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

et al. (2014) In Vitro Interaction of Pseudomonas aeruginosa with Human Middle Ear Epithelial Cells. PLoS ONE 9(3): e91885. doi:10.1371/journal.pone.0091885 In Vitro Interaction of Pseudomonas aeruginosa with Human Middle Ear Epithelial Cells Rahul Mittal 0 M'hamed Grati 0 Robert Gerring 0 Patricia Blackwelder 0 Denise Yan 0 Jian-Dong Li 0 Xue Zhong Liu 0 Min Wu, University of North Dakota, United States of America 0 1 Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, Florida, United States of America, 2 Center for Advanced Microscopy, University of Miami, Coral Gables, Florida, United States of America , 3 RSMAS , University of Miami , Key Biscayne , Florida, United States of America, 4 Center for Inflammation, Immunity, and Infection and Department of Biology, Georgia State University , Atlanta , Georgia , United States of America, 5 Department of Human Genetics, 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 Factin 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. - Funding: The research work in Dr. Lius laboratory is supported by the National Institutes of Health grants R01DC005575, R01DC012546, and R01DC012115. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Chronic suppurative otitis media (CSOM) is a frequently encountered chronic inflammation of the middle ear and mastoid process characterized by both tympanic membrane perforation and discharge [1]. CSOM is one of the most common chronic infectious diseases worldwide. CSOM affects diverse racial and cultural groups in both developing and developed countries and occurs frequently in children [2]. When it occurs during the first two years of life, the consequent hearing loss is likely to have serious effects on the critical period of a young childs development, and may have long term effects on language development, early communication, auditory processing, psychosocial and cognitive development, as well as educational progress and achievement [3,4]. CSOM has been associated with considerable morbidity and substantial healthcare costs [5]. Without treatment, there is continuous or intermittent purulent ear discharge for months or even years with destruction of the bones of the middle ear and increasing hearing impairment [6]. The presence of mucus prevents the transmission of sound waves from middle ear to inner ear leading to conductive hearing loss. Chronic infection of the middle ear leads to oedema of the middle-ear lining and discharge, tympanic membrane perforation, and possibly ossicular chain disruption that further aggravates the problem of hearing loss in CSOM patients [7]. CSOM can also cause sensorineural hearing loss [810]. It has been shown that inflammatory mediators generated during CSOM can penetrate from the round window into the inner ear causing loss of hair cells in the cochlea leading to sensorineural hearing loss in animal models [1113]. Human studies have also demonstrated the loss of outer and inner hair cells in the basal turn of the cochlea in CSOM patients [14]. The pathogenesis of CSOM is multifactorial including abnormal function of the eustachian tube (resulting from small size, genetic syndromes, viral respiratory infections, functional immaturity, allergy, and environmental smoke exposure), invasion of the middle ear by bacteria and/or viruses, and inflammation [15,16]. The bacterial infection of the middle ear is the most common cause of CSOM. Antibiotics and surgery are the only treatment options for CSOM, but have only moderate efficacy against the disease. The excessive use of the antibiotics has led to the emergence of resistant bacteria that has further complicated the treatment of CSOM. Antibiotics can also have severe ototoxic effects, especially in children, which should also be taken into consideration [17,18]. In addition, antibiotics cause lysis of bacteria with subsequent release of endotoxin and consequent triggering of inflammatory processes that can further aggravate inflammation. Therefore, alternative treatment strategies against CSOM are warranted for which understanding the pathogenesis of disease is of utmost importance. The colonization of host mucosal surfaces is the first and essential step in the infectious process [19]. The infection of a host by a pathogenic microorganism triggers complex cascades of events that influence the immediate and long-term outcome of this interaction [2022]. One of the most important initial signaling events involves interaction of epithelial cells with the pathogen [23,24]. The surface exposed moieties on pathogens like outer membrane proteins (OMPs) have been shown to play an important role in mediating this interaction [25,26]. The middle ea (...truncated)