In Vitro Interaction of Pseudomonas aeruginosa with Human Middle Ear Epithelial Cells

Abstract

BackgroundOtitis 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).Methodology and FindingsIn 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 and SignificanceThis 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.

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network

“…Although many pathogenic bacteria modulate the actin cytoskeleton of eukaryotic cells to facilitate the invasion of epithelial cells, some pathogens are capable of modulating the dynamics of host microtubules as well. Few studies have examined the requirement for microtubules during P. aeruginosa PAO1 strain internalization, and it seems uncertain since it is independent for corneal epithelial cell infection ( 18 ) and dependent for middle ear epithelial cells ( 45 ). Hence we studied P. aeruginosa uptake by HeLa cells in the presence of colchicine or nocodazole, two microtubule-destabilizing agents.…”

Otopathogenic Pseudomonas aeruginosa Enters and Survives Inside Macrophages

“…It was concluded that hydrophobicity is a necessary surface condition for effective phagocytosis of S. aureus by J774A macrophage cell line. Our previous studies have demonstrated that P. aeruginosa can invade human middle ear epithelial cells (HMEECs) (Mittal et al, 2014). However, the interaction of otopathogenic P. aeruginosa with primary macrophages has never been explored in previous studies.…”

Role of innate immunity in the pathogenesis of otitis media

“…We have observed, using scanning electron microscopy, that P. aeruginosa binds and adheres to HMEECs (Figure 2). 45 Transmission electron microscopy demonstrated P. aeruginosa internalization inside HMEECs enclosed in membrane-bound vacuoles (Figure 3). 45 However, how this adhesion and subsequent internalization leads to mucin up-regulation needs to be explored in future studies.…”