Potent modulation of the CepR quorum sensing receptor and virulence in a Burkholderia cepacia complex member using non-native lactone ligands

The Burkholderia cepacia complex (Bcc) is a family of closely related bacterial pathogens that are the causative agent of deadly human infections. Virulence in Bcc species has been shown to be controlled by the CepI/CepR quorum sensing (QS) system, which is mediated by an N-acyl L-homoserine lactone (AHL) signal (C8-AHL) and its cognate LuxR-type receptor (CepR). Chemical strategies to block QS in Bcc members would represent an approach to intercept this bacterial communication process and further delineate its role in infection. In the current study, we sought to identify non-native AHLs capable of agonizing or antagonizing CepR, and thereby QS, in a Bcc member. We screened a library of AHL analogs in cell-based reporters for CepR, and identified numerous highly potent CepR agonists and antagonists. These compounds remain active in a Bcc member, B. multivorans, with one agonist 250-fold more potent than the native ligand C8-AHL, and can affect QS-controlled motility. Further, the CepR antagonists prolong C. elegans survival in an infection model. These AHL analogs are the first reported non-native molecules that both directly modulate CepR and impact QS-controlled phenotypes in a Bcc member, and represent valuable chemical tools to assess the role of QS in Bcc infections.


Contents.
• Figure S1. Structures of AHLs and other QS modulators examined in this study.
• Table S2. CepR agonism data for all compounds in the E. coli reporter.
• Table S3. CepR antagonism data for all compounds in the E. coli reporter.
• Table S4. CepR agonism data for selected compounds in the B. multivorans reporter.
• Table S5. CepR antagonism data for selected compounds in the B. multivorans reporter.
• Figure S2. Dose-response data for lead compounds examined in this study.
• Table S6. Strains and plasmids used in this study.  Figure S1. Structures of AHLs and other QS modulators examined in this study. Abbreviations: i =inverted lactone head group stereochemistry; ii =phenyl head group; iii =ethyl ester head group; iv =3-nitro phenyl head group; TL=thiolactone head group; CPA=cyclopentyl amine head group.

S-4
Notes on compounds in Figure S1.
The compound numbering in Figure S1 matches that used in our previous reports. Compounds labeled as "control" compounds (e.g., Control 5) were from a collection of compounds we previously investigated for their activity against LasR in P. aeruginosa. 1 The A library (A1-A7 and A11-A13) was composed of straight-chain AHLs of varied acyl chain length (4-16 carbons) and 3'-position oxidation state (methylene and oxo). 2 The B library (B1 and B3-B14) contained cycloalkyl-and aromatic-containing AHLs and analogs of the AHLs with one-carbon insertions or deletions in the acyl chain or opposite lactone stereochemistry. 2 The C library (C1-C25) was composed of phenylacetanoyl homoserine lactones (PHLs) with substituents on the aryl ring of varying electronics and size, and in varying positions. 2 The D library (D1-D4, D6-D7, D9, D10, D13-D16, and D20) was composed of a wide variety of AHLs intended to broadly examine the effect of acyl chain structure on AHL activity. 3 The E library (E1-E39) contained additional PHLs and phenoxyacetanoyl homoserine lactones (POHLs) and phenylpropionyl HLs (PPHLs) with aryl ring substituents complementary to those in the C library. 4 The F library (F2, F3, F10-F13, F16, F18, F20, F21, F25, F26, F31, F39, F40, F45, F47, F50, and F55) contained a variety of AHL analogs, including several with non-native headgroups in place of the lactone. 5 The H library (H2, H4-H6, H15, H23, H25, and H26) contained AHLs with long acyl chains, aryl side chains, and/or 3'-hydroxyl groups. 6 The Q library (Q1-Q13) was composed of benzoyl homoserine lactones with varied substituents on the aryl ring. 7 The R library (R1-R5) contained para-alkoxy substituted benzoyl homoserine lactones of varying chain lengths. 7 The S library (S1-S4, S6, and S7) contained AHLs with either branched alkyl chains or cycloalkyl rings. 7 We selected 169 compounds from these libraries for the current study.

Referred to in text as:
Alternative name 1 Alternative name 2 Ref.   S-16 Figure S2. Dose-response data for compounds examined in this study.

Ec-reporter CepR antagonist dose-response curves for compounds displaying upturn doseresponse behavior
CepR antagonism dose-response curves for AHLs C4-AHL, A12, B12, C6, C8, C9, E3, E7, E10, and E25 in the Ec-reporter are provided below. Two curves are shown for each AHL; the left curve incorporates all data points measured (showing CepR inhibition at lower concentrations and then inversion to CepR activation at higher concentrations), while the right curve incorporates only the data points where CepR inhibition was noted. The right curves were used to calculate IC50 and maximum inhibition values for each compound. Investigating the origins of this non-monotonic behavior is currently ongoing.

Bm-reporter CepR antagonist dose-response curves for compound displaying upturn doseresponse behavior
CepR antagonism dose-response curves for C4-AHL in the Bm-reporter are provided below. Two curves are shown; the left curve incorporates all data points measured (showing CepR inhibition at lower concentrations and then inversion to CepR activation at higher concentrations), while the right curve incorporates only the data points where CepR inhibition was noted. The right curve was used to calculate IC50 and maximum inhibition value for C4-AHL. Again, investigating the origins of this non-monotonic behavior is currently ongoing.