantified by densitometry of immuno-dot blot. C7 treatment increases C3 binding to levels similar to an unencapsulated cell (K1 SYNTHESIS mutant; p = 0.0054).

Figure 5. C7 sensitizes K1 encapsulated UPEC to human serum.

Three ml cultures of the K1 encapsulated UPEC strain UTI89, a capsule synthesis mutant (K1 SYNTHESIS), and wild-type cells treated with 100 µM C7 were grown in LB +1% DMSO or C7 for ∼3 hrs with shaking until reaching an OD₆₀₀ of 0.8. Human serum from at least two individuals was heat inactivated (HIS) at 55°C for 45 min or maintained on ice (Normal Serum, NS). Bacterial cells were resuspended in PBS, diluted to 5–9×10³ and incubated in RPMI/20% serum supplemented with 1% DMSO or 100 µM C7 at 37°C for 2.0 hrs. Duplicate independent cultures for each strain were tested and the ratio of NS/HIS CFU/ml is shown. The graph represents the average of two experiments.

Figure 6. C7 is active against other clinical K1 UPEC strains.

A panel of K1F phage sensitive clinical E. coli strains was tested for phage sensitivity in the presence or absence of 100 µM C7. Strains were grown in 1% DMSO or C7 and infected with K1F phage where indicated. Relative OD₆₀₀ change after 3 hrs post infection (normalized to the growth of UPEC strain UTI89) is shown. Addition of 100 µM C7 inhibits lysis of tested clinical strains. P value: <0.0001 no C7 (phage only) compared to C7 treatment. Arrow indicates two clinical strains in which C7 only partially restored growth.