• In-depth Profiling of MvfR-Regulated Small Molecules in Pseudomonas aeruginosa after Quorum Sensing Inhibitor Treatment.

      Allegretta, Giuseppe; Maurer, Christine K; Eberhard, Jens; Maura, Damien; Hartmann, Rolf W; Rahme, Laurence; Empting, Martin; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland 9 University, 66123 Saarbrücken, Germany. (2017)
      Pseudomonas aeruginosa is a Gram-negative bacterium, which causes opportunistic infections in immuno-compromised individuals. Due to its multiple resistances toward antibiotics, the development of new drugs is required. Interfering with Quorum Sensing (QS), a cell-to-cell communication system, has shown to be highly efficient in reducing P. aeruginosa pathogenicity. One of its QS systems employs Pseudomonas Quinolone Signal (PQS) and 4-hydroxy-2-heptylquinoline (HHQ) as signal molecules. Both activate the transcriptional regulator MvfR (Multiple Virulence Factor Regulator), also called PqsR, driving the production of QS molecules as well as toxins and biofilm formation. The aim of this work was to elucidate the effects of QS inhibitors (QSIs), such as MvfR antagonists and PqsBC inhibitors, on the biosynthesis of the MvfR-regulated small molecules 2'-aminoacetophenone (2-AA), dihydroxyquinoline (DHQ), HHQ, PQS, and 4-hydroxy-2-heptylquinoline-N-oxide (HQNO). The employed synthetic MvfR antagonist fully inhibited pqs small molecule formation showing expected sigmoidal dose-response curves for 2-AA, HQNO, HHQ and PQS. Surprisingly, DHQ levels were enhanced at lower antagonist concentrations followed by a full suppression at higher QSI amounts. This particular bi-phasic profile hinted at the accumulation of a biosynthetic intermediate resulting in the observed overproduction of the shunt product DHQ. Additionally, investigations on PqsBC inhibitors showed a reduction of MvfR natural ligands, while increased 2-AA, DHQ and HQNO levels compared to the untreated cells were detected. Moreover, PqsBC inhibitors did not show any significant effect in PA14 pqsC mutant demonstrating their target selectivity. As 2-AA is important for antibacterial tolerance, the QSIs were evaluated in their capability to attenuate persistence. Indeed, persister cells were reduced along with 2-AA inhibition resulting from MvfR antagonism, but not from PqsBC inhibition. In conclusion, antagonizing MvfR using a dosage capable of fully suppressing this QS system will lead to a favorable therapeutic outcome as DHQ overproduction is avoided and bacterial persistence is reduced.
    • N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral PathogenTannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism.

      Hottmann, Isabel; Mayer, Valentina M T; Tomek, Markus B; Friedrich, Valentin; Calvert, Matthew B; Titz, Alexander; Schäffer, Christina; Mayer, Christoph; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018)
      Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugarN-acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of theEscherichia colietherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described forT. forsythia.In between the respective genes on theT. forsythiagenome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104-fold higher catalytic efficiency (kcat/Km) for MurNAc than forN-acetylglucosamine (GlcNAc) withkcatvalues of 10.5 s-1and 0.1 s-1andKmvalues of 200 μM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S]= 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism ofT. forsythia, a kinase deletion mutant (ΔTf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, theΔTf_murK::ermmutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 μg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 μg/ml MurNAc for optimal growth. These data reveal thatT. forsythiareadily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion ofTf_murKblocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of theT. forsythiacell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.