Interactions between exeA and peptidoglycan in the type II secretion system of aeromonas hydrophila
Aeromonas hydrophila uses the type II secretion system to transport protein toxins across the outer membrane. The trans-envelope system is comprised of more than ten proteins, including ExeA and ExeB, which form a complex in the inner membrane and are required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan binding domain (Pfam protein families database number PF01471) is present in the periplasmic region of ExeA (pExeA), leading to the hypothesis that ExeA generates gaps in peptidoglycan, a barrier for trans-envelope transport and apparatus assembly, to allow ExeD to assemble into the outer membrane. In this study, interactions between ExeA and peptidoglycan were examined both in vivo and in vitro. Wild type ExeA, but not the mutants containing substitution mutations of three highly conserved amino acid residues in the putative peptidoglycan binding domain, was cross-linked to peptidoglycan in vivo with DTSSP. Furthermore, the presence of wild type ExeA was also required for co-crosslinking of ExeB and ExeC to peptidoglycan. In vitro cosedimentation revealed that purified pExeA was able to bind to highly purified peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in cross-linking and co-gel filtration experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at temperatures above 25 °C. Two pExeA constructs, which disrupted the putative peptidoglycan binding domain, greatly reduced the cosedimentation, accompanied by decreased multimerization in the presence of peptidoglycan fragments. These results provide evidence that the putative peptidoglycan binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions cause ExeA to multimerize, possibly forming a ring-like structure on the peptidoglycan, to generate a gap large enough to accommodate the secretion apparatus and/or to form an assembly scaffold. The putative peptidoglycan binding domain of ExeA was also analyzed by comparing its amino acid sequence with that of other homologues. The highly conserved amino acid residues were found to cluster at one pocket on the surface in the crystal structure of hydrolase metallo (Zn) DD-peptidase that also contains this domain. We propose that this pocket is the binding site for the peptidoglycan ligand.
DegreeDoctor of Philosophy (Ph.D.)
DepartmentMicrobiology and Immunology
ProgramMicrobiology and Immunology
SupervisorHoward, S. Peter
CommitteeLuo, Yu; Goldie, Hughes; Delbaere, Louis T. J.; Clarke, A. J.; Xiao, Wei
Type II secretion