Actinobacillus pleuropneumoniae exotoxin ApxI induces cell death via attenuation of FAK through LFA-1

Siou-Cen Li,Yu-Tsen Cheng,Zeng-Weng Chen,Jyh-Perng Wang,Shih-Ling Hsuan,Jiunn-Horng Lin,Ching-Yang Wang,Jia-Ying Wu

doi:10.1038/s41598-021-81290-9

Abstract

ApxI exotoxin is an important virulence factor derived from Actinobacillus pleuropneumoniae that causes pleuropneumonia in swine. Here, we investigate the role of lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), a member of the β2 integrin family, and the involvement of the integrin signaling molecules focal adhesion kinase (FAK) and Akt in ApxI cytotoxicity. Using Western blot analysis, we found that ApxI downregulated the activity of FAK and Akt in porcine alveolar macrophages (AMs). Preincubation of porcine AMs with an antibody specific for porcine CD18 reduced ApxI-induced cytotoxicity as measured by a lactate dehydrogenase release assay and decreased ApxI-induced FAK and Akt attenuation, as shown by Western blot analysis. Pretreatment with the chemical compounds PMA and SC79, which activate FAK and Akt, respectively, failed to overcome the ApxI-induced attenuation of FAK and Akt and death of porcine AMs. Notably, the transfection experiments revealed that ectopic expression of porcine LFA-1 (pLFA-1) conferred susceptibility to ApxI in ApxI-insensitive cell lines, including human embryonic kidney 293T cells and FAK-deficient mouse embryonic fibroblasts (MEFs). Furthermore, ectopic expression of FAK significantly reduced ApxI cytotoxicity in pLFA-1-cotransfected FAK-deficient MEFs. These findings show for the first time that pLFA-1 renders cells susceptible to ApxI and ApxI-mediated attenuation of FAK activity via CD18, thereby contributing to subsequent cell death.

Highlights

Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes severe hemorrhagic and necrotizing pleuropneumonia in pigs, leading to great economic loss in industry
To the best of our knowledge, the present study is the first to show that ApxI induces cytotoxicity via the β2 integrin lymphocyte function-associated antigen 1 (LFA-1) and attenuation of the focal adhesion kinase (FAK) survival signaling protein
LFA-1 mediates ApxI-induced cell death, as our study showed that cotransfection of porcine cd11a and cd[18] conferred susceptibility to ApxI in Human embryonic kidney (HEK) 293T cells and mouse embryonic fibroblasts (MEFs)

Summary

Introduction

Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes severe hemorrhagic and necrotizing pleuropneumonia in pigs, leading to great economic loss in industry. The CD18 subunit of lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) results in ApxIII-induced cell death[15], and CD18 mediates ApxI-induced activation of the p38, c-Jun N-terminal kinase, and nuclear factor-κB pathways for the expression of proinflammatory cytokines[16,17]. The role of the FAK signaling pathway in β1 integrin-mediated cell survival is well defined, and Akt modulates diverse prosurvival and antideath functions[20]. Previous studies reported that CyaA, Hly, and Lkt attenuated Akt activity[22,23,24], suggesting that RTX toxins downregulate cell survival pathways. Whether LFA-1 mediates the cytotoxic effects of ApxI via FAK and Akt is unclear. The present study examined the role of LFA-1 in ApxI cytotoxicity and delineated the possible involvement of FAK and Akt in this event

Methods

Results

Conclusion