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PLoS One ; 11(3): e0150109, 2016.
Article in English | MEDLINE | ID: mdl-27031335

ABSTRACT

Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10-80 fold increase, termed "swarming"), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds.


Subject(s)
Bacterial Adhesion/physiology , Cystic Fibrosis/pathology , Pseudomonas aeruginosa/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Chemotaxis , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Membrane Proteins/deficiency , Membrane Proteins/genetics , Microscopy, Fluorescence , Microscopy, Video , Pseudomonas aeruginosa/genetics , Receptors, Amino Acid/deficiency , Receptors, Amino Acid/genetics , Respiratory Mucosa/cytology
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