Synthesis of N -Acyl Homoserine Lactone Analogues Reveals Strong Activators of SdiA, the Salmonella enterica Serovar Typhimurium LuxR Homologue

Abstract

N-Acyl homoserine lactones (AHLs) are molecules that are synthesized and detected by many gram-negative bacteria to monitor the population density, a phenomenon known as quorum sensing. Salmonella enterica serovar Typhimurium is an exceptional species since it does not synthesize its own AHLs, while it does encode a LuxR homologue, SdiA, which enables this bacterium to detect AHLs that are produced by other species. To obtain more information about the specificity of the ligand binding by SdiA, we synthesized and screened a limited library of AHL analogues. We identified two classes of analogues that are strong activators of SdiA: the N-(3-oxo-acyl)-homocysteine thiolactones (3O-AHTLs) and the N-(3-oxo-acyl)-trans-2-aminocyclohexanols. To our knowledge, this is the first report of compounds (the 3O-AHTLs) that are able to activate a LuxR homologue at concentrations that are lower than the concentrations of the most active AHLs. SdiA responds with greatest sensitivity to AHTLs that have a keto modification at the third carbon atom and an acyl chain that is seven or eight carbon atoms long. The N-(3-oxo-acyl)-trans-2-aminocyclohexanols were found to be less sensitive to deactivation by lactonase and alkaline pH than the 3O-AHTLs and the AHLs are. We also examined the activity of our library with LuxR of Vibrio fischeri and identified three new inhibitors of LuxR. Finally, we performed preliminary binding experiments which suggested that SdiA binds its activators reversibly. These results increase our understanding of the specificity of the SdiA-ligand interaction, which could have uses in the development of anti-quorum-sensing-based antimicrobials.

Comparative Analyses of N‐Acylated Homoserine Lactones Reveal Unique Structural Features that Dictate Their Ability to Activate or Inhibit Quorum Sensing

“…This result was remarkable, and explained the unusual inhibition trends for the nitro PHL series in LuxR (C13-C15; see above). Few superactivators of R proteins have been reported; [24,25] therefore, our discovery of 3-nitro PHL C14 as a superactivator of LuxR is significant. Additional studies in our laboratory have shown that PHL C14 can also superactivate LuxR in wildtype V. fischeri and is tolerated in invertebrate model systems; this suggests that this compound could have considerable value as a probe to study V. fischeri-host symbioses.…”

“…Likewise, this dearth of SAR data has also protracted the design of non-native AHL activators of QS. [19,24,25] The use of different assay procedures to assess agonistic or antagonistic activities against the same R protein has further complicated comparisons between past studies.…”

Are There Acyl-Homoserine Lactones within Mammalian Intestines?

“…The Salmonella LuxR homolog SdiA can detect acyl-HSL variants at concentrations ranging from 1 nM to 1 M depending on the variant (24,39). This is similar to the sensitivities of other LuxR homologs, although SdiA seems to detect a broader range of variants than other LuxR family members.…”

“…The Salmonella SdiA reporter system did not respond to this strain of A. baumannii (M. C. Swearingen and B. M. M. Ahmer, unpublished data). It is somewhat surprising that SdiA did not respond to the A. baumannii acyl-HSL 3-OH-C12 because it does respond to 3-oxo-C12 at a concentration of 60 nM (39). SdiA responds to hydroxy variants with roughly 10-foldless sensitivity than oxo variants, so while 3-OH-C12 has not yet been tested, one can surmise that it may be detected at a concentration of roughly 600 nM (39).…”

“…It is somewhat surprising that SdiA did not respond to the A. baumannii acyl-HSL 3-OH-C12 because it does respond to 3-oxo-C12 at a concentration of 60 nM (39). SdiA responds to hydroxy variants with roughly 10-foldless sensitivity than oxo variants, so while 3-OH-C12 has not yet been tested, one can surmise that it may be detected at a concentration of roughly 600 nM (39). More work is needed to determine the detection limit of Salmonella SdiA with regard to synthetic 3-OH-C12 and the concentrations of acyl-HSLs produced by A. baumannii in the gut and in culture.…”