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This research project aims to characterise the expression of bacterial adhesin(s) under environmental conditions that resemble the physiological conditions encountered in the host tissue.
Study Duration : March 1996 to May 1999
Contractor : University of Warwick
Verocytotoxin producing Escherichia coli (VTEC) is recognised as an important human pathogen. Strains belonging to serotype O157:H7 have been predominantly associated with outbreaks of haemorrhagic colitis and haemolytic uremic syndrome, two potentially life-threatening conditions. VTEC produces one or more cytotoxins, known as verocytotoxins (VTs). In addition to toxin production, adherence of these organisms to intestinal mucosal cells is thought to be an important primary event in pathogenesis of VTEC infection.
Since the laboratory culture conditions are not the normal environment that pathogenic organisms find during infection, where growth rate is often restricted by nutrient limitation and host defences, this project was designed to investigate bacterial adherence under conditions which are as close as possible to the in vitro situation in the human colon.
The main objectives of this study were to:
The adherence of verocylotoxin producing E. coli (VTEC) O157:H7 to HeLa (human cervical epithelium carcinoma) and HEp-2 (human laryngeal carcinoma) cells was investigated under variable culture conditions. In vitro parameters, such as bacterial growth phase, oxygen limitation, iron restriction and pH of the culture medium, influenced adherence of VTEC strain to both cell lines. Actively growing bacteria in the exponential growth phase were found to be more adherent than cells in the stationary phase. Differences in the ability to adhere were observed due to changes in pH of the culture media, with maximum adherence at pH 6-7. However, a more significant increase in adherence of VTEC strains occurred under anaerobic culture conditions. VTEC grown in low iron media were found to be less adherent than the same strains grown in standard media. These findings are the first to demonstrate that the adherence of VTEC to human epithelial cells is influenced by such environmental factors.
Ultrastructural investigations of the bacterial cell components, which have been implicated as adherence factors e.g. fimbriae, flagellae, lipopolysaccharides (LPS), and outer membrane proteins (OMPs), were investigated by negative staining and immunogold labelling techniques which identified the H-7 flagellar antigen, the O157 antigen of the LPS and the antigenic OMPs. An exhaustive search by electron microscopy did not reveal fimbriation in any of the strains studied. In addition, all strains failed to agglutinate red blood cells from different sources, supporting the conclusion that the bacterial strains under study were non-fimbriated. It was therefore concluded that these strains express adherence through non-fimbrial adhesins.
The second approach taken to characterise the cellular adhesins was through the use of competitive inhibitors of bacterial adherence, which included purified LPS, OMPs and antibodies specific to the H-7 flagella, O157 antigen and the OMPs. The highest degree of inhibition was obtained with the OMPs. The expression of these proteins under variable culture conditions was investigated by electrophoresis. SDS-PAGE analysis of OMPs demonstrated the expression and repression of specific protein bands when bacteria were grown under anaerobic conditions, low iron, different growth phases and variable pH values of the culture medium. It is likely that these bacteria, in response to particular environmental conditions, synthesise specific adhesin(s) which mediate adherence to mucosal surfaces.
Microscopic studies of the adherence mechanism have demonstrated two patterns of adherence. All strains attached to HeLa and HEp-2 cells in a diffuse form. However, the same strains demonstrated an aggregative pattern of adherence with the colonic carcinoma cell lines Caco-2 and HT-29. When examined by scanning electron microscopy, VTEC strains were clearly binding to the extracellular microfilaments of HeLa and HEp-2 cells, whilst the same strains appeared to be invading Caco-2 and HT-29 cells. Further investigation of this observation by gentamicin survival assays confirmed that these strains of O157:H7 only adhere to HeLa and HEp-2 cells but are adherent/invasive to HT-29 and Caco-2 cells.
The final report is available from the FSA Library and Information centre.
To obtain a copy, please contact the Enquiry Desk,
Dr Elsie Widdowson Library and Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email:
library&info@foodstandards.gsi.gov.uk
).
Contact : For any enquiries concerning this research project, please contact the relevant Programme contact or email science@foodstandards.gsi.gov.uk
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