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VIDYARANI MOHANKUMAR, LakshmiPriya Jeganathan, Lalitha Prajna, Chidambaranathan Gowri Priya; Pseudomonas aeruginosa induces autophagy in human corneal epithelial cells . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4051.
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© ARVO (1962-2015); The Authors (2016-present)
Keratitis caused by Pseudomonas aeruginosa is a serious ocular infection which may lead to corneal perforation if the intracellular bacteria are not cleared completely. Autophagy, a normal catabolic process, has been shown to play a major role in the clearance of intracellular pathogens. We propose that autophagy induced by P. aeruginosa in human corneal epithelial cells (HCET), may have a role in the clearance of intracellular bacteria.
HCET cells transfected with LC3-GFP plasmid were infected with three different ocular isolates of P. aeruginosa and autophagy was monitored after 1h using a Leica TCS SP8 confocal microscope. EBSS treated (amino acid starvation) HCET cells were used as positive control for autophagy. Another set of HCET cells were infected with P. aeruginosa to study the mRNA expression of autophagy related protein, beclin1 by real time PCR. To study the intracellular survival and replication of the bacteria, HCET cells were infected with P. aeruginosa in the presence of EBSS or 3 -methyl adenine (3mM in EBSS), an inhibitor of autophagosome formation. After 3h, the extracellular bacteria were killed with gentamicin and the cells were incubated for another three hours to allow for intracellular bacterial replication. Diluted cell lysates were plated on to MacConkey agar, and the colonies were counted after an overnight incubation.
The HCET cells upon infection with three different P. aeruginosa isolates showed an increased LC3 punctation, which is a classical marker for autophagosome formation. The total number of LC3 positive cells and the relative number of LC3-puncta per cell varied upon infection with the different isolates which may be due to the difference in the intracellular bacterial load. Correspondingly, the mRNA expression of beclin1 was higher in P. aeruginosa infected cells compared to uninfected controls.The ocular isolates were able to efficiently invade and replicate inside the epithelial cells and the bacterial load was relatively higher when the cells were pretreated with 3-methyl adenine.
Altogether, the results suggest that P. aeruginosa induces autophagy in human corneal epithelial cells, which in-turn may limit the intracellular bacterial load. Since the ocular P. aeruginosa isolates can efficiently invade and replicate inside the epithelial cells, autophagy may represent a host defensive mechanism to curtail infection in P. aeruginosa keratitis.
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