Everiss, and K

Everiss, and K. types or in healthy controls. These data demonstrate that TagA is expressed in vivo and provide circumstantial evidence for a role in the pathogenesis of the disease. The gene is present only in STEC strains belonging to serogroup O157, and so antibodies to TagA are a potentially useful serological marker for infections due to such strains. Shiga toxigenic (STEC) organisms are an important cause of gastrointestinal disease in humans, particularly since these infections may result in life-threatening sequelae such as the hemolytic-uremic syndrome (HUS) (13, 17, 23). The STEC family is very Tariquidar (XR9576) diverse, and strains belonging to a broad range of O:H Rabbit Polyclonal to Desmin serotypes have been associated with human disease. However, certain STEC subsets account for a disproportionately high number of serious infections. Members of one such subset have the capacity to produce attaching and effacing (A/E) lesions on intestinal mucosa, a property encoded on a pathogenicity island termed the locus for enterocyte effacement (LEE). LEE encodes a type III secretion system and enterohemolysin (EhxA) (26) and an extracellular serine protease (EspP) (2), both of which may be accessory virulence factors. STEC strains belonging to serogroup O157 appear to be of particular virulence for humans. Although epidemiological data may have been skewed by the fact that they are much easier to detect than other STEC strains (because they are sorbitol negative), this serogroup (particularly serotype O157:H7) has been historically responsible for most major outbreaks of serious human STEC disease (13, 17, 23). For this reason, O157:H7 STEC strains have been the subject of intensive study in recent years. Indeed, the complete genome sequences of two O157:H7 STEC strains have recently been published (9, 24); the sequences of the large plasmids (designated pO157) from the same two strains had been reported separately (3, 16). The sequenced strains were EDL933, which was responsible for an outbreak of hemorrhagic colitis in 1983, and RIMD0509952, which was associated with a massive outbreak of hemorrhagic colitis and HUS in Sakai, Japan, in 1996. These studies have provided a valuable resource for STEC research. In particular, they have demonstrated that the O157:H7 STEC genome contains approximately 1,400 genes not present in the genome of K-12. However, determining which of these, including many with no homology to known virulence genes of other bacteria, actually function in the pathogenesis of human disease is a difficult undertaking, particularly given the paucity of suitable Tariquidar (XR9576) animal models. One potentially useful approach to the identification of virulence-related gene products is to determine which STEC-specific proteins elicit a host immune response during Tariquidar (XR9576) infection. Indeed, convalescent-phase sera from HUS patients have been shown to contain antibodies to several proteins already strongly implicated in pathogenesis, including the LEE-encoded proteins intimin, Tir, EspA, and EspB (11, 15, 20, 28), as well as the plasmid-encoded hemolysin EhxA and the serine protease EspP (2, 26). In an attempt to identify additional virulence-related gene products of O157:H7 STEC, we have screened a cosmid library of EDL933 DNA for clones reacting with convalescent HUS patient sera. Mapping of sequence data generated from these clones on the genome facilitates characterization of the full repertoire of targets of the Tariquidar (XR9576) human immune response to STEC infection. MATERIALS AND METHODS Bacterial strains and cloning vectors. The O157:H7 STEC strain EDL933 and a sorbitol-fermenting, nontoxigenic O157:H20 isolate were provided by R. Robins-Browne, Royal Children’s Hospital, Melbourne, Australia. All other strains used in this study were clinical isolates from the Women’s and Children’s Hospital, North Adelaide, Australia. K-12 strains DH1 and JM109 have been described previously (7, 29). The cosmid vector pHC79 has also been described previously (10). The phagemids pBluescript KS (encoding ampicillin resistance) and pBC SK (encoding chloramphenicol resistance) were obtained from Stratagene, La Jolla, Calif. All strains were routinely grown in Luria-Bertani (LB) medium with or without 1.5% Bacto-Agar (Difco Laboratories, Detroit, Mich.). Where appropriate, ampicillin or chloramphenicol were added to growth media at a concentration of 50 or 40 g/ml, respectively. Construction of EDL933 cosmid bank. To construct a cosmid gene bank of EDL933 DNA, high-molecular-weight genomic DNA was digested partially with DH1, which had been grown in.