LDH release by HMEECs infected with <i>P. aeruginosa</i>.

<p>HMEECs were infected with <i>P. aeruginosa</i> at an MOI of 10 for varying time periods and LDH levels were determined in the culture supernatants of infected cells. Results were expressed as the percentage compared with maximum LDH release by lysed cells. Data represents mean ± SD and is representative of five individual experiments carried out in triplicate. # P<0.05 or *P<0.001 compared to WT or pOprF.</p

Invasion of <i>P. aeruginosa</i> in HMEECs is dose and time dependent.

<p>HMEECs were infected with <i>P. aeruginosa</i> at different MOI for 2h and invasion was determined by gentamicin protection assay (A). In separate experiments, HMEECs were infected with <i>P. aeruginosa</i> at an MOI of 10 for varying time periods and bacterial invasion was determined by gentamicin protection assay (B). Data represents mean ± SD. Results are representative of five independent experiments carried out in triplicate.</p

OprF expression in <i>P. aeruginosa</i> is required for HMEECs invasion.

<p>HMEECs were infected with <i>P. aeruginosa</i> strains at an MOI of 10 for 2h and bacterial invasion was determined by gentamicin protection assay (A). The binding of <i>P. aeruginosa</i> to HMEECs was also determined (B). In separate experiments HMEECs were pretreated with exogenous OprF (C) or bacteria were pretreated with anti-OprF monoclonal antibody (mAb) (D) and then performed invasion assay. Data represents mean ± SD. Results were expressed as percentage compared to the invasion or binding of the wild-type strain. <b>^#</b> P<0.05 or * P<0.01 or ** P<0.001 or <b>^†</b> P>0.05 compared to WT or pOprF.</p

Scanning electron micrographs demonstrating interaction of <i>P. aeruginosa</i> with HMEECs.

<p>Epithelial cells were infected with <i>P. aeruginosa</i> for 30 min (A), 1h (B), 1.5h (C), 2h (D), 4h (E) and 8h (F) and then subjected to SEM. Large number of bacteria were seen on the surface of HMEECs at 8h post-infection. Results are representative of four independent experiments carried out in triplicate. Scale bars 2 μm.</p

<i>In Vitro</i> Interaction of <i>Pseudomonas aeruginosa</i> with Human Middle Ear Epithelial Cells

<div><p>Background</p><p>Otitis media (OM) is an inflammation of the middle ear which can be acute or chronic. Acute OM is caused by <i>Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis</i> whereas <i>Pseudomonas aeruginosa</i> 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 <i>P. aeruginosa</i> with human middle ear epithelial cells (HMEECs).</p><p>Methodology and Findings</p><p>In the present investigation, we demonstrate that <i>P. aeruginosa</i> 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 <i>P. aeruginosa</i>. 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 <i>P. aeruginosa</i> located in the plasma membrane-bound vacuoles. We observed a significant decrease in cell invasion of <i>OprF</i> mutant compared to the wild-type strain. <i>P. aeruginosa</i> 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, <i>OprF</i> mutant showed little cell damage compared to wild-type <i>P. aeruginosa</i>.</p><p>Conclusions and Significance</p><p>This study deciphered the key events in the interaction of <i>P. aeruginosa</i> with HMEECs <i>in vitro</i> 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.</p></div

<i>P. aeruginosa</i> causes epithelial cell damage.

<p>HMEECs were infected with <i>P. aeruginosa</i> at an MOI of 10 for varying time periods. Cell damage was examined using the LIVE/DEAD fluorescent assay where uptake of green dye indicates live cells and red staining corresponds to dead cells. Results are representative of four independent experiments carried out in triplicate. Scale bars 10 μm.</p

Confocal Microscopy of HMEECs infected with <i>P. aeruginosa</i>.

<p>HMEECs were infected with <i>P. aeruginosa</i> at an MOI of 10 for 2h and then bacteria were stained with anti-<i>P. aeruginosa</i> antibody followed by a secondary Alexa Fluor® 488 antibody. The slides were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and visualized by confocal laser fluorescence microscope (A). The analytical sectioning was performed from top to bottom of cells and orthogonal panels were prepared demonstrating bacterial invasion of HMEECs (B). Results are representative of four independent experiments carried out in triplicate. Scale bars 10 μm.</p