The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri.

Xia Wang,Jian Yang,Lihong Chen,Bo Liu,Feng Jiang,Lilian Sun,Jianhua Zheng,Guowei Yang,Jie Dong,Qi Jin,Yafang Zhu

doi:10.1098/rsob.160073

Abstract

Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

Highlights

The outer membrane proteins (OMPs) of Gram-negative bacteria are unique membrane proteins that generally contain a b-barrel fold and range in size from 8 to 26 strands [1,2]
We report a comprehensive validation of how S. flexneri PldA functions under standard growth conditions using proteomic and transcriptomic approaches
We characterized the extracellular proteome for both S. flexneri WT and DpldA strains during the exponential growth phase through mass spectrometry (MS) to detect the membrane integrity and cellular content leakage

Summary

Introduction

The outer membrane proteins (OMPs) of Gram-negative bacteria are unique membrane proteins that generally contain a b-barrel fold and range in size from 8 to 26 strands [1,2]. Outer membrane phospholipase A (OMPLA) comprises a family of b-barrel proteins embedded in the OM of bacteria that hydrolyse membrane PLs and remove the ester bonds at the stereochemical numbering (sn) positions sn-1 (first carbon) or sn-2 (second carbon) from the glycerophosphodiester backbone of both PLs and lysophospholipids [9,10]. The crystal structure of the OMPLA isolated from E. coli indicated that this enzyme is a serine hydrolase with a His142-Ser144-Asn156 catalytic triad located on the exterior of the b-barrel. The activity of this enzyme is regulated by reversible dimerization and requires calcium as a cofactor [11,12]

Methods

Results

Conclusion