title: 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.
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Vanderlinde
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Su
authors.initials: S
authors.lastname: Zhong
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Martynowski
authors.affiliation: Canadian Light Source, Saskatoon, Saskatchewan, Canada; College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Pawel
authors.initials: P
authors.lastname: Grochulski
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: S Peter
authors.initials: SP
authors.lastname: Howard
mesh_terms: D016980:Aeromonas hydrophila / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; C090403:ExeB protein, Aeromonas hydrophila; C094204:ExeD protein, Aeromonas; D008565:Membrane Proteins