These compounds have potent antibacterial activity. Structure resembles known antibacterials and antifungals.

Transcription

1 1 CELL TO CELL SIGNALLING III Additional AHL Functions (Kaufmann et al, Jan 2005 PNAS) During a study to determine the lifespan of Pa. LasI AHL in water, it was discovered that 3oxoC12 underwent an unexpected reaction to a compound belonging to a class of compounds called tetramic acids. (compound 4) These compounds have potent antibacterial activity. Structure resembles known antibacterials and antifungals. A variety of AHLs were subjected to the same conditions and all formed tetramic acids irrespective of chain length. Significant antibacterial activity was found for compound 4 towards all tested Gram + bacteria, and none was found towards any Gram bacteria. The activity is biologically relevant because AHL concentrations are high and other antibacterials have similar activities.

2 2 Bacillus lactonase (Aii) activity protected from compound 1 (AHL), but not compound 4. Maybe this activity is useful not only to disrupt QS, but also to reduce toxicity. Lactobacillus reutercyclin is bactericidal as it acts as a proton-ionophore (carries ions), dissipating the transmembrane change in ph and leading to cell lysis (probably due to its high hydrophobicity). Structural resemblance between compound 4 and antibacterials is shaded. Interestingly, 3oxoC12 also demonstrated cytotoxicity. Dual role: Not due to tetramic acid formed since the experiment did not run long enough for spontaneous conversion. B. cereus strains producing a penicillinase were resistant to C12 AHL and not to compound 4.

3 3 Because B. cereus produce lactonases, perhaps signaling interference or quorum quenching is not the real goal of these enzymes Instead they may permit survival of cytotoxicity. This would eliminate toxic effects of 1 and prevent formation of 4. And maybe 3 oxoc12 AHL evolved as signaling molecules because of their fortuitous cytotoxic abilities, and their ability to convert to tetramic acids, which are resistant to lactonase degradation. But wait Compound 4 also has the structure of a simple siderophore, complexing 1 Fe 3+ for every 3 tetramic acids

4 4 The affinity constant for compound 4 is quite high, much higher than one of Pa. s normal siderophores, and similar to other iron chelators. Whether iron complexation is required for cytotoxic activity is unknown. What about other molecules: (IL Lamont et al 2002, 2003) Numerous bacteria secrete low molecular weight compounds that have high affinity for iron siderophores In addition to its role as an iron scavenger for Pa., pyoverdine plays a role in controlling gene expression of 3 virulence factors. Exotoxin A, endoprotease, & pyoverdine Regulation occurs through a transmembrane signaling system that includes the outer MB receptor for ferri-pyoverdine, a signaltransducing protein that extends from periplasm to cytoplasm, and a sigma factor. Two sigma factors! PvdS and FpvI Gene expression is triggered by ferri-pyoverdine, which therefore is a signaling molecule. Like Rse (captures sigma)

5 5 Used dot blot and reporter fusion to test for the production of Exotoxin A. Showed that it was dependant on pyoverdine. Overexpression of FpvR led to reduced production of pyoverdine > reduced growth in iron limiting conditions (with EDDA). Consistent with repression of pvd genes and the anti-sigma mechanism Ferri-pyoverdine binds outer MB protein FpvA FpvA interacts with the periplasmic domain of FpvR (anti sigma factor) > trans-mb signal of FpvR releases sigma PvdS (thanks to proteolysis of FpvR) > transcription of pyoverdine and synthesis of endo/exo toxins.

6 6 A second interaction shows FpvR can also bind another σ factor called FpvI, which when released caused transcription of the ferri-pyoverdine receptor FpvA. Both secreted and MB proteins produced, and 1 anti-σ for 2 different σ factors! Similar siderophore signaling pathways have been identified in E. coli (FecARI = Ferric citrate) and P. putida (PupBRI), but significant differences exist. There are over 70 uncharacterized homologues of FpvR in a wide range of bacterial species; over 500 specific siderophores, many of which probably regulate their own synthesis, and probably other products as well. What advantage would there be for having a siderophore as a signaling molecule? It would be inneficient for a bacterium to secrete a siderophore if there is a low probability of it being recaptured as a ferri-siderophore, as in an aquatic environment.

7 7 However, if growth occurs where there is a limited space and lots of bacteria (as in a biofilm), then siderophores represent an efficient mechanism of iron uptake. Coupling secreted protein prod. to siderophore reception makes sense.