title: Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System.
abstract: In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. InAeromonas hydrophila, outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth ofA. hydrophilain 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects inexeAstrains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system inEscherichia coliand showed that ExeAB and ExeC are the onlyA. hydrophilaproteins required for the assembly of the ExeD secretin inE. coliand that ExeAB-independent assembly of ExeD can occur upon overexpression of the d,d-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS.IMPORTANCEThe PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG cross-linking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated inA. hydrophilaIn a heterologous assembly model inE. coli, expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly.
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Vanderlinde
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Strozen
authors.affiliation: Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Center for Microbial Research, Umeå University, Umeå, Sweden.
authors.forename: Sara B
authors.initials: SB
authors.lastname: Hernández
authors.affiliation: Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Center for Microbial Research, Umeå University, Umeå, Sweden.
authors.forename: Felipe
authors.initials: F
authors.lastname: Cava
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada peter.howard@usask.ca.
authors.forename: S Peter
authors.initials: SP
authors.lastname: Howard
mesh_terms: D016980:Aeromonas hydrophila / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D030781:Organisms, Genetically Modified; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism*; D012633:Secretin / Q000737:chemistry / Q000378:metabolism*; D000069178:Type II Secretion Systems / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D010457:Peptidoglycan; D000069178:Type II Secretion Systems; C090745:global stress protein A, bacteria; D012633:Secretin
keywords: peptidoglycan; secretin assembly; type II secretion system