title: Biochemical characterization of the enterotoxigenic Escherichia coli LeoA protein.
abstract: Enterotoxigenic Escherichia coli (ETEC) causes enterotoxin-induced diarrhoea and significant mortality. The molecular mechanisms underlying how the heat-labile enterotoxin (LT) is secreted during infection are poorly understood. ETEC produce outer-membrane vesicles (OMVs) containing LT that are endocytosed into host cells. Although OMV production and protein content may be a regulated component of ETEC pathogenesis, how LT loading into OMVs is regulated is unknown. The LeoA protein plays a role in secreting LT from the bacterial periplasm. To begin to understand the function of LeoA and its role in ETEC H10407 pathogenesis, a site-directed mutant lacking the putative GTP-binding domain was constructed. The ability of wild-type and mutant LeoA to hydrolyse GTP in vitro was quantified. This domain was found to be responsible for GTP binding; it is important to LeoA's function in LT secretion, and may play a modest role in the formation and protein content of OMVs. Deletion of leoA reduced the abundance of OmpX in outer-membrane protein preparations and of LT in OMVs. Immunoprecipitation experiments revealed that LeoA interacts directly with OmpA, but that the GTP-binding domain is non-essential for this interaction. Deletion of leoA rendered ETEC H10407 non-motile, through apparent periplasmic accumulation of FliC.
authors.affiliation: Center for Infectious Disease Research and Vaccinology, South Dakota State University, Brookings, SD 57007, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Brown
authors.affiliation: Center for Infectious Disease Research and Vaccinology, South Dakota State University, Brookings, SD 57007, USA.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Hardwidge
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D054307:Enterotoxigenic Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004768:Enterotoxins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D046529:Microscopy, Electron, Transmission; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000378:metabolism; D022161:Transport Vesicles; D020798:Two-Hybrid System Techniques
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C078167:OMPA outer membrane proteins; D006160:Guanosine Triphosphate; C027538:heat-labile enterotoxin, E coli; D020558:GTP Phosphohydrolases