Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
2001-01-24
2003-10-21
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S185100, C424S190100, C530S350000, C530S402000, C435S007100, C435S007320, C435S007370, C435S007200, C435S006120
Reexamination Certificate
active
06635259
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel proteins which are secreted from enterohemorrhagic
Escherichia coli
(EHEC), a pathogen responsible for bloody diarrhea and hemolytic uremic syndrome (HUS) in humans. The invention relates to serodiagnostic techniques of these conditions using these proteins.
BACKGROUND OF THE INVENTION
The most common cause of bloody diarrhea and hemolytic uremic syndrome (HUS) in North America is infection by enterohemorrhagic
E. coli
(EHEC) (1). Alternative names for EHEC are Shiga toxin-producing
E. coli
(STEC), Shiga-like toxin-producing
E. coli
(SLTEC), Verocytotoxin-producing
E. coli
(VTEC), or Verotoxin-producing
E.coli
. In the United States, this food-borne
E. coli
is the most common infectious cause of bloody diarrhea in individuals of all ages. HUS is the most common cause of kidney failure in children in the U.S. and Canada.
This organism was the cause of the infamous “Jack-in-the-Box” food-poisoning outbreak in Seattle in 1993 which infected over 500 people and resulted in 4 deaths and many cases of long-term kidney damage. In 1996, this organism caused an enormous outbreak involving more than 8,000 people in Japan, resulting in 7 deaths. In late 1996, EHEC again caused an outbreak of food-poisoning in Scotland which affected 250 people and killed 18 people.
The most important virulence factor of
E. coli
associated with HUS is a potent cytotoxin known as Shiga toxin, Shiga-like toxin, verocytotoxin, or verotoxin, also called Stx. After production by
E. coli
colonizing the large intestine, Shiga toxin is absorbed into the circulation and eventually affects the kidney. The evidence linking Stx to HUS is both epidemiological and experimental. The first epidemiological association between Stx-producing
E. coli
and HUS was made by Karmali et al. (2) in 1983, and numerous studies since then have supported this association. Subsequent in vitro studies have shown that purified Stx has profound effects on renal endothelial cells resulting in cell death (3).
However, besides the clear-cut involvement of Stx in pathogenesis, little is known about other bacterial virulence factors involved in this disease. The lipopolysaccharide (LPS) of EHEC has been reported to enhance the effect of the Stx on human vascular endothelial cells (4), although the exact mechanism is not known. The other virulence factor of this organism that has been implicated in animal models is the 94 kilodalton outer membrane protein (OMP) known as intimin, which was discovered in the inventor's laboratory (5, 6). Intimin is involved in the colonization of the intestinal tract, which is apparently necessary for disease, but there is no evidence that intimin is directly involved in the renal disease. Since oral ingestion of preformed toxin is apparently not sufficient for causing HUS, other bacterial virulence factors clearly must be involved in the pathogenesis of this disease.
Two Distinct Sites of Disease, Intestinal and Renal
Disease due to EHEC starts by ingestion of meat, water, or other items that are contaminated with this organism. The organism then colonizes the large bowel where it can produce non-bloody diarrhea or bloody diarrhea (hemorrhagic colitis). In the colon, EHEC produces mucosal edema, erythema, ulceration and hemorrhage. A characteristic histopathology known as attaching and effacing (AE) results which is characterized by effacement of intestinal microvilli, intimate adherence of bacteria to enterocytes, and accumulation of polymerized actin and other cytoskeletal components in the epithelial cell directly beneath the adherent bacteria. The AE lesion has been repeatedly demonstrated in animals infected with EHEC and in cultured human epithelial cells. AE is assumed to occur in the colon early during the course of human infection, although direct evidence is lacking, probably because patients with EHEC infections undergo colonoscopy relatively late in the infection when the colonic surface has been denuded of epithelial cells. The AE histopathology is similar to that seen with enteropathogenic
E. coli
(EPEC), which do not produce Stx. We have shown that formation of the AE lesion by both EPEC and EHEC is mediated by the gene products of a 35 kilobase region of chromosomal DNA present in these strains but absent from normal flora
E. coli
(5, 7).
The pathogenic mechanisms by which EHEC produces non-bloody diarrhea and bloody diarrhea (hemorrhagic colitis) are largely unknown. Formation of the AE lesion in the absence of Stx is believed to be sufficient to cause non-bloody diarrhea in EPEC infections. Pure Stx can act as an enterotoxin, and in studies using rabbit jejunal tissue mounted in Ussing chambers, Stx selectively kills the absorptive tip cells while not affecting the secretory crypt cells, thereby changing the net balance of secretory/absorptive processes towards secretion (8). The bloody diarrhea is presumably due at least in part to the powerful cytotoxic effects of Stx, although the contribution of other bacterial factors and the host inflammatory response is not known. In at least two animal systems, gnotobiotic piglets and rabbits, Stx was not required for EHEC strains to alter secretory activity or cause severe histological changes (9, 10). O'Loughlin and colleagues (9, 11) have shown that disruption of colonic epithelium and changes in electrolyte transport during EHEC infection in rabbits are mediated by the host inflammatory response and that bacterial products other than Stx and factors encoded on the 60 MDa plasmid (see below) are necessary for the intestinal manifestations of EHEC disease.
The classic HUS triad includes microangiopathic hemolytic anemia, thrombocytopenia, and renal failure and may be accompanied by central nervous system manifestations in 30-50% of patients (12). Although hemolytic uremic syndrome (HUS) occurs in only 2-7% of all EHEC infections (while bloody diarrhea occurs in 90% of all infections (1,13)), HUS is associated with the greatest mortality due to this organism. The Shiga toxin produced in the bowel reaches the circulation and produces vascular endothelial damage that results in occlusion of the renal glomerular microvasculature by fibrin and platelets. Induction of inflammatory cytokines has also been suggested to contribute to the disease process (14-16). Although the preeminence of Stx in the disease process is accepted, the role of other bacterial factors in producing host damage or facilitating the delivery of Stx is unknown.
Virulence Factors of
E. coli
O157:H7
The majority of work on pathogenic factors of EHEC has focused on the Shiga toxins, which are encoded on bacteriophage inserted into the chromosome. Additional potential virulence factors are encoded in the chromosome and on a 60 MDa plasmid found in most strains of EHEC
Toxins Stx occurs in two major forms, Stx1 and Stx2, which share 55 and 57% sequence identity in the A and B subunits, respectively (17). While Stx1 is highly conserved, sequence variation exists within Stx2. The toxins consist of a single A subunit of ca. 32 kDa and 5 identical B subunits of ca. 7.7 kDa (18) The B subunit serves to bind the toxin to a specific glycolipid receptor, globotriaosylceramide or Gb
3
, while the A subunit is internalized and cleaves N-glycoside bonds on the 28S rRNA of the 60S ribosome. The resulting disruption of protein synthesis leads to death of renal endothelial cells, intestinal epithelial cells, vero or Hela cells, or any cell which possesses the Gb
3
receptor.
The 60 MDa plasmid commonly found in EHEC strains contains genes encoding an alpha-hemolysin (19). Although this alpha hemolysin is widely distributed among Stx-producing strains of
E. coli
, there are no data indicating that it is expressed in vivo or involved in pathogenesis of disease. Two other distinctly different phage-encoded hemolysins, termed enterohemolysins, are produced by many Stx-producing
E. coli
(20, 21) but again, there are no data to suggest in vivo expression or any role in pathogenesis.
Intestinal Adherence Factors
Jarvis Karen
Kaper James B.
Doering Andrea E.
Marks David L.
Portner Ginny Allen
Smith Lynette R. F.
University of Maryland Baltimore
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