Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB100

Poster Communications

A Bacterial Signal Peptide Increases Mucociliary Clearance in Explanted Mouse Trachea by Stimulating Solitary Chemosensory Cells and Paracrine Cholinergic Signaling

A. Perniss1, B. Bufe2, G. Krasteva-Christ3, S. Appenzeller4, S. Liu5, S. Offermanns5, J. Klein6, I. Wessler7, U. Boehm8, F. Zufall9, W. kummer1

1. Institute of Anatomy and Cell Biology, German Center for Lung Research, Justus Liebig University, Giessen, Germany. 2. Molecular Immunology Section, Faculty of Computer Science and Microsystems Engineering, University of Applied Sciences Kaiserslautern, Zweibrücken, United Kingdom. 3. Department of Anatomy and Cell Biology, Saarland University, Homburg, Germany. 4. Core Unit systems Medicine, Julius-Maximilians-University, Würzburg, United Kingdom. 5. Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany. 6. Department of Pharmacology, School of Pharmacy, Goethe-University Frankfurt, Frankfurt am Main, Germany. 7. Institute of Pathology, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany. 8. Experimental Pharmacology, Center for Molecular Signaling (PZMS), Saarland University, Homburg, Germany. 9. Center for Integrative Physiology and Molecular Medicine (CIPMM), Department of Physiology, Saarland University, Homburg, Germany.

Objective: Mucociliary clearance (MC) is a major innate defense mechanism, driven by the coordinated beating of ciliated epithelial cells. Bacterial products such as quorum sensing molecules (QSM) alter MC (1,2). A subpopulation of airway epithelial cells, called solitary chemosensory cells (SCC), expresses taste receptors (TasR) and contributes to the recognition of these bacterial products (3,4). SCC recognize QSM via the classical taste transduction pathway involving PLCβ2 (phospholipase C β2), a rise in intracellular calcium and TRPM5 (transient receptor potential cation channel subfamily M member 5), leading to release of acetylcholine (ACh), an known activator of MC. We here asked whether formylated bacterial signal peptides modulate MC, and assessed ciliary beat frequency (CBF) and particle transport speed (PTS) on the mucosal surface of explanted murine tracheas and release of ACh. Methods: The transcriptome of single tracheal ciliated and SCC was analyzed by deep sequencing. PTS was studied in wildtype C57Bl6 (WT) mice, and mice lacking components of the taste transduction cascade (TRPM5; ITPR3 (inositol 1,4,5-triphosphate receptor 3); PLCβ2; and Tas2r143/Tas2r135/Tas2r126 triple KO-mice (3 of 35 bitter taste receptors)) or lacking SCC (TRPM5-DTA). ACh was measured by HPLC. Results: The N-formylated bacterial signal peptide FL185 (10 µM), produced by various pathogens, e.g. E. coli and Salmonella typhimurium, increased PTS from 44±2 to 75±3 µm/s (mean±SEM; p<0.0001; n=23; paired t-test). Deep sequencing showed FPR expression in both ciliated and SCC, and presence of TRPM5, PLCβ2, ITPR3 and several chemoreceptors including bitter receptors in SCC. FPR1/2 inhibitors cyclosporine H (1 µM) and t-BOC2 (10 µM) did not reduce the effect 74±8 to 62±3 % increase in PTS (p=0.5454; n=6; unpaired t-test). The effect was reduced in TRPM5- (74±8 to 3±2 %; p=0.0011; n=6), PLCβ2- (71±17 to 0.45±3 %; p=0.0018; n=7), ITPR3- (34±1 to 2±1 %; p=0.0009; n=5), Tas2r143/Tas2r135/Tas2r126-deficient mice (37±5 to 22±5 %; p=0.0484; n=12) and TRPM5-DTA mice (92±2 to 9±1 %; p=0.0012; n=5). Atropine (1 µM; 74±8 to 18±7 %; p=0.0025; n=7) and 4-DAMP (1 µM; 66±16 to 11±4 %; p=0.0037; n=6), muscarinic receptor antagonists, diminished the effect of FL185. ACh was increased in supernatants of tracheas after stimulation with FL185 (70±17 to 158±36 nM; p=0.0203; n=6; paired t-test). This was not the case in TRPM5-KO mice (112±70 to 60±20 nM); p=0.5000; n=3; Wilcoxon matched-pairs signed rank test). Conclusion: A bacterial signal peptide stimulates MC independently of FPR. Instead, this effect involves typical taste transduction elements, solitary chemosensory cells, and subsequent cholinergic signaling to ciliated cells. Thus, detection of a defined set of bacterial signal peptides by SCC provides a novel defense mechanism against bacteria.

Where applicable, experiments conform with Society ethical requirements