Twitching Motility Contributes to the Role of Pili in Corneal Infection Caused by Pseudomonas aeruginosa

ABSTRACT Twitching motility is a form of surface-associated bacterial movement mediated by type IV pili of Pseudomonas aeruginosa. Others have shown that pilT and pilU mutants, which are piliated but defective in twitching motility, display reduced cytotoxic capacity towards epithelial cells in vitro. Although these mutants efficiently infected lungs in vivo, they were defective in dissemination to the liver. In this study the role of twitching motility in P. aeruginosa epithelial cell invasion and corneal disease pathogenesis was explored. pilU and pilT mutants of P. aeruginosa strain PAK were compared to a nonpiliated pilA mutant and to wild-type bacteria in their ability to associate with and to invade corneal epithelial cells in vitro and to cause disease in a murine model of corneal infection. As expected, the pilA mutant demonstrated reduced association and invasion of corneal epithelial cells (P < 0.05 in both cases). The pilT mutant, but not the pilU mutant, was less invasive than wild-type PAK was (P < 0.05 versus P = 0.43), while both pilU and pilT mutants exhibited association levels similar to those of the wild type (P = 0.31 and 0.52, respectively). In vivo, all mutants were markedly attenuated in virulence and showed reduced ability to colonize the cornea at 4 and 48 h (all P values < 0.02). Thus, twitching motility contributed to the role of pili in corneal disease but was not involved in the role of pili in adherence to or invasion of corneal epithelial cells.

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FlhA, a Component of the Flagellum Assembly Apparatus of Pseudomonas aeruginosa, Plays a Role in Internalization by Corneal Epithelial Cells

Infection and Immunity ◽

10.1128/iai.69.8.4931-4937.2001 ◽

2001 ◽

Vol 69 (8) ◽

pp. 4931-4937 ◽

Cited By ~ 55

Author(s):

Suzanne M. J. Fleiszig ◽

Shiwani K. Arora ◽

Rajana Van ◽

Reuben Ramphal

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cell ◽

Epithelial Cells ◽

Vector Control ◽

Plasmid Vector ◽

Wild Type ◽

Corneal Epithelial Cells ◽

Corneal Epithelial

ABSTRACT Pseudomonas aeruginosa invades various epithelial cell types in vitro and in vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to invA of Salmonella spp. Because invA is required for invasion ofSalmonella spp., a role for flhA in P. aeruginosa invasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be nonmotile. Complementation with flhA in transrestored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the flhA mutant, the flhA mutant complemented with flhA in trans, anflhA mutant containing the plasmid vector control, or anfliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mutants invaded at a lower level than the wild-type strain did, suggesting that both fliC andflhA played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by flhAmutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the flhA mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the fliC mutant did. The percentage of adherent bacteria that invaded was reduced by ∼80% for the flhAmutant and ∼50% for the fliC mutant, showing that only part of the role of flhA in invasion involvesfliC. Invasion was restored by complementing theflhA mutant with flhA in trans but not by the plasmid vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although flhAand fliC mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in P. aeruginosainvasion of epithelial cells. Since the flhA mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, or to survive inside epithelial cells after entry, the role of flhA in invasion of epithelial cells is likely to occur during the process of bacterial internalization.

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Anti-Inflammatory and Antioxidative Effects of Camellia japonica on Human Corneal Epithelial Cells and Experimental Dry Eye: In Vivo and In Vitro Study

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.16-20634 ◽

2017 ◽

Vol 58 (2) ◽

pp. 1196 ◽

Cited By ~ 13

Author(s):

Hyo Seok Lee ◽

Joo-Hee Choi ◽

Lian Cui ◽

Ying Li ◽

Jee Myung Yang ◽

...

Keyword(s):

Epithelial Cells ◽

Dry Eye ◽

In Vitro Study ◽

Corneal Epithelial Cells ◽

Camellia Japonica ◽

Corneal Epithelial ◽

Antioxidative Effects ◽

Human Corneal Epithelial Cells

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Calcitriol inhibits apoptosis via activation of autophagy in hyperosmotic stress stimulated corneal epithelial cells in vivo and in vitro

Experimental Eye Research ◽

10.1016/j.exer.2020.108210 ◽

2020 ◽

Vol 200 ◽

pp. 108210

Author(s):

Ning Lyu ◽

Jing Zhang ◽

Yiqin Dai ◽

Jun Xiang ◽

Yue Li ◽

...

Keyword(s):

Epithelial Cells ◽

Hyperosmotic Stress ◽

Corneal Epithelial Cells ◽

Corneal Epithelial

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Morphogenic Effects of Ezrin Require a Phosphorylation-Induced Transition from Oligomers to Monomers at the Plasma Membrane

The Journal of Cell Biology ◽

10.1083/jcb.150.1.193 ◽

2000 ◽

Vol 150 (1) ◽

pp. 193-204 ◽

Cited By ~ 198

Author(s):

Alexis Gautreau ◽

Daniel Louvard ◽

Monique Arpin

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Wild Type ◽

Erm Proteins ◽

Threonine Residue ◽

Phosphorylation Event ◽

Membrane Bound

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

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Atropine Differentially Modulates ECM Production by Ocular Fibroblasts, and Its Ocular Surface Toxicity Is Blunted by Colostrum

Biomedicines ◽

10.3390/biomedicines8040078 ◽

2020 ◽

Vol 8 (4) ◽

pp. 78

Author(s):

Martina Cristaldi ◽

Melania Olivieri ◽

Salvatore Pezzino ◽

Giorgia Spampinato ◽

Gabriella Lupo ◽

...

Keyword(s):

Epithelial Cells ◽

Test Results ◽

Corneal Epithelial Cells ◽

Corneal Epithelial ◽

Atropine Treatment ◽

Progressive Myopia ◽

Methyl Xanthine

Background: The etiology and the mechanism behind atropine treatment of progressive myopia are still poorly understood. Our study addressed the role of scleral and choroidal fibroblasts in myopia development and atropine function. Methods: Fibroblasts treated in vitro with atropine or 7-methylxanthine were tested for ECM production by Western blotting. Corneal epithelial cells were treated with atropine in the presence or absence of colostrum or fucosyl-lactose, and cell survival was evaluated by the MTT metabolic test. Results: Atropine and 7-methyl-xanthine stimulated collagen I and fibronectin production in scleral fibroblasts, while they inhibited their production in choroidal fibroblasts. Four days of treatment with atropine of corneal epithelial cells significantly decreased cell viability, which could be prevented by the presence of colostrum or fucosyl-lactose. Conclusions: Our results show that atropine may function in different ways in different eye districts, strengthening the scleral ECM and increasing permeability in the choroid. The finding that colostrum or fucosyl-lactose attenuate the corneal epithelial toxicity after long-term atropine treatment suggests the possibility that both compounds can efficiently blunt its toxicity in children subjected to chronic atropine treatment.

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A synthetic metalloporphyrin SOD mimetic protects corneal epithelial cells from oxidative stress‐induced damage in vitro and in vivo

Acta Ophthalmologica ◽

10.1111/aos.13972_463 ◽

2018 ◽

Vol 96 (S261) ◽

pp. 124-124

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Corneal Epithelial Cells ◽

Corneal Epithelial

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The Role of Twitching Motility inPseudomonas aeruginosaExit from and Translocation of Corneal Epithelial Cells

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.08-2785 ◽

2009 ◽

Vol 50 (5) ◽

pp. 2237 ◽

Cited By ~ 31

Author(s):

Irania Alarcon ◽

David J. Evans ◽

Suzanne M. J. Fleiszig

Keyword(s):

Epithelial Cells ◽

Twitching Motility ◽

Corneal Epithelial Cells ◽

Corneal Epithelial

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Roles of TRIM32 in Corneal Epithelial Cells After Infection with Herpes Simplex Virus

Cellular Physiology and Biochemistry ◽

10.1159/000481563 ◽

2017 ◽

Vol 43 (2) ◽

pp. 801-811 ◽

Cited By ~ 4

Author(s):

Hao Cui ◽

Ying Liu ◽

Yifei Huang

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Epithelial Cells ◽

Microbial Pathogens ◽

Corneal Epithelial Cells ◽

Corneal Epithelial ◽

Simplex Virus ◽

Hsv 1

Background: Epithelial cells play important roles as a critical barrier in protecting the cornea from microbial pathogens infection. Methods: In this study, we were aiming to investigate the role of E3 ubiquitin ligase tripartite motif protein 32 (TRIM32) in corneal epithelial cells in response to Herpes Simplex Virus type 1 (HSV-1) infection and to elucidate the underlying mechanisms. Results: We found the expression of TRIM32 was increased after infected with HSV-1 both in murine corneas and cultured human epithelial (HCE) cells. Furthermore, knockdown of the expression of TRIM32 significantly aggravated HSV-1 induced herpetic stromal keratitis (HSK) in mice and promoted the replication of HSV-1 in cultured HCE cells. We also observed that silencing of TRIM32 resulted in the decreased expression of IFN-β and suppressed activation of interferon regulatory factor 3 (IRF3) both in vivo and in vitro. Finally, we found TRIM32 positively regulate IFN-β production in corneal epithelial cells through promoting K63-linked polyubiquitination of stimulator of interferon genes (STING). Conclusion: In conclusion, our data suggested that TRIM32 as a crucial positive regulator of HSV-1 induced IFN-β production in corneal epithelial cells, and it played a predominant role in clearing HSV-1 from the cornea.

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