Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD

Abstract

The type two secretion system is a large, trans-envelope apparatus that secretes toxins across the outer membrane of many Gram-negative bacteria. In Aeromonas hydrophila, ExeA interacts with peptidoglycan and forms a heteromultimeric complex with ExeB that is required for assembly of the ExeD secretin of the secretion system in the outer membrane. While the peptidoglycan-ExeAB (PG-AB) complex is required for ExeD assembly, the assembly mechanism remains unresolved. We analyzed protein-protein interactions to address the hypothesis that ExeD assembly in the outer membrane requires direct interaction with the PG-AB complex. Yeast and bacterial two hybrid analyses demonstrated an interaction between the periplasmic domains of ExeB and ExeD. Two-codon insertion mutagenesis of exeD disrupted lipase secretion, and immunoblotting of whole cells demonstrated significantly reduced secretin in mutant cells. Mapping of the two-codon insertions and deletion analysis showed that the ExeB-ExeD interaction involves the N0 and N1 subdomains of ExeD. Rotational anisotropy using the purified periplasmic domains of ExeB and ExeD determined that the apparent dissociation constant of the interaction is 1.19±0.16 µM. These results contribute important support for a putative mechanism by which the PG-AB complex facilitates assembly of ExeD through direct interaction between ExeB and ExeD. Furthermore, our results provide novel insight into the assembly function of ExeB that may contribute to elucidating the role of homologous proteins in secretion of toxins from other Gram negative pathogens.

Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System

“…Previously, we provided evidence that suggests that ExeB acts as a scaffold by interacting directly with both ExeA and ExeD (22). In addition, PG binding by ExeA induces multimerization of ExeA (20).…”

“…Gly-mediated assembly of the secretin ExeD occurs by an ExeAB-independent mechanism. In addition to the PG-binding function of ExeA, the assembly factor complex ExeAB also contains a cytoplasmic ATPase domain associated with ExeA, whose function is essential for secretin assembly (17), and an ExeD-binding function associated with ExeB (18,22). Growth in Gly restored ExeD secretin assembly in the C5.84 exeA mutant; however, the amount of assembled secretin observed was significantly less than that in the wild type and in several strains expressing ExeA mutants with point mutations in the PG-binding domain (Fig.…”

“…Our current working model for the assembly of the ExeD secretin in A. hydrophila is that the ExeAB complex interacts with PG through the PG-binding domain of ExeA (20,32) and recruits ExeD for assembly through interaction with ExeB (22). Since interactions between PG, ExeAB, and ExeD are the main components of this model, we sought to establish a heterologous secretin assembly system in E. coli by coexpressing only ExeAB and ExeD.…”

“…Point mutations in conserved residues within the PG-binding domain of ExeA demonstrated that PG binding is required for ExeD assembly (20). Recent results have also indicated that ExeB directly interacts with the amino terminal domain of ExeD (22).…”

“…Our current working model for ExeAB-mediated assembly of ExeD is that the ExeAB complex in the inner membrane binds to PG through interactions involving ExeA and recruits ExeD for assembly via interactions involving ExeB (18,20,22). Given the structural barrier imposed by the PG sacculus, we further hypothesize that the function of the ExeA PG-binding domain is to modify the pore size of the PG, possibly by locally perturbing the growth of the sacculus, to allow the transport and assembly of ExeD in the outer membrane.…”