New clues as to the virulence of the potentially lethal bacterium Escherichia coli O157:H7 has emerged from structural and functional relationship studies of its autotransport and proteolytic EspP proteins. A comparison with X-ray diffraction results reveals important clues about these proteins.

E. coli O157:H7 hit the headlines again recently, with several children who had visited petting farms in the south of England succumbing to infection and several cases reported in Canada and elsewhere.

This strain of E. coli is usually found in cattle waste, and can be transferred to humans through direct contact or via contaminated food or water, in particular beef or dairy products. Infection with the bacterium causes a range of health problems through the release of Shiga toxins.

Infection causes diverse symptoms, including abdominal pain and intestinal bleeding (haemorrhagic colitis) or even just uncomplicated diarrhoea. In between 2 and 7 percent of cases, however it can cause potentially fatal kidney problems (haemolytic uraemic syndrome) that are often chronically debilitating if not lethal, especially in young children and the elderly.

Now, Jens Brockmeyer, Thorsten Kuczius, Martina Bielaszewska, and Helge Karch of the Institute for Hygiene and the National Consulting Laboratory on Hemolytic Uremic Syndrome, and Sabrina Spelten of the Department of Medicine B, University Hospital Münster, Münster, in Germany, have looked closely at the protein chemistry of this bacterium.

Gram-negative bacteria, such as the rod-shaped O157:H7 have evolved several methods for secreting proteins into their surroundings. According to Brockmeyer, the autotransporter, or Type V secretion pathway, is the simplest known. Serine protease autotransporters from O157:H7 and its ilk, the enterobacteriaceae, are a subfamily with serine protease activity, and appear to be present only in pathogenic bacteria.

One member of this family is EspP, extracellular serine protease, plasmid-encoded found in O157:H7. This protein is an important virulence factor in the spread of this emergent pathogen because it cleaves coagulation factor V in human plasma. According to Brockmeyer and colleagues, they have previously demonstrated that EspP is one of the most abundant proteins in the liquid extracted from a bacterial culture of O157:H7.

The team points out that there are four sub-types of the protein - alpha, beta, gamma, and delta, which differ significantly in structure and function. The alpha form is produced mainly by the strains associated with severe disease, including haemolytic uraemic syndrome.

The fact that the others have different function and form and are not active in causing such symptoms suggested to the researchers a possible approach to determining which features of the protein are key to its devastating impact on patients.

They have now used genetic engineering techniques - transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain - to uncover the underlying differences between the different forms of EspP and the links to virulence of O157:H7. Comparison of their findings with known crystal structures also provided additional information.

Their results suggest that the correct formation of the tertiary structure of the protein's passenger domain is essential for efficient autotransport. They also identified an elastase-like serine protease domain in the N-terminal Domain 1 that is responsible for its proteolytic behaviour. When stabilizing interactions are missing, as is the case with the beta and delta forms, the protein has shows no proteolytic activity.

The team also points out that their experiments confirm that three amino acid residues - His¹²⁷, Asp¹⁵⁶ and Ser²⁶³ - are key to forming the proteolytically active site in EspP, which in turn, supports their hypothesis that Domain 1 comprises a classical serine protease with a catalytic triad, it is this feature that endows the protein with its proteolytic activity.

The next step will be to investigate whether or not EspP not only shares structural features with elastases but also functional aspects of that serine protease family.

"We intended to analyse EspP as an additional virulence factor of EHEC (the major bona fide known virulence factor are Shiga toxins, which are toxic to renal cells and lead to the known severe clinical outcomes of EHEC infections," Brockmeyer told SpectroscopyNOW, "In our work presented in PLos ONE we first of all tried to enhance the understanding of the structural properties of EspP and to investigate the differences between the EspP subtypes from virulent and less virulent EHEC strains. In our current work we are at the moment analysing in more detail the functional properties of this additional virulence factor."

He points out that this work cannot be translated directly into the development of new antibiotics, because antibiotic therapy itself often triggers the expression of the major virulence factor Shiga toxin in this infection. "Due to this fact, it is most important to investigate the pathoomechanisms of this pathogen in great detail to develop alternative therapeutic strategies for the treatment of EHEC infections," he says, "We believe that EspP in its active form contributes to the pathogenicity of EHEC infections and that EHEC contain a large repertoire of virulence factors which might act in concert." Any one of these mechanisms might one day provide a target for non-antibiotic therapy. 

Related links:

• PLoS One, 2009, 4, e6100

Article by David Bradley