Knockdown of IQGAP-1 Enhances Tight Junctions and Prevents P. aeruginosa Invasion of Human Corneal Epithelial Cells

ABSTRACT

Purpose

To determine the role of IQ-domain GTPase-activating protein1 (IQGAP-1) in tight junctions of human corneal epithelial cells (HCECs) and its effect against P. aeruginosa (PAK) invasion.

Material and Methods

Primary human corneal epithelial cells (HCECs), immortalized HCECs, and IQGAP-1 RNA knockdown HCECs (siHCECs) were used. Confocal microscopy, transepithelial electrical resistance (TER), trypan blue exclusion assay and gentamicin invasion assay were done.

Results

In primary and immortalized HCECs, IQGAP-1 co-localized with zonular occludin-1 (ZO-1) and actin. Enhanced actin and ZO-1 aggregation were seen in siHCECs. IQGAP-1 knockdown significantly increased TER of immortalized HCECs (P < .0001). Cell viability after PAK infection increased for siHCECs for up to 4 h after infection. PAK intracellular invasion was significantly lowered by 50% in siHCECs at 1 h post-infection.

Conclusion

IQGAP-1 knockdown increased the strength and integrity of tight junctions and may provide an early protective effect against P. aeruginosa invasion.

Keywords:

• IQGAP-1, P. aeruginosa
• tight junction
• Type III secretion system
• ZO-1

Acknowledgments

The authors thank Professor Stephen Lory (Department of Microbiology and Immunobiology, Harvard Medical School) for providing the invasive PAK P. aeruginosa strain. We thank the Second Core Lab of the National Taiwan University, College of Medicine for technical assistance. In addition, we are deeply grateful to Professor Ching-Hwa Tsai (Graduate Institute of Microbiology, College of Medicine, National Taiwan University) for offering insightful advice for this study.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The authors have no proprietary or commercial interest in the research presented herein. Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Honolulu, Hawaii, USA, April 29–May 3, 2018.

Additional information

Funding

This work was supported, in part, by Ministry of Science and Technology, Taiwan (MOST-105-2314-B-002-167-MY3, MOST106-2314-B303-011MY3), and a grant from the Taipei Tzuchi Hospital, The Buddhist Tzuchi Medical Foundation (TCRD-TPE-106-041), and Excellent Translational Medicine Research Projects of National Taiwan University, College of Medicine and National Taiwan University Hospital (103C101-A2).