In vitro interaction of Pseudomonas aeruginosa with human middle ear epithelial cells
Rahul Mittal1, M'hamed Grati1, Robert Gerring1
1Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
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).
Methodology And Findings:
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 And Significance:
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.
Insights
Pseudomonas aeruginosa invades human middle ear cells using actin and microtubules. The outer membrane protein OprF is crucial for this invasion and subsequent cell damage, offering potential therapeutic targets for chronic suppurative otitis media.
Area of Science:
- Microbiology
- Cell Biology
- Pathogenesis
Background:
- Otitis media (OM) encompasses acute and chronic forms, with Pseudomonas aeruginosa being a primary cause of chronic suppurative otitis media (CSOM).
- CSOM leads to middle ear infection, discharge, hearing loss, and neurological issues.
- The interaction mechanisms between P. aeruginosa and human middle ear epithelial cells (HMEECs) remain largely uncharacterized.
Purpose of the Study:
- To elucidate the interaction between P. aeruginosa and HMEECs.
- To identify the role of bacterial factors in P. aeruginosa entry and pathogenesis within HMEECs.
- To explore potential therapeutic targets for CSOM.
Main Methods:
- Investigated P. aeruginosa invasion into HMEECs using actin and microtubule inhibitors.
- Utilized confocal and electron microscopy (SEM, TEM) to visualize bacterial entry and localization.
- Assessed cytotoxicity via lactate dehydrogenase release and fluorescent dye assays.
- Compared invasion and cytotoxicity of wild-type P. aeruginosa with an OprF mutant.
Main Results:
- P. aeruginosa invades HMEECs via an actin and microtubule-dependent mechanism.
- F-actin condensation was observed during bacterial entry.
- Internalization occurred within membrane-bound vacuoles.
- An OprF mutant showed significantly reduced invasion and cytotoxicity compared to the wild-type strain.
Conclusions:
- P. aeruginosa utilizes an active invasion pathway into HMEECs involving cytoskeletal elements.
- The bacterial outer membrane protein OprF plays a critical role in P. aeruginosa adherence, invasion, and virulence.
- Understanding these interactions is vital for developing novel therapeutic strategies against CSOM and preventing hearing loss.
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