Protective efficacy and pharmacokinetics of human/mouse chimeric anti-Stx1 and anti-Stx2 antibodies in mice.

In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.

Enhanced virulence of the Escherichia coli O157:H7 spinach-associated outbreak strain in two animal models is associated with higher levels of Stx2 production after induction with ciprofloxacin.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). STEC strains may produce Stx1a and/or Stx2a or variants of either toxin. A 2006 spinach-associated outbreak of STEC O157:H7 resulted in higher hospitalization and HUS rates than previous STEC outbreaks. The spinach isolate, strain K3995, contains both stx2a and stx2c. We hypothesized that the enhanced virulence of K3995 reflects the combination of stx2 alleles (carried on lysogenic phages) and/or the amount of Stx2 made by that strain. We compared the virulence of K3995 to those of other O157:H7 isolates and an isogenic Stx2 mutant in rabbits and mice. We also measured the relative levels of Stx2 produced from those strains with or without induction of the stx-carrying phage. Some rabbits infected with K3995 exhibited intestinal pathology and succumbed to infection, while none of those infected with O157:H7 strain 2812 (Stx1a(+) Stx2a(+)) died or showed pathological signs. Rabbits infected with the isogenic Stx2a mutant K3995 stx2a::cat were not colonized as well as those infected with K3995 and exhibited no signs of disease. In the streptomycin-treated mouse model, more animals infected with K3995 died than did those infected with O157:H7 strain 86-24 (Stx2a(+)). Additionally, K3995 produced higher levels of total Stx2 and toxin phage DNA in cultures after phage induction than did 86-24. Our results demonstrate the greater virulence of K3995 compared to other O157:H7 strains in rabbits and mice. We conclude that this enhanced virulence is linked to higher levels of Stx2 expression as a consequence of increased phage induction.

Oral intoxication of mice with Shiga toxin type 2a (Stx2a) and protection by anti-Stx2a monoclonal antibody 11E10.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB5 toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD50) of 2.9 µg, but no morbidity occurred after oral intoxication with up to 157 µg of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.

Helmet-Cam: tool for assessing miners' respirable dust exposure.

Video technology coupled with datalogging exposure monitors have been used to evaluate worker exposure to different types of contaminants. However, previous application of this technology used a stationary video camera to record the worker's activity while the worker wore some type of contaminant monitor. These techniques are not applicable to mobile workers in the mining industry because of their need to move around the operation while performing their duties. The Helmet-Cam is a recently developed exposure assessment tool that integrates a person-wearable video recorder with a datalogging dust monitor. These are worn by the miner in a backpack, safety belt or safety vest to identify areas or job tasks of elevated exposure. After a miner performs his or her job while wearing the unit, the video and dust exposure data files are downloaded to a computer and then merged together through a NIOSH-developed computer software program called Enhanced Video Analysis of Dust Exposure (EVADE). By providing synchronized playback of the merged video footage and dust exposure data, the EVADE software allows for the assessment and identification of key work areas and processes, as well as work tasks that significantly impact a worker's personal respirable dust exposure. The Helmet-Cam technology has been tested at a number of metal/nonmetal mining operations and has proven to be a valuable assessment tool. Mining companies wishing to use this technique can purchase a commercially available video camera and an instantaneous dust monitor to obtain the necessary data, and the NIOSH-developed EVADE software will be available for download at no cost on the NIOSH website.

Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia.

Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ΔAmes strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies.

Shiga Toxins: Potent Poisons, Pathogenicity Determinants, and Pharmacological Agents.

The Shiga toxins (Stxs), also known as Vero toxins and previously called Shiga-like toxins, are a family of potent protein synthesis inhibitors made by Shigella dysenteriae type 1 and some serogroups of Escherichia coli that cause bloody diarrhea in humans. Stxs act as virulence factors for both S. dysenteriae and E. coli and contribute to the disease process initiated by those organisms both directly and indirectly. A handful of methods exist for toxin purification, and the toxins can now even be purchased commercially. However, the Stxs are now classified as select agents, and specific rules govern the distribution of both the toxin and clones of the toxin. Toxin delivery into the host in S. dysenteriae type 1 is most likely aided by the invasiveness of that organism. Although the Stxs are made and produced by bacteria, they do not appear to act against either their host organism or other bacteria under normal circumstances, most likely because the A subunit is secreted from the cytoplasm as soon as it is synthesized and because the holotoxin cannot enter intact bacterial cells. The effectiveness of antibiotic therapy in patients infected with Stx-producing E. coli (STEC) such as O157:H7 as well as the potential risks of such treatment are areas of controversy. Several studies indicate that the course of the diarrhea stage of the disease is unaltered by antibiotic treatment. Several groups anticipate that a therapy that targets the Stxs is an important component of trying to alleviate disease caused by Stx-producing bacteria.

Cytotoxic necrotizing factor type 1-positive Escherichia coli causes increased inflammation and tissue damage to the prostate in a rat prostatitis model.

Infection of rat prostates with cytotoxic necrotizing factor type 1 (CNF1)-positive uropathogenic Escherichia coli caused more inflammation-mediated morphological and histological tissue damage than did infection with isogenic CNF1-negative mutants. These striking differences occurred despite the finding that bacterial counts for the strain pairs were indistinguishable. We conclude that CNF1 contributes to E. coli virulence in a model of acute prostatitis. To our knowledge, the results of this study provide the first demonstration of a role for any uropathogenic E. coli virulence factor in acute prostatitis.

Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis.

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.

Mutation of the gene encoding cytotoxic necrotizing factor type 1 (cnf(1)) attenuates the virulence of uropathogenic Escherichia coli.

Cytotoxic necrotizing factor type 1 (CNF1) is a 115-kDa toxin that activates Rho GTPases and is produced by uropathogenic Escherichia coli (UPEC). While both epidemiological studies that link CNF1 production by E. coli with urinary tract disease and the cytopathic effects of CNF1 on cultured urinary tract cells are suggestive of a role for the toxin as a UPEC virulence factor, few in vivo studies to test this possibility have been reported. Therefore, in this investigation, we evaluated the importance of CNF1 in a murine model of urinary tract infection (UTI) by comparing the degree of colonization and damage induced by three different CNF1-producing E. coli strains with isogenic CNF1-deficient derivatives. The data from single-strain challenge experiments with C3H/HeOuJ mice indicated a trend toward higher counts of the wild-type strains in the urine and bladders of these animals up to 3 days after challenge in two of three strain pairs. Furthermore, this difference was statistically significant at day 2 of infection with one strain pair, C189 and C189cnf(1). To control for the animal-to-animal variability inherent in this model, we infected C3H/HeOuJ mice with a mixture of CNF1-positive and -negative isogenic derivatives of CP9. The CNF1-positive strain was recovered in higher numbers than the CNF1-negative strain in the urine, bladders, and kidneys of the mice up to 9 days postinfection. These striking coinfection findings, taken with the trends observed in single-strain infections, led us to conclude that CNF1-negative strains were generally attenuated compared to the wild type in the C3H/HeOuJ mouse model of UTI. Furthermore, histopathological examination of bladder specimens from mice infected with CNF1-positive strains consistently showed deeper, more extensive inflammation than in those infected with the isogenic mutants. Lastly, we found that CNF1-positive strain CP9 was better able to resist killing by fresh human neutrophils than were CP9cnf(1) bacteria. From these data in aggregate, we propose that CNF1 production increases the capacity of UPEC strains to resist killing by neutrophils, which in turn permits these bacteria to gain access to deeper tissue and persist better in the lower urinary tract.

Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis.

Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC(+) phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

Epitope mapping of monoclonal antibodies capable of neutralizing cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli.

Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.

Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response.

This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.

Cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli kills cultured human uroepithelial 5637 cells by an apoptotic mechanism.

Pathogenic Escherichia coli associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of E. coli-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with uropathogenic E. coli.

Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.

Evidence derived from human and animal studies strongly supports the notion that dysfunctional alveolar epithelial cells (AECs) play a central role in determining the progression of inflammatory injury to pulmonary fibrosis. We formed the hypothesis that impaired production of the regulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) by injured AECs plays a role in the development of pulmonary fibrosis. To test this hypothesis, we used the well-characterized model of bleomycin-induced pulmonary fibrosis in rats. GM-CSF mRNA is expressed at a constant high level in the lungs of untreated or saline-challenged animals. In contrast, there is a consistent reduction in expression of GM-CSF mRNA in the lung during the first week after bleomycin injury. Bleomycin-treated rats given neutralizing rabbit anti-rat GM-CSF IgG develop increased fibrosis. Type II AECs isolated from rats after bleomycin injury demonstrate diminished expression of GM-CSF mRNA immediately after isolation and in response to stimulation in vitro with endotoxin compared with that in normal type II cells. These data demonstrate a defect in the ability of type II epithelial cells from bleomycin-treated rats to express GM-CSF mRNA and a protective role for GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.

Escherichia coli O157:H7 causes more-severe systemic disease in suckling piglets than in colostrum-deprived neonatal piglets.

Our objective was to determine if suckling neonatal piglets are susceptible to enterohemorrhagic Escherichia coli (EHEC) O157:H7 disease. Surprisingly, EHEC O157:H7 caused more-rapid and more-severe neurological disease in suckling neonates than in those fed an artificial diet. Shiga toxin-negative O157:H7 did not cause neurological disease but colonized and caused attaching-and-effacing intestinal lesions.

Elastase in intestinal mucus enhances the cytotoxicity of Shiga toxin type 2d.

Shiga toxin variant type 2d (Stx2d) produced by some strains of Shiga toxin-producing Escherichia coli is composed of an enzymatically active A subunit and a B (binding) pentamer. The cytotoxicity of Stx2d is increased (activated) 10-1000-fold for Vero cells when the toxin is incubated with mucus obtained from the small intestine of mice. In this study we isolated an Stx2d activator and identified it as a mouse elastase with strong homology to human elastase IIIB. Moreover, commercially available porcine pancreatic elastase preparations also activated Stx2d cytotoxicity although with a lower specific activity than isolated mouse elastase. Elastase directly nicked the Stx2d A subunit to A(1) and A(2), an event that did not correlate with activation. However, elastase also reduced the size and changed the isoelectric point of the A(2) peptide, as determined by SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis followed by Western immunoblot analysis. This elastase-mediated size and charge shift in the A(2) peptide of Stx2d occurred concurrently with activation of the toxin. Both the reduction in size of the Stx2d A(2) peptide by incubation with elastase as well as the associated activation of Stx2d cytotoxicity were fully inhibited by elastatinal, an elastase-specific inhibitor.

Decreased adherence of enterohemorrhagic Escherichia coli to HEp-2 cells in the presence of antibodies that recognize the C-terminal region of intimin.

Antiserum raised against intimin from enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 has been shown previously by our laboratory to inhibit adherence of this strain to HEp-2 cells. In the present study, we sought to identify the region(s) of intimin important for the effect of anti-intimin antisera on EHEC adherence and to determine whether antisera raised against intimin from an O157:H7 strain could reduce adherence of other strains. Compared to preimmune serum controls, polyclonal sera raised against the histidine-tagged intimin protein RIHisEae (intimin(O157)) or against His-tagged C-terminal fragments of intimin from strain 86-24 reduced adherence of this strain. Furthermore, an antibody fraction purified from the anti-RIHisEae serum that contained antibodies to the C-terminal third of intimin, the putative receptor-binding domain, also reduced adherence of strain 86-24, but a purified fraction containing antibodies to the N-terminal two-thirds of intimin did not inhibit adherence. The polyclonal anti-intimin(O157) serum raised against RIHisEae inhibited, to different degrees, the adherence of another O157:H7 strain, an EHEC O55:H7 strain, one of two independent EHEC O111:NM isolates tested, and one of two EHEC O26:H11 strains tested. Adherence of the other O26:H11 and O111:NM strains and an EPEC O127:H6 strain was not reduced. Finally, immunoblot analysis indicated a correlation between the antigenic divergence in the C-terminal third of intimins from different strains and the capacity of anti-intimin(O157) antiserum to reduce adherence of heterologous strains. Taken together, these data suggest that intimin(O157) could be used as an immunogen to elicit adherence-blocking antibodies against O157:H7 strains and closely-related EHEC.

Role of alveolar epithelial cell intercellular adhesion molecule-1 in host defense against Klebsiella pneumoniae.

Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells (AEC) in the normal alveolar space. We postulate that AEC ICAM-1 enhances the antimicrobial activity of macrophages and neutrophils in the alveolar space. Wild-type and mutant mice deficient in ICAM-1 were inoculated intratracheally with Klebsiella pneumoniae. After 10 days, 43% of the ICAM-1 mutant mice had died compared with 14% of the wild-type controls (P = 0.003). Significantly more bacteria were isolated from lungs of ICAM-1 mutant mice than controls 24 h after inoculation (log colony-forming units 5.14 +/- 0.21 vs. 3.46 +/- 0. 16, P = 0.001). However, neutrophil recruitment to the lung was not different. In similar experiments in the rat, inhibition of alveolar ICAM-1 by intratracheal administration of antibody resulted in significantly impaired clearance of K. pneumoniae. The role of phagocyte interactions with AEC ICAM-1 for antimicrobial activity was investigated in vitro using primary cultures of rat AEC that express abundant ICAM-1. Alveolar macrophage phagocytosis and killing of K. pneumoniae were increased significantly in the presence of AEC; these effects were inhibited significantly (47.5 and 52%, respectively) when AEC ICAM-1 was blocked. Similarly, neutrophil phagocytic activity for K. pneumoniae in the presence of AEC in vitro was decreased when ICAM-1 on the AEC surface was blocked. Thus in the absence of ICAM-1, there is impaired ability to clear K. pneumoniae from the lungs, resulting in increased mortality. These studies indicate that AEC ICAM-1 plays an important role in host defense against K. pneumoniae by determining the antimicrobial activity of phagocytes within the lung.

Bacterial toxins: friends or foes?

Many emerging and reemerging bacterial pathogens synthesize toxins that serve as primary virulence factors. We highlight seven bacterial toxins produced by well-established or newly emergent pathogenic microbes. These toxins, which affect eukaryotic cells by a variety of means, include Staphylococcus aureus alpha-toxin, Shiga toxin, cytotoxic necrotizing factor type 1, Escherichia coli heat-stable toxin, botulinum and tetanus neurotoxins, and S. aureus toxic-shock syndrome toxin. For each, we discuss the information available on its synthesis and structure, mode of action, and contribution to virulence. We also review the role certain toxins have played in unraveling signal pathways in eukaryotic cells and summarize the beneficial uses of toxins and toxoids. Our intent is to illustrate the importance of the analysis of bacterial toxins to both basic and applied sciences.