Involvement of theEscherichia coliO157:H7(pO157)ecfOperon and Lipid A Myristoyl Transferase Activity in Bacterial Survival in the Bovine Gastrointestinal Tract and Bacterial Persistence in Farm Water Troughs

Abstract

Escherichia coli O157:H7 is an important food-borne pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome in humans. Recently, we reported that the pO157 ecf (E. coli attaching and effacing gene-positive conserved fragments) operon is thermoregulated by an intrinsically curved DNA and contains the genes for bacterial surface-associated proteins, including a second copy of lipid A myristoyl transferase, whose chromosomal copy is the lpxM gene product. E. coli O157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tracts (GIT) to nutrient-dilute farm water troughs. Transcriptional regulation of the ecf operon by intrinsic DNA curvature and the genetic redundancy of lpxM that is associated with lipid A modification led us to hypothesize that the pO157 ecf operon and lpxM are associated with bacterial survival and persistence in various in vivo and ex vivo environments by optimizing bacterial membrane structure and/or integrity. To test this hypothesis, three isogenic ecf operon and/or lpxM deletion mutants of E. coli O157:H7 ATCC 43894 were constructed and analyzed in vitro and in vivo. The results showed that a double mutant carrying deletions in the ecf and lpxM genes had an altered lipid A structure and membrane fatty acid composition, did not survive passage through the bovine GIT, did not persist well in farm water troughs, had increased susceptibility to a broad spectrum of antibiotics and detergents, and had impaired motility. Electron microscopic analyses showed gross changes in bacterial membrane structure.The outer membrane (OM) of gram-negative bacteria provides an effective physical barrier to various environments (39). An important component for this function is the lipopolysaccharide (LPS), which is a highly ordered and rigid quasicrystalline structure occupying the outer leaflet of the bacterial OM (29). The inner leaflet of the OM is composed of glycerophospholipids. It is well known that under stress conditions, bacteria can alter their membrane lipid composition (52). An example of such an alteration is the homeoviscous adaptation that occurs with heat stress (47). Also, increasing evidence suggests that structural modification of the OM can change membrane functions by regulating the expression, transportation, or secretion of the membrane-associated proteins (3, 62). Pathogens that thrive in diverse habitats likely adapt their membrane structure to optimize survival under various in vivo and ex vivo conditions. Escherichia coli O157:H7 is the most common serotype of Shiga toxin-producing E. coli (STEC) associated with hemorrhagic colitis and the hemolytic-uremic syndrome in the United States and other countries around the world (36). Healthy cattle are the principal reservoir of this microorganism, and a bacterial colonization site has been recently identified at the most distal part of the bovine gastrointestinal tract (GIT), the recto-anal junction (RAJ) mucosa (21, 37). Major virulence factors identified in animal models incl...

Role of Escherichia coli O157:H7 Virulence Factors in Colonization at the Bovine Terminal Rectal Mucosa

“…Its product is similar to that of the chromosomal gene lpxM. Deletion of both genes leads to reduced survival in the bovine GIT and reduced persistence in water troughs (68). Deletion of toxB results in reduced adherence to cultured epithelial cells and influenced the expression and secretion of proteins encoded by LEE but did not affect the colonization of E. coli O157:H7 in calves (60,62).…”

“…Three of four steers given E. coli O157:H7 continued to be culture positive on day 32, whereas only one of four steers given E. coli K-12 continued to be culture positive on day 25 and all four animals in this group eliminated the E. coli K-12 by day 32 (Fig. 1) (57,58,68), and showed a distinct colonization pattern compared with the nonpathogenic E. coli K-12.…”

Phosphoethanolamine substitution in the lipid A of Escherichia coli O157 : H7 and its association with PmrC

“…In addition, WabB shows a UDP-Nacetylglucosamine : (heptose) LPS a-1,7-N-acetylglucosamine transferase that is responsible for a non-stoichiometric Nacetylglucosamine substitution in the heptose-III residue (Kaniuk et al, 2004). Therefore, the shf locus seems to be a module involved in O157 LPS lipid A-core modifications, and is also suggested to play a role in the survival and persistence of E. coli O157 : H7 in cattle and in the environment (Yoon et al, 2005).…”

pO157_Sal, a Novel Conjugative Plasmid Detected in Outbreak Isolates of Escherichia coli O157:H7

“…pO157, a 92-kb F-like plasmid, is found almost invariably in all E. coli O157:H7 clinical isolates (18) and shares sequence similarities with plasmids present in other EHEC serotypes (15,23). A number of virulence factors encoded by pO157, including an enterohemolysin (ehxA), a type II secretion system, a serine protease (espP), a catalase-peroxidase (katP), a potential adhesin (toxB), a Cl esterase inhibitor (stcE), and attaching and effacing gene-positive conserved fragments (ecf), were identified (2,25). ecf encodes a functional LpxM homologue that catalyzes the addition of myristate to the lipid A moiety of lipopolysaccharide (LPS) (25).…”

“…A number of virulence factors encoded by pO157, including an enterohemolysin (ehxA), a type II secretion system, a serine protease (espP), a catalase-peroxidase (katP), a potential adhesin (toxB), a Cl esterase inhibitor (stcE), and attaching and effacing gene-positive conserved fragments (ecf), were identified (2,25). ecf encodes a functional LpxM homologue that catalyzes the addition of myristate to the lipid A moiety of lipopolysaccharide (LPS) (25). Earlier plasmid-profiling studies showed the variable presence in O157:H7 isolates of, in addition to pO157, several plasmids ranging from 2 to 6 kb (19,21).…”