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A Parallel Clinical Pattern to O104:H4

The O104:H4 serotype has an unusual clinical pattern for Shiga toxin-producing E. coli (STEC) pathogens, including bloody diarrhea in adults followed by a high conversion rate to adult hemolytic uremic syndrome (HUS) or to severe non-HUS enterohemorrhagic symptoms in adults.

There is another STEC group that parallels the unusual pattern of the current outbreak in Germany. It as characterized by a form of Shiga toxin that becomes more deadly after it is attacked by the patient’s body’s defense mechanisms.

Most STEC like O147:H7 have a class of Shiga toxin known as Stx2. But there are a variety of subtypes of Stx2 such as Stx2a, Stx2b, Stx2c Stx 2d and others. There is a form of Shiga toxin Stx2d that becomes more virulent following attack by the mucosal enzyme elastase, whether from mice or humans.

Elastase attacks the toxin by cleaving off two amino acids from one of its components, at the C-terminal end of the A2 peptide (short protein) of Stx 2d. In lab tests with mouse models in 2002, as well as other tests, conducted by Dr. Alison O’Brien’s group at the Uniformed Services University of the Health Sciences (Bethesda, Maryland), these E. coli with toxins activated by exposure to mucosal preparations or the specific enzyme elastase, were more virulent than any other (STEC) Stx E.coli strains [1].

This type of Shiga toxin was then named “Stx 2d activatable” because instead of damaging the toxin by splitting it, the elastase, or crude mucosal preparation containing elastase, made it worse — “activating” it.

Professor Dr. Helge Karch and associated researchers at the National Consulting Laboratory on Hemolytic Uremic Syndrome, University of Muenster and the Interdisciplinary Center for Clinical Research, Muenster, wanted to study what the clinical implications of this kind of strain were for humans, rather than mice. They did a stratified analysis of 922 human STEC strains from patients with HUS, bloody or nonbloody diarrhea and asymptomatic carriers, looking at different kinds of STEC strains and disease course outcomes.

It takes more than Shiga toxin to have a virulent STEC pathogen. In particular, in “normal” STEC strains, tissue invasion is aided by an adhesive protein called intimin, encoded by the eae gene. In most STEC descriptions, having the eae gene for intimin is a crucial component for pathogenicity.

The Muenster researchers found that Stx2d activatable strains were also highly virulent in human cases, not just in mice or the in vitro model of Vero cells (the basis of calling Shiga toxins “verotoxins” is based on this kind of test). This was despite being eae (-), lacking the production of intimin. They also found the unusual pattern of adults and older patients with bloody diarrhea and HUS, as in the O104:H4 outbreak [2,3].

There were several alternative hypotheses to explain this. One was that the increased virulence of the activated toxin was a compensation for lack of intimin, or that toxin delivery into tissue was actually aided by the peptide editing caused by human mucosal elastase. A second possibility was that there was overexpression of the Stx gene to compensate for lack of intimin. A third possibility was that Stx2d activatable STEC strains had an unknown virulence factor that replaced the intimin functionality.

The first characterization by the Robert Koch Institute of the German O104:H4 outbreak strain included the notation that it was also eae (-), lacking intimin. Rather like the third hypothesis for Stx2d activatable strains, the outbreak strain had different adhesion genes characteristic of the entero-aggregative types of pathogenic E. coli, not seen in a “normal” STEC outbreak, whether in O157:H7 or pathogenic non-O157s.

Here the close parallel ends.

The O104:H4 outbreak strain, has no need for intimin, one might say, since it has other adhesion and invasion factors from its enteroaggregative genetic background. O104:H4 appears to be an enteroaggregative type of E. coli with Shiga toxin functionality from normal STEC. It is in a sense, a Shiga toxin containing enteroaggregative strain type. It could be

described as a STEC “armed” enteroaggregative E. coli or as a hybrid of STEC and enteraggregative types.

Virulence and adult bloody diarrhea and adult HUS at a higher rate of conversion may be due entirely to these enteroaggregative derived genes, not due to increased virulence of the Shiga toxin subtype, or increased virulence due to peptide editing by elastase, or some other enzyme.

The DNA sequence of the toxin from O104:H4 is supposed to be the same as Stx2, not Stx2d-activatable.

However, the 5-day time course from bloody diarrhea to HUS is somewhat suggestive of a pathogen-human-pathogen interaction like that of mucosal elastase and Stx2d activatable strains. Indicators of patient status slightly improve on the third day, and some patients think they are recovering just before plunging into a rapid development of HUS.

It is possible that this reflects a similar trigger, where toxin is functionally enabled for increased virulence and rapidly deployed in response to the same human defenses that give patients or physicians the illusion of pos- sible improvement.

STEC strains can change and evolve even during the infection course of a single patient (3), losing or gaining plasmids and phages, with their associated functionalities for Shiga toxin or intimin or drug resistance, for example. The parallel unusual clinical patterns between the O104:H4 outbreak strain and STEC Stx2d activatable pathogenic strains — severity of adult clinical symptoms — may be an example of convergent evolution, getting to the same results by completely different mechanisms. The mechanism could also be analogous or very similar, if not identical.

Regardless of mechanism, two parallel types of cases, with similar clinical pattern and outcomes, can still give clinicians and researchers sugges- tions for approaches, context and comparisons for treating the disease course.

Daniel B. Cohen

Maccabee Seed Company

Davis, CA

[1] Martina Bielaszewska, Alexander W. Friedrich, Thomas Aldick, Robin Schürk-Bulgrin, and Helge Karch Shiga Toxin Activatable by Intestinal Mucus in Escherichia coli Isolated from Hu- mans: Predictor for a Severe Clinical Outcome Clin Infect Dis. (2006) 43(9): 1160-1167 doi:10.1086/508195

[2] “A subset of eae-negative STEC strains (i.e., STEC that produce Stx2dactivatable) differ from other eae-negative STEC strains by their strong association with severe disease… By its association with severe outcome of the infection caused by eae-negative STEC, Stx2dactivatable parallels the pathogenetic significance of Stx2 among eae-positive STEC … However, in contrast to Stx2, which is associated with HUS development in children <5 years old…, most of the patients with HUS from whom we isolated Stx2dactivatable-producing STEC were adults.” From Bielaszewska et al., next citation.

[3] Activation of Shiga toxin type 2d (Stx2d) by elastase involves cleavage of the C-terminal two amino acids of the A2 peptide in the context of the appropriate B pentamer. Melton-Celsa AR, Kokai-Kun JF, O’Brien AD Mol Microbiol. 2002 Jan;43(1):207-15.

[4] Alexander W. Friedrich, Wenlan Zhang, Martina Bielaszewska, Alexander Mellmann, Robin Köck, Angelika Fruth, Helmut Tschäpe, and Helge Karch  Prevalence, Virulence Profiles, and Clinical Significance of Shiga Toxin-Negative Variants of Enterohemorrhagic Escherichia coli O157 Infection in Humans. Clin Infect Dis. (2007) 45(1): 39-45 doi:10.1086/518573

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