Use of In Vivo-Induced Antigen Technology for Identification of Escherichia coli O157:H7 Proteins Expressed during Human Infection

Abstract

Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumventsthe need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 (O157) is a uniquely human pathogen that causes disease ranging from acute, self-resolving watery diarrhea to hemorrhagic colitis and the potentially fatal hemolytic-uremic syndrome (HUS). Currently, no therapies are available to lessen the potential morbidity and mortality of this infection.E. coli O157 is thought to have evolved from a strain of enteropathogenic E. coli (EPEC) O55:H7 bearing the pathogenicity island termed the locus for enterocyte effacement (LEE), through the acquisition of bacteriophages encoding Shiga toxins type 1 (stx 1 ) and/or 2 (stx 2 ), acquisition of a virulence plasmid (pO157), transition of somatic antigen O55 to O157, and loss of sorbitol fermentation and ␤-glucuronidase activity (21). HUS as a complication of E. coli O157 infection has been associated with the presence of the stx 2 gene or its variant stx 2c in the infecting E. coli O157 strain (21). In addition, the characteristic attaching and effacing (A/E) lesions produced by this organism on the human colonic epithelium are a result of proteins encoded on the LEE, including the adhesion molecule intimin-␥ (Eae), its receptor (Tir), the type III protein secretion system, which secretes a variety of LEEencoded translocator proteins (EspA, EspB, and EspD) that translocate effectors into host cells, and effector proteins (Tir, EspG, EspF, Map, and EspH) that modulate the host cell cytoskeleton (21). The type III secretion system translocates Tir into the host cell, with subsequent trafficking to the host cell membrane. Intimin binding of Tir leads to host cell actin rearrangement and formation of A/E lesions. Other putative virulence factors are encoded on pO157 and include an enterohemolysin (Ehx), an immunomodulator (Lif), and a serine protease (EspP) (21). Hence several factors may be involved in E. coli O157 pathogenesis, and research is ongoing to understand the complexity of this infection.The sequenc...

Proteomics-based Expression Library Screening (PELS)

“…Here we wish to emphasize that further experimentation is required to ascertain whether the rest of the PELS-identified proteins are unique to the O157 immunome in cattle. This is because the immunoproteome defined by IVIAT is a partial one in that it includes only proteins that are expressed either uniquely during human infection or at significantly higher levels in vivo than during in vitro growth because of a series of adsorptions of human convalescent sera against O157 grown in standard laboratory media (10). In contrast, the unadsorbed cattle hyperimmune sera used in PELS facilitated the definition of a more complete immunoproteome.…”

“…Vector and insert DNA in the size range of 0.5-3.0 kbp were prepared as described previously (10). Because accuracy of protein identification using tandem MS/MS data and SEQUEST database searching increases with the number of peptides generated following trypsin digestion of the cognate protein (8), we sought to preferentially ligate insert DNA fragments that were larger than the average size of ORFs in this pathogen.…”

Identification of Salmonella enterica Serovar Pullorum Antigenic Determinants Expressed In Vivo

“…The PhoP-activated gene pagC regulated by the PhoP/ PhoQ system was identified as an IVI virulence protein in S. Typhi, and the PagC antibody was detected in sera from 11 of 14 patients, which shows that it has the potential to be developed as a diagnostic antigen (12). Interestingly, PhoQ was also confirmed to be an IVI protein of E. coli O157:H7, as identified by the use of IVIAT (18). In this study, PhoQ was detected in 68% of serum samples, which implies that the protein is not an ideal diagnostic antigen candidate or that the N-terminal amino acids that we did not express have the potential to be antigenic determinants.…”

“…Of the 45 proteins identified in this study, many proteins were screened from other Gram-negative and Gram-positive bacteria by IVIAT (12,(17)(18)(19)(20)(21), such as ShdA in S. Typhi, PhoQ and FolE in E. coli O157:H7, and AdhE in E. coli CFT073 (a uropathogenic E. coli [UPEC] strain). In addition, some genes with similar function or involved in the same pathway were identified in these pathogens.…”

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part II: Vaccines forShigella,Salmonella,enterotoxigenicE. coli(ETEC) enterohemorragicE. coli(EHEC) andCampylobacter jejuni

“…The characterization of STEC antigens using human serum, naturally and artificially infected cattle and other animal models, has allowed the identification of different immunogenic proteins that have been proposed as targets for the development of vaccines. [130][131][132][133][134][135] The antigens best characterized immunologically, and that have been used systematically in different vaccine candidates, include: the Shiga toxins Stx1 and Stx2, LPS, flagellin (FliC -H7) and virulence factors encoded in the LEE and secreted by a TTSS, such as Tir, intimin, EspA, EspB and EspD. The use of effectors coded by the LEE has been particularly attractive, given their importance in STEC pathogenesis and due to the possibility of generating cross-protection against enteropathogenic Escherichia coli (EPEC) [reviewed in 136 ]; this is another important pathotype associated with infantile diarrhea that presents an LEE locus with a high degree of homology.…”