Otopathogenic Pseudomonas aeruginosa Enters and Survives Inside Macrophages

Abstract

Otitis media (OM) is a broad term describing a group of infectious and inflammatory disorders of the middle ear. Despite antibiotic therapy, acute OM can progress to chronic suppurative otitis media (CSOM) characterized by ear drum perforation and purulent discharge. Pseudomonas aeruginosa is the most common pathogen associated with CSOM. Although, macrophages play an important role in innate immune responses but their role in the pathogenesis of P. aeruginosa-induced CSOM is not known. The objective of this study is to examine the interaction of P. aeruginosa with primary macrophages. We observed that P. aeruginosa enters and multiplies inside human and mouse primary macrophages. This bacterial entry in macrophages requires both microtubule and actin dependent processes. Transmission electron microscopy demonstrated that P. aeruginosa was present in membrane bound vesicles inside macrophages. Interestingly, deletion of oprF expression in P. aeruginosa abrogates its ability to survive inside macrophages. Our results suggest that otopathogenic P. aeruginosa entry and survival inside macrophages is OprF-dependent. The survival of bacteria inside macrophages will lead to evasion of killing and this lack of pathogen clearance by phagocytes contributes to the persistence of infection in CSOM. Understanding host–pathogen interaction will provide novel avenues to design effective treatment modalities against OM.

Killing from the inside: Intracellular role of T3SS in the fate ofPseudomonas aeruginosawithin macrophages revealed bymgtCandoprFmutants

“…Our previous results based on gentamicin protection assay on J774 infected macrophages and counting of colony forming units (CFU) indicated that mgtC mutant in the PAO1 background survived to a lesser extent than the wild-type strain [23]. More recently, an oprF mutant in a P. aeruginosa otopathogenic strain was also found to be defective in intracellular survival in mouse bone marrow macrophages based on gentamicin protection assay [24]. To analyze phenotypes of oprF mutant towards J774 macrophages and compare with mgtC mutant, we used here a previously described oprF mutant in the PAO1 parental background [23,32].…”

“…However, P. aeruginosa has been shown to be phagocytosed by macrophages in an acute model of infection in zebrafish embryos [20] and has been reported to be engulfed by alveolar macrophages in the lungs of mice early after pulmonary infection [21], suggesting that P. aeruginosa may have to escape macrophage killing. Although P. aeruginosa has been localized within cultured macrophages during gentamicin protective assays [22–24], the intramacrophage fate of the bacteria has not been characterized and bacterial factors involved in this step remain largely unexplored.…”

“…MgtC is known to promote intramacrophage growth in several classical intracellular bacteria, including Salmonella typhimurium where it inhibits bacterial ATP synthase and represses cellulose production [26–28], and is considered as a clue to reveal bacterial pathogens adapted to an intramacrophage stage [28,29]. In addition, a recent study has implicated OprF in the ability of otopathogenic P. aeruginosa strains to survive inside macrophages [24]. OprF is an outer membrane porin that can modulate the production of various virulence factors of P. aeruginosa [30,31].…”

Panel 7 – Pathogenesis of otitis media – a review of the literature between 2015 and 2019

“…Mittal et al have described that otopathogenic P. aeruginosa entry and survival inside macrophages is OprF-dependent and that this mechanism leads to evasion of killing and persistence of infection in CSOM [184]. The bacteria activate the PKC pathway through phosphorylation of PKC-alpha (PKC-α) in human middle ear epithelial cells (HMEECs) and this is dependent on bacterial OprF expression [185]. Blocking the PKC pathway attenuated the ability of bacteria to invade HMEECs [185].…”

“…The bacteria activate the PKC pathway through phosphorylation of PKC-alpha (PKC-α) in human middle ear epithelial cells (HMEECs) and this is dependent on bacterial OprF expression [185]. Blocking the PKC pathway attenuated the ability of bacteria to invade HMEECs [185]. and this pathway should be considered in the development of improved treatments for this severe form of OM.…”