title: Characterization of the Direct Interaction between Hybrid Sensor Kinases PA1611 and RetS That Controls Biofilm Formation and the Type III Secretion System in Pseudomonas aeruginosa.
abstract: One of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients is pulmonary infection with Pseudomonas aeruginosa, and the pathophysiology of pulmonary infection in CF is affected by the lifestyle of this micro-organism. RetS-GacS/A-RsmA is a key regulatory pathway in P. aeruginosa that determines the bacterium's lifestyle choice. Previously, we identified PA1611, a hybrid sensor kinase, as a new player in this pathway that interacts with RetS and influences biofilm formation and type III secretion system. In this study, we explored the structural and mechanistic basis of the interaction between PA1611 and RetS. We identified the amino acid residues critical for PA1611-RetS interactions by molecular modeling. These residues were then targeted for site-directed mutagenesis. Amino acid substitutions were carried out at seven key positions in PA1611 and at six corresponding key positions in RetS. The influence of such substitutions in PA1611 on the interaction was analyzed by bacterial two-hybrid assays. We carried out functional analysis of these mutants in P. aeruginosa for their effect on specific phenotypes. Two residues, F269 and E276, located within the histidine kinase A and histidine kinase-like ATPase domains of PA1611 were found to play crucial roles in the PA1611-RetS interaction and had profound effects on phenotypes. Corresponding mutations in RetS demonstrated similar results. We further confirmed that these mutations in PA1611 function through the GacS/GacA-RsmY/Z signaling pathway. Collectively, our findings provide a noncognate sensor kinase direct interaction model for a signaling pathway, key for lifestyle selection in P. aeruginosa, and targeting such interaction may serve as a novel way of controlling infections with P. aeruginosa.
authors.affiliation: Department of Oral Biology and Manitoba Chemosensory Biology Research Group, College of Dentistry, Rady Faculty of Health Sciences , 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W2, Canada.|Biology of Breathing Group, Children's Hospital Research Institute of Manitoba , 715 McDermot Avenue, Winnipeg, Manitoba R3E  3P4, Canada.
authors.forename: Anjali Y
authors.initials: AY
authors.lastname: Bhagirath
authors.affiliation: Department of Oral Biology and Manitoba Chemosensory Biology Research Group, College of Dentistry, Rady Faculty of Health Sciences , 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W2, Canada.
authors.forename: Sai P
authors.initials: SP
authors.lastname: Pydi
authors.affiliation: Department of Oral Biology and Manitoba Chemosensory Biology Research Group, College of Dentistry, Rady Faculty of Health Sciences , 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W2, Canada.|Biology of Breathing Group, Children's Hospital Research Institute of Manitoba , 715 McDermot Avenue, Winnipeg, Manitoba R3E  3P4, Canada.
authors.forename: Yanqi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Oral Biology and Manitoba Chemosensory Biology Research Group, College of Dentistry, Rady Faculty of Health Sciences , 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W2, Canada.
authors.forename: Chen
authors.initials: C
authors.lastname: Lin
authors.affiliation: Department of Oral Biology and Manitoba Chemosensory Biology Research Group, College of Dentistry, Rady Faculty of Health Sciences , 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W2, Canada.
authors.forename: Weina
authors.initials: W
authors.lastname: Kong
authors.affiliation: Department of Oral Biology and Manitoba Chemosensory Biology Research Group, College of Dentistry, Rady Faculty of Health Sciences , 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W2, Canada.|Biology of Breathing Group, Children's Hospital Research Institute of Manitoba , 715 McDermot Avenue, Winnipeg, Manitoba R3E  3P4, Canada.
authors.forename: Prashen
authors.initials: P
authors.lastname: Chelikani
authors.affiliation: Department of Oral Biology and Manitoba Chemosensory Biology Research Group, College of Dentistry, Rady Faculty of Health Sciences , 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W2, Canada.|Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, Rady Faculty of Health Sciences , 745 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0J9, Canada.|Biology of Breathing Group, Children's Hospital Research Institute of Manitoba , 715 McDermot Avenue, Winnipeg, Manitoba R3E  3P4, Canada.
authors.forename: Kangmin
authors.identifier: 0000-0002-3016-786X
authors.initials: K
authors.lastname: Duan
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D018441:Biofilms; D015964:Gene Expression Regulation, Bacterial; D000071677:Histidine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D000072417:Protein Domains; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D000069296:Type III Secretion Systems / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C507835:RetS protein, Pseudomonas aeruginosa; D000069296:Type III Secretion Systems; D000071677:Histidine Kinase
keywords: Pseudomonas aeruginosa; bacterial two-hybrid assays (BTH); biofilms; site-directed mutagenesis (SDM); two-component regulatory systems (TCS); type three secretion system (T3SS)