Inhibition of autotransporter biogenesis by small molecules

Abstract

SummaryDisarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram‐negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane‐based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway, we used transcriptomics analysis to develop a fluorescence‐based high‐throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen‐43. VUF15259 also impairs β‐barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam‐complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug‐like compounds.

A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier

“…Recently, a small molecule inhibiting autotransporter assembly was described. Although this molecule may inhibit BAM function, the target has yet to be identified (12).…”

Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

“…We have shown previously that the σ E and heat-shock stress responses in E. coli cells can be monitored by placing the gene encoding the green-fluorescent protein mNeonGreen (mNG) in pUA66 under control of the stress-regulated rpoE or groES promoter, respectively [ 9 ]. To monitor Rcs and Cpx stress in E. coli we used the same strategy and placed the mNG-encoding gene under control of the rprA or cpxP promoter, respectively.…”

“…To test the resulting P cpxP -mNG reporter construct it was transformed into E. coli cells together with pEH3-Hbp110C/348C that encodes a translocation incompetent derivative of the autotransporter hemoglobin protease (Hbp) [ 26 ]. We have shown previously that Hbp110C/348C accumulates in the periplasm inducing Cpx stress response [ 9 , 26 ]. Indeed, as shown in Figure 2 B, expression of Hbp110C/348C increased fluorescence by ~2.5 fold compared to cells expressing wild-type Hbp, indicating that this reporter construct can be used to detect Cpx activation.…”

“…To examine how E. coli cells respond to antibacterial agents with known targets in the cell envelope and cytosol we monitored activation of the Rcs, σ E and Cpx stress responses using the P rprA -mNG, P rpoE -mNG and P cpxP -mNG reporters, respectively. To obtain insight in the specificity of the reactions to different cues we also monitored induction of the cytosolic heat-shock stress response using a P groES -mNG reporter construct [ 9 ]. E. coli cells harboring the reporter constructs were grown in 96-well plates containing a two-fold increasing concentration of the antibacterial agents that are listed in Table 1 .…”

“…Considering the stressors of the σ E , Rcs and Cpx systems we reasoned that monitoring these responses in E. coli cells may help to identify novel agents that affect diverse aspects of cell envelope biogenesis and integrity. Recently, we described fluorescence-based high-throughput screening (HTS) assays that report on σ E cell envelope stress and cytosolic heat-shock stress as a control [ 9 ]. In the current study, we developed similar assays that signal activation of the Rcs and Cpx responses.…”