Bacterial protein AvrA stabilizes intestinal epithelial tight junctions via blockage of the C-Jun N-terminal kinase pathway

Abstract

The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junctions (TJ) and epithelial functions in intestinal infection and inflammation. In a previous study, we have demonstrated that a bacterial effector AvrA plays a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. However, the molecular mechanisms by which AvrA-modulates TJ protein expression remain unknown. AvrA possesses acetyltransferase activity toward specific mitogen-activated protein kinase kinases (MAPKKs) and potently inhibits the c-Jun N-terminal kinase (JNK) pathway in inflammation. Inhibition of the JNK pathway is known to inhibit the TJ protein disassemble. Therefore, we hypothesize that AvrA stabilizes intestinal epithelial TJs via c-Jun and JNK pathway blockage. Using both in vitro and in vivo models, we showed that AvrA targets the c-Jun and JNK pathway that in turn stabilizes TJ protein ZO-1. Inhibition of JNK abolished the effect of AvrA on ZO-1. We further determined that AvrA suppressed the transcription factor activator protein-1, which was regulated by activated JNK. Moreover, we identified the functional domain of AvrA that directly regulated TJs using a series of AvrA mutants. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampens the inflammatory response of the host. Our findings have uncovered a novel role of the bacterial protein AvrA in suppressing the inflammatory response of the host through JNK-regulated blockage of epithelial cell barrier function.

Keywords:

• AvrA
• c-Jun
• epithelial cell
• inflammation
• intestine
• JNK
• permeability
• Salmonella
• tight junction
• ZO-1

Abbreviations:

• JNK, c-jun N-terminal kinase
• TTSS III, type III secretory system
• TJ, tight junctions
• MAPKKs, mitogen-activated protein kinase kinases
• ZO, zonula occludens
• IBD, inflammatory bowel diseases
• CD, crohn's disease
• TER, transepithelial resistance
• CFU, colony-forming unit
• FBS, fetal bovine serum
• HBSS, Hank's balanced salt solutions
• DMEM, Dulbecco's modified eagle's medium
• PBS, phosphate buffered saline
• AP-1, activator protein-1

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the NIDDK (KO1 DK075386 and 1R03DK089010-01), the American Cancer Society (RSG-09-075-01-MBC), and Swim Across America Cancer Research Award to JS and 1R01HL113640 to AX.