Dataset Information

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).

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.

SUBMITTER: Mittal R

PROVIDER: S-EPMC3954863 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

In vitro interaction of Pseudomonas aeruginosa with human middle ear epithelial cells.

Mittal Rahul R Grati M'hamed M Gerring Robert R Blackwelder Patricia P Yan Denise D Li Jian-Dong JD Liu Xue Zhong XZ

PloS one 20140314 3

<h4>Background</h4>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 avai ...[more]

PMID: 24632826

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Project description:IntroductionAir pollutants such as Asian sand dust (ASD) and Streptococcus pneumoniae are risk factors for otitis media (OM). In this study, we evaluate the role of ASD in pneumococcal in vitro biofilm growth and colonization on human middle ear epithelium cells (HMEECs) and rat middle ear using the rat OM model.MethodsS. pneumoniae D39 in vitro biofilm growth in the presence of ASD (50-300 μg/ml) was evaluated in metal ion-free BHI medium using CV-microplate assay, colony-forming unit (cfu) counts, resazurin staining, scanning electron microscopy (SEM), and confocal microscopy (CF). Biofilm gene expression analysis was performed using real-time RT-PCR. The effects of ASD or S. pneumoniae individually or on co-treatment on HMEECs were evaluated by detecting HMEEC viability, apoptosis, and reactive oxygen species (ROS) production. In vivo colonization of S. pneumoniae in the presence of ASD was evaluated using the rat OM model, and RNA-Seq was used to evaluate the alterations in gene expression in rat middle ear mucosa.ResultsS. pneumoniae biofilm growth was significantly (P < 0.05) elevated in the presence of ASD. SEM and CF analysis revealed thick and organized pneumococcal biofilms in the presence of ASD (300 μg/ml). However, in the absence of ASD, bacteria were unable to form organized biofilms, the cell size was smaller than normal, and long chain-like structures were formed. Biofilms grown in the presence of ASD showed elevated expression levels of genes involved in biofilm formation (luxS), competence (comA, comB, ciaR), and toxin production (lytA and ply). Prior exposure of HMEECs to ASD, followed by treatment for pneumococci, significantly (P < 0.05) decreased cell viability and increased apoptosis, and ROS production. In vivo experiment results showed significantly (P < 0.05) more than 65% increased bacteria colonization in rat middle ear mucosa in the presence of ASD. The apoptosis, cell death, DNA repair, inflammation and immune response were differentially regulated in three treatments; however, number of genes expressed in co-treatments was higher than single treatment. In co-treatment, antimicrobial protein/peptide-related genes (S100A family, Np4, DEFB family, and RATNP-3B) and OM-related genes (CYLD, SMAD, FBXO11, and CD14) were down regulated, and inflammatory cytokines and interleukins, such as IL1β, and TNF-related gene expression were elevated.ConclusionASD presence increased the generation of pneumococcal biofilms and colonization.

Project description:The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was <10% upon 24 h incubation of PBMC with P. aeruginosa biofilms. PBMC incubated for 24 h with preformed P. aeruginosa biofilms were significantly more activated compared to PBMC incubated alone. Interestingly, a marked activation of CD56+CD3- natural killer (NK) cells was observed that reached 60% of NK cells as an average of different donors. In the culture supernatants of PBMC co-cultured with P. aeruginosa biofilms, not only pro-inflammatory (IL-1β, IFN-γ, IL-6, and TNF-α) but also anti-inflammatory (IL-10) cytokines were significantly increased as compared to PBMC incubated alone. Furthermore, incubation of biofilms with PBMC, caused a statistically significant increase in the CFU number of P. aeruginosa, as compared to biofilms incubated without PBMC. In order to assess whether PBMC products could stimulate the growth of P. aeruginosa biofilms, we incubated preformed P. aeruginosa biofilms with or without supernatants obtained from the co-cultures of PBMC with biofilms. In the presence of the supernatants, the CFU count of biofilm-derived P. aeruginosa, was two to seven times higher than those of biofilms incubated without supernatants (P < 0.01). Overall, the results obtained shed light on the reciprocal interaction between human PBMC and P. aeruginosa biofilms. P. aeruginosa biofilms induced PBMC activation and cytokine secretion but, in turn, the presence of PBMC and/or PBMC-derived components enhanced the number of P. aeruginosa biofilm associated bacteria. This may indicate a successful bacterial defensive/persistence strategy against immune response.

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Dataset Information

In vitro interaction of Pseudomonas aeruginosa with human middle ear epithelial cells.

Background

Methodology and findings

Conclusions and significance

Publications

In vitro interaction of Pseudomonas aeruginosa with human middle ear epithelial cells.

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