Fatal hemorrhage induced by subtilase cytotoxin from Shiga-toxigenic Escherichia coli

Subtilase cytotoxin (SubAB) is an AB₅ type toxin produced by a subset of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone BiP. The B subunit binds to a receptor on the cell surface. Although SubAB is lethal for mice, the cause of death is not clear. In this study, we demonstrate in mice that SubAB induced small bowel hemorrhage and a coagulopathy characterized by thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time. SubAB also induced inflammatory changes in the small intestine as detected by ¹⁸F-fluoro-2-deoxy-d-glucose positron emission tomography imaging and histochemical analysis. Using RT-PCR and ELISA, SubAB was shown to increase interleukin-6 in a time-dependent manner. Thus, our results indicate that death in SubAB-treated mice may be associated with severe inflammatory response and hemorrhage of the small intestine, accompanied by coagulopathy and IL6 production.     

Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli

Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.     

Prevalence of subtilase cytotoxin in verocytotoxin-producing Escherichia coli isolated from humans and raw meats in Belgium

Recently, subtilase cytotoxin (SubAB) was detected in verocytotoxin-producing Escherichia coli (VTEC) that do not carry the Locus of Enterocyte Effacement (LEE) pathogenicity island. The distribution of the subA gene in VTEC isolated from patients with the hemolytic uremic syndrome, patients with diarrheal disease and raw meats from ruminants and wildlife in Belgium was investigated with PCR. The subA gene was detected more frequently (χ ² = 10.2; d.f. = 1; P = 0.001) in VTEC from raw meats (10 of 87 strains) than in those from humans (8 of 274 strains), and never in serogroups O157, O26, O103, O111 and O145. This virulence marker could play a role in the development of HUS after infection with LEE-negative VTEC but was only found in one O178:H19 isolate out of 36 HUS-associated VTEC strains.     

Protective immunization of mice with an active-site mutant of subtilase cytotoxin of Shiga toxin-producing Escherichia coli

We have recently described a novel AB(5) cytotoxin produced by certain Shiga toxin-producing Escherichia coli strains. The A subunit of this toxin is a subtilase-like serine protease, while the B pentamer mediates binding to host cell glycolipid receptors. The subtilase cytotoxin is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidneys, and liver. In the present study, we have immunized mice with a purified derivative of the toxin with a Ser272 --> Ala mutation in the A subunit which abolishes cytotoxicity. This elicited strong antibody responses, as judged by enzyme-linked immunosorbent assay, which conferred protection against intraperitoneal challenge with purified toxin. Immunized mice were also protected from weight loss resulting from oral challenge with an E. coli K-12 clone expressing the active toxin.     

Multiplex PCR for direct detection of Shiga toxigenic Escherichia coli strains producing the novel subtilase cytotoxin

We have recently described a novel AB₅ subtilase cytotoxin produced by certain Shiga toxigenic Escherichia coli (STEC) strains. This potentially lethal toxin may contribute to severe gastrointestinal and systemic disease in humans. In this study we have developed a trivalent PCR assay for the detection of the novel toxin A subunit gene subA, as well as stx₁ and stx₂. The three primer pairs used in the assay do not interfere with each other and generate amplification products of 556, 180, and 255 bp, respectively. The assay can be used for determining the toxin genotype of STEC isolates, as well as for direct detection of toxin genes in primary fecal culture extracts.     

Shiga-like cytotoxin production by enteropathogenic Escherichia coli serogroups.

The mechanism by which enteropathogenic Escherichia coli (EPEC) cause disease remains to be defined. We studied EPEC and non-EPEC strains of E. coli from stool specimens obtained from infants and adults for production of Shiga-like cytotoxin. Although it was common for healthy infants and adults to have cytotoxin-producing E. coli as part of the fecal flora, Shiga-like cytotoxin was detected more commonly and in greater amounts among EPEC than among other fecal E. coli. These results suggest a role for Shiga-like cytotoxin in the pathogenesis of EPEC-related gastroenteritis.     

Enterotoxin and cytotoxin production by enteroinvasive Escherichia coli.

It has long been suspected that besides their ability to invade enterocytes, enteroinvasive Escherichia coli (EIEC) strains have the ability to elaborate an enterotoxin. We tested 35 EIEC strains for cytotoxins and 9 (1 per serogroup) for enterotoxins. All 35 strains exhibited low levels of Vero cell cytotoxins that are immunologically and genetically distinct from Shiga-like toxin I or II of enterohemorrhagic E. coli. Sterile supernatants and cell lysates of two EIEC strains were tested in rabbit ileal loops, and both stimulated moderate fluid accumulation (circa 0.5 ml/cm) without tissue damage; secretory activity was confirmed in Ussing chambers, where these two strains and the seven others tested significantly increased short circuit current without altering tissue conductance. Curing the 140-MDa invasiveness plasmid from an EIEC strain did not diminish enterotoxin production. Culture in minimal Fe2+ medium is necessary to detect expression of the enterotoxin which is circa 68 to 80 kDa in size and is distinct from the EIEC cytotoxin.     

Properties of an Escherichia coli cytotoxin.

Isoelectric focusing of a heat-labile cytotoxin of Escherichia coli H30 revealed the presence of two molecular variants, pI 7.2 and a comparatively small quantity of pI 6.8. Predominant component pI 7.2 had a molecular weight of 28,000, induced some fluid accumulation in rabbit ileal loops, and showed no morphological response in Y-1 cells but a strong cytotoxic effect on Vero cells.     

Lack of Shiga-like cytotoxin production by enteroinvasive Escherichia coli.

Enteroinvasive Escherichia coli has not been extensively studied for cytotoxin production. We evaluated 30 well-characterized enteroinvasive E. coli strains of all the known invasive serogroups from several geographic regions for their ability to produce Shiga-like cytotoxic activity assayed in a HeLa cell system. None of these strains produced cytotoxic activity that was neutralizable with antibody to Shiga-like toxin I or II.     

Experimental and Mathematical Models of Escherichia coli Plasmid Transfer In Vitro and In Vivo

Little is known about the factors that govern plasmid transfers in natural ecosystems such as the gut. The consistent finding by earlier workers that plasmid transfer in the normal gut can be detected only at very low rates, if at all, has given rise to numerous speculations concerning the presence in vivo of various inhibitors of plasmid transfer. Plasmids R1, R1drd-19, and pBR322 were studied in Escherichia coli K-12 and wild-type E. coli hosts in two experimental systems: (i) gnotobiotic mice carrying a synthetic indigenous microflora (F-strains) which resemble in their function the normal indigenous microflora of the mouse large intestine, and (ii) anaerobic continuous-flow cultures of indigenous large intestinal microflora of the mouse, which can simulate bacterial interactions observed in the mouse gut. Mathematical models were developed to estimate plasmid transfer rates as a measure of the “fertility,” i.e., of the intrinsic ability to transfer the plasmid under the environmental conditions of the gut. The models also evaluate the effects of plasmid segregation, reduction of the growth rates of plasmid-bearing bacterial hosts, repression of transfer functions, competition for nutrients, and bacterial attachment to the wall of the gut or culture vessel. Some confidence in the validity of these mathematical models was gained because they were able to reproduce a number of known phenomena such as the repression of fertility of the R1 plasmid, as well as known differences in the transmission and mobilization of the plasmids studied. Interpretation of the data obtained permitted a number of conclusions, some of which were rather unexpected. (i) Fertility of plasmid-bearing E. coli in the normal intestine was not impaired. The observed low rates of plasmid transfer in the normal gut can be explained on quantitative grounds alone and do not require hypothetical inhibitory mechanisms. (ii) Conditions for long-term spread and maintenance throughout human or animal populations of a diversity of conjugative and nonconjugative plasmids may be optimal among E. coli strains of low fertility, as are found among wild-type strains. (iii) E. coli strains carrying plasmid pBR322 plus R1drd-19 were impaired in their ability to transfer R1drd-19, but strains carrying pBR322 were significantly better recipients of R1drd-19 than a plasmid-free recipient E. coli. (iv) Long-term coexistence of plasmid-bearing and plasmid-free E. coli, in spite of undiminished fertility, appeared to be due to a detrimental effect of the plasmid on the growth rate of its host bacterium, rather than due to high rates of plasmid segregation. (v) Mathematical analysis of experimental data published by earlier investigators is consistent with the conclusion that plasmid transfer occurs consistently in the human gut, but that the resulting transconjugant E. coli populations are too small to be detected regularly with the culture methods used by earlier investigators. It is concluded that the long-term interactions observed were often the consequences of minor differences in parameters such as growth rates, fertility, rates of segregation, etc., which were too small to be detected except by precise mathematical analysis of long-term experiments, but which were nevertheless decisive determinants of the ultimate fates of the plasmids and their hosts.     

Membrane changes induced by exposure of Escherichia coli to human serum.

The effect of bactericidal concentrations of lysozyme-free human serum on parameters of membrane integrity has been studied in serum-susceptible and serum-resistant Escherichia coli strains. Serum treatment released all of the alkaline phosphatase from the periplasmic space of two rapidly serum-susceptible strains but did so at different rates. In contrast, no periplasmic enzyme was released from two serum-resistant strains or from one moderately susceptible smooth strain. Lysozyme-free serum and heat-inactivated serum released comparable amounts of 86Rb+ from preloaded cells at comparable rates, regardless of serum susceptibility. Serum decreased the rate of phospholipid biosynthesis in both serum-susceptible and serum-resistant strains. In susceptible but not in resistant strains, intracellular ATP pools were depleted after serum exposure. Outer membranes and cytoplasmic membranes were prepared from serum-treated E. coli, and assays for C3 and C5b-9(m) were performed. With rapidly susceptible strains, C3 deposition on the outer membrane without attachment of C5b-9(m) occurred during the short prekilling phase. Subsequent bacterial killing was accompanied by deposition of C5b-9(m), which was recovered with C3 exclusively in outer membrane fractions with increased density and by eventual total loss of recoverable cytoplasmic membranes. Minimal deposition of complement components, without accompanying cytoplasmic membrane loss, occurred with serum-resistant strains. Loss of recoverable cytoplasmic membrane was not due to the action of either serum or bacterial phospholipase A. The results raise the possibilities that C5b-9(m) primarily damages the outer membrane and that the bacteria themselves actively participate in the ensuing, as yet unclarified, metabolic reactions that finally lead to their death.     

Vero response to a cytotoxin of Escherichia coli.

A cytotoxin was found in culture filtrates of a number of Escherichia coli strains that differed from the known heat-stable and heat-labile enterotoxins of E. coli. It was cytotoxic for Vero but not for Y-1 or CHO cells, and its effect on Vero was distinctly different from that of heat-labile enterotoxin. It was labile to heat and antigenically different from heat-labile enterotoxin, and membrane filtration indicated a molecular weight of 10,000 to 30,000.     

Mannose-sensitive stimulation of human leukocyte chemiluminescence by Escherichia coli.

Escherichia coli organisms with mannose-sensitive adherence factors (adhesins) are known to associate with human peripheral leukocytes (WBCs) in vitro in the absence of serum. To determine whether the WBC respiratory burst is activated during the interaction with E. coli, WBC chemiluminescence was measured. E. coli with mannose-sensitive adhesins stimulated a sharp burst of chemiluminescence which peaked 15 to 30 min after the bacteria and WBCs were mixed. Stimulation of chemiluminescence could be abrogated by including 10 mM alpha-methyl-D-mannoside in the test suspension. The addition of alpha-methyl-D-mannoside up to 20 min after the E. coli and WBCs were combined caused a rapid decrease in chemiluminescence. E. coli stimulation of chemiluminescence could not be inhibited by pretreating the WBCs with purified type 1 pili (fimbriae). E. coli lacking mannose-sensitive adhesins failed to stimulate chemiluminescence. The results emphasize the importance of mannose-sensitive adhesins in the association of E. coli with WBCs and suggest that the E. coli-WBC interaction system may be a useful tool for studying the mechanisms involved in the activation of the respiratory burst during phagocytosis.     

Enterohemorrhagic Escherichia coli Colonization of Human Colonic Epithelium In Vitro and Ex Vivo

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect the in vivo situation.     

Aberrant Forms of Escherichia coli in Blood Cultures: In Vitro Reproduction of an In Vivo Observation

Aberrant filamentous forms of Escherichia coli were observed on direct Gram stain of blood cultures from a patient being treated with the beta-lactam antibiotic cephalexin. After the institution of an alternative antibiotic regimen which included a different cell wall-active agent, E. coli of normal morphology was detected in blood cultures for an additional 48 h. Filamentous forms of E. coli could be reproduced reliably in vitro by incubating the organism in Mueller-Hinton broth containing various concentrations of cephalexin. Both supra- and subinhibitory concentrations of cephalexin resulted in filament formation after 4 h of incubation, whereas 24 h of incubation yielded intact filaments at only a narrow range of subinhibitory concentrations of cephalexin. In vitro comparison of the ability of cephalexin, cephalothin, ampicillin, and gentamicin to cause filamentous forms of E. coli showed that cephalexin and cephalothin produced pure filament formation after 4 h of incubation at subinhibitory concentrations of as low as one-fourth the minimum inhibitory concentration of the antibiotic. Ampicillin was not associated with pure filament formation at concentrations below the minimum inhibitory concentration, and gentamicin produced no filaments at any concentration. The effect of preincubation of E. coli with subinhibitory concentrations of cephalexin on subsequent minimum inhibitory concentrations of ampicillin was examined in an effort to develop an explanation for the persistent sepsis exhibited by the patient. No diminution of the activity of ampicillin by preincubation with cephalexin could be demonstrated. Other possible clinical implications of filamentous forms of gram-negative bacilli are discussed.     

Specificity of mutational DNA sequence changes induced by X-rays in the cloned Escherichia coli crp gene

Plasmid DNA carrying the adenosine 3′,5′-cyclic monophosphate receptor protein (crp) gene of Escherichia coli was irradiated, in solution, with X-rays, and the mutations produced in the crp gene were assayed by transforming the recipient E. coli cells. Ninety-six mutant clones were isolated, and mutational changes were determined by DNA sequencing. Of the 92 mutations thus detected, 74 represented base substitution mutations and the remaining 18 were frameshifts. The base substitutions included 56 G:C to A:T transitions, 10 G:C to T:A transversions and 7 G:C to C:G transversions. An A:T to G:C transition was found only once, and neither an A:T to T:A nor an A:T to C:G transversion was detected. The frameshift mutations consisted of 11 one-base deletions and 7 one-base insertions. Accordingly, G:C to A:T transition was the predominant type of mutation, which constituted 76% (56/74) of the total base substitutions and 60% (56/92) of all detected mutations. Furthermore, of the 56 transitions, about three-quarters (41 clones) clustered at an indentical site, a cytosine residue at the 706 position, demonstrating that this site is a distinct hot spot for X-ray mutagenesis. These results raise the possibility that radiation-induced mutations may not necessarily occur randomly, at least in certain cases.     

Clinical and pathological variability of infection by enterohaemorrhagic (Vero cytotoxin producing) Escherichia coli.

The clinical and pathological features of five sporadic cases of enteric infection caused by Escherichia coli O157 (enterohaemorrhagic or Vero cytotoxin-producing E coli showed a range of features. These included one case with pseudomembranous colitis, one with an acute exacerbation of ulcerative colitis, and three with enterocolitis. Diagnostic difficulties encountered initially in four of the five cases were finally resolved by correlating the results of microbiological with histopathological investigations. In view of the heterogeneity of clinical and histological signs and symptoms, it is concluded that all patients with abdominal pain and diarrhoea or rectal bleeding should have early microbiological investigation.     

Global changes in gene expression in Escherichia coli K12 induced by bacteriophage Mu Gem protein.

We have studied the growth properties of some Mu lysogens with respect to the non-lysogenic strain and have observed that the division time in minimal medium was increased over 4-fold when the bacteria carried the prophage mutated in the gem gene (Mu gem3). Since this phage gene has previously been shown to be involved in modulation of expression of host genes, we have analysed the proteins extracted from lysogens and non-lysogens as a rapid assay of global gene expression. The pattern of proteins extracted showed marked quantitative variations between non-lysogens, lysogens for wild-type Mu and lysogens for phage Mu gem3. These effects were no longer as evident when the strains were grown in rich medium. This dramatic change in the physiological state of the lysogenic strain versus the non-lysogenic in particular growth conditions extends the concept of lysogeny. For many years, the prophage has been considered only as a potentially lethal factor, while here it also appears as a genetic element capable of profoundly modifying host biology.