In vitro Effects of Bacterial Exposure on Secretion of Zonulin Family Peptides and Their Detection in Human Tissue Samples.

Commercially available ELISAs for zonulin (pre-haptoglobin 2), a protein with tight junction regulatory activity in the epithelia, were recently shown to recognize other proteins that are structurally and functionally related to zonulin, termed zonulin family peptides (ZFPs). With little or no information about the identity and property of ZFPs, various commercial zonulin ELISA kits are widely utilized in research as a marker of intestinal permeability. Bacterial exposure is a known trigger for the secretion of zonulin, but it remains unclear whether distinct bacteria differ in their capability to stimulate zonulin secretion. We hypothesized that ZFPs are similar to zonulin regarding response to bacterial exposure and aimed to compare the effects of non-pathogenic, Gram-negative bacteria (Escherichia coli RY13 and E. coli K12 DH5α) and probiotic, Gram-positive bacteria (Lactobacillus rhamnosus GG and Bifidobacterium bifidum) on ZFP secretion in an in vitro model. Additionally, utilizing samples from human clinical trials, we correlated circulating levels of ZFPs to the gut bacteria and determined the presence of ZFPs in various human tissues. Unexpectedly, we found that the ZFPs quantified by the widely used IDK® Zonulin ELISA kits are specifically triggered by the exposure to live Lactobacillus rhamnosus GG in HT-29 cells, associated with absolute abundances of intestinal Lactobacillus and Bifidobacterium in adults, and are copious in the small intestine but undetectable in the liver or adipose tissue. These characteristics appear to be different from zonulin and highlight the need for further characterization of ZFPs recognized by commercially available and widely used "zonulin" ELISAs.

Flavones as Quorum Sensing Inhibitors Identified by a Newly Optimized Screening Platform Using Chromobacterium violaceum as Reporter Bacteria.

Quorum sensing (QS) is the process by which bacteria produce and detect signal molecules to coordinate their collective behavior. This intercellular communication is a relevant target for anti-biofilm therapies. Here we have optimized a screening-applicable assay to search for new quorum sensing inhibitors from natural compound libraries. In this system, QS is correlated with the production of violacein, which is directly controlled by the LuxI/LuxR system in Chromobacterium violaceum ATCC 31532. The parallel use of C. violaceum Tn5-mutant CV026, which depends on auto-inducer addition, allows simultaneous discrimination of compounds that act as quenchers of the AHL signal (quorum quenchers). The incorporation of a redox stain into the platform allowed further distinction between QS inhibitors, quorum quenchers and antibacterial compounds. A pilot screening was performed with 465 natural and synthetic flavonoids. All the most active compounds were flavones and they displayed potencies (IC50) in the range of 3.69 to 23.35 µM. These leads were particularly promising as they inhibited the transition from microcolonies into mature biofilms from Escherichia coli and Pseudomonas aeruginosa strains. This approach can be very effective in identifying new antimicrobials posing lesser risks of resistance.