title: In vitro interaction of Pseudomonas aeruginosa with human middle ear epithelial cells.
abstract: BACKGROUND
  Otitis media (OM) is an inflammation of the middle ear which can be acute or chronic. Acute OM is caused by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis whereas Pseudomonas aeruginosa is a leading cause of chronic suppurative otitis media (CSOM). CSOM is a chronic inflammatory disorder of the middle ear characterized by infection and discharge. The survivors often suffer from hearing loss and neurological sequelae. However, no information is available regarding the interaction of P. aeruginosa with human middle ear epithelial cells (HMEECs).
  
  
  RESULTS
  In the present investigation, we demonstrate that P. aeruginosa is able to enter and survive inside HMEECs via an uptake mechanism that is dependent on microtubule and actin microfilaments. The actin microfilament disrupting agent as well as microtubule inhibitors exhibited significant decrease in invasion of HMEECs by P. aeruginosa. Confocal microscopy demonstrated F-actin condensation associated with bacterial entry. This recruitment of F-actin was transient and returned to normal distribution after bacterial internalization. Scanning electron microscopy demonstrated the presence of bacteria on the surface of HMEECs, and transmission electron microscopy confirmed the internalization of P. aeruginosa located in the plasma membrane-bound vacuoles. We observed a significant decrease in cell invasion of OprF mutant compared to the wild-type strain. P. aeruginosa induced cytotoxicity, as demonstrated by the determination of lactate dehydrogenase levels in culture supernatants of infected HMEECs and by a fluorescent dye-based assay. Interestingly, OprF mutant showed little cell damage compared to wild-type P. aeruginosa.
  
  
  CONCLUSIONS
  This study deciphered the key events in the interaction of P. aeruginosa with HMEECs in vitro and highlighted the role of bacterial outer membrane protein, OprF, in this process. Understanding the molecular mechanisms in the pathogenesis of CSOM will help in identifying novel targets to design effective therapeutic strategies and to prevent hearing loss.
authors.affiliation: Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
authors.forename: Rahul
authors.initials: R
authors.lastname: Mittal
authors.affiliation: Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
authors.forename: M'hamed
authors.initials: M
authors.lastname: Grati
authors.affiliation: Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Gerring
authors.affiliation: Center for Advanced Microscopy, University of Miami, Coral Gables, Florida, United States of America; RSMAS, University of Miami, Key Biscayne, Florida, United States of America.
authors.forename: Patricia
authors.initials: P
authors.lastname: Blackwelder
authors.affiliation: Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
authors.forename: Denise
authors.initials: D
authors.lastname: Yan
authors.affiliation: Center for Inflammation, Immunity, and Infection and Department of Biology, Georgia State University, Atlanta, Georgia, United States of America.
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Li
authors.affiliation: Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida, United States of America; Department of Human Genetics, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
authors.forename: Xue Zhong
authors.initials: XZ
authors.lastname: Liu
mesh_terms: D008841:Actin Cytoskeleton / Q000382:microbiology; D001426:Bacterial Proteins / Q000378:metabolism; D004432:Ear, Middle / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology / Q000382:microbiology*; D015964:Gene Expression Regulation, Bacterial; D054884:Host-Pathogen Interactions; D006801:Humans; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; C547605:OprF protein, Pseudomonas aeruginosa