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 nonessential for this interaction. Deletion of leoA rendered ETEC H10407 non-motile, through apparent periplasmic accumulation of FliC.

LeoA, B and C from Enterotoxigenic Escherichia coli (ETEC) Are Bacterial Dynamins

“…The observation that LeoA is a large GTPase (64.2 kDa), with a putative involvement in membrane vesicle (MV) secretion [12], [13] prompted us to question whether LeoA could be an as yet unrecognised dynamin-like protein (DLP).…”

“…This is important because LeoA has previously been linked with the secretion of heat labile enterotoxin (LT) through membrane vesicle (MV) biogenesis and release from the bacterial cell surface [12], [13]. Currently there are very few reports indicating functional roles of bacterial dynamins [14].…”

A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa

“…All of these observations suggest a periplasmic function of FliC and DnaK. Such a periplasmic FliC function was described in the context of the E. coli LeoA protein activity during enterotoxin secretion (49). Deletion of leoA led to reduced toxin export, accumulation of FliC in the periplasm, and a nonmotile phenotype (49).…”

“…Such a periplasmic FliC function was described in the context of the E. coli LeoA protein activity during enterotoxin secretion (49). Deletion of leoA led to reduced toxin export, accumulation of FliC in the periplasm, and a nonmotile phenotype (49). Moreover, FliC function in adhesion and virulence has been described in detail before (50,51).…”

Distribution of Classical and Nonclassical Virulence Genes in Enterotoxigenic Escherichia coli Isolates from Chilean Children and tRNA Gene Screening for Putative Insertion Sites for Genomic Islands

“…LeoA is a cytoplasmic protein with GTPase activity, required for maximal LT secretion. An isogenic H10407 leoA mutant strain produces smaller amounts of LT membrane vesicles and shows less fluid accumulation in the rabbit ileal loop model than those of wild-type controls (5,11). The gene coding for LeoA is located in a 46-kb pathogenicity island (PAI) that also carries the tia gene.…”