title: Deciphering the role of Burkholderia cenocepacia membrane proteins in antimicrobial properties of chitosan.
abstract: Chitosan, a versatile derivative of chitin, is widely used as an antimicrobial agent either alone or mixed with other natural polymers. Burkholderia cenocepacia is a multidrug-resistant bacteria and difficult to eradicate. Our previous studies shown that chitosan had strong antibacterial activity against B. cenocepacia. In the current study, we have investigated the molecular aspects for the susceptibility of B. cenocepacia in response to chitosan antibacterial activity. We have conducted RNA expression analysis of drug efflux system by RT-PCR, membrane protein profiling by SDS-PAGE, and by LC-MS/MS analysis following the validation of selected membrane proteins by real-time PCR analysis. By RT-PCR analysis, it was found that orf3, orf9, and orf13 were expressed at detectable levels, which were similar to control, while rest of the orf did not express. Moreover, shotgun proteomics analysis revealed 21 proteins in chitosan-treated cells and 16 proteins in control. Among them 4 proteins were detected as shared proteins under control and chitosan-treated cells and 17 proteins as uniquely identified proteins under chitosan-treated cells. Among the catalog of uniquely identified proteins, there were proteins involved in electron transport chain and ATP synthase, metabolism of carbohydrates and adaptation to atypical conditions proteins which indicate that utilization and pattern of chitosan is diverse which might be responsible for its antibacterial effects on bacteria. Moreover, our results showed that RND drug efflux system, which display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents, was not determined to play its role in response to chitosan. It might be lipopolysaccharides interaction with chitosan resulted in the destabilization of membrane protein to membrane lyses to cell death. Membrane proteome analysis were also validated by RT-qPCR analysis, which corroborated our results that of membrane proteins.
authors.affiliation: State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, Zhejiang University, 388 Yuhangtang Road, Hangzhou, 310058, People's Republic of China.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ibrahim
authors.forename: Zhongyun
authors.initials: Z
authors.lastname: Tao
authors.forename: Annam
authors.initials: A
authors.lastname: Hussain
authors.forename: Yang
authors.initials: Y
authors.lastname: Chunlan
authors.forename: Mehmoona
authors.initials: M
authors.lastname: Ilyas
authors.forename: Abdul
authors.initials: A
authors.lastname: Waheed
authors.forename: Fang
authors.initials: F
authors.lastname: Yuan
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Guan-Lin
authors.initials: GL
authors.lastname: Xie
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D057508:Burkholderia cenocepacia / Q000187:drug effects*; D048271:Chitosan / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000502:physiology; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*
chemical_list: D000890:Anti-Infective Agents; D008565:Membrane Proteins; D048271:Chitosan