Infect Immun

Effect of prior experimental human enteropathogenic Escherichia coli infection on illness following homologous and heterologous rechallenge.

Donnenberg MS. Tacket CO. Losonsky G. Frankel G. Nataro JP. Dougan G. Levine MM.
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201, USA. mdonnenb@umabnet.ab.umd.edu
Two studies of adult volunteers were performed to determine whether prior enteropathogenic Escherichia coli (EPEC) infection confers protective immunity against rechallenge. In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain. In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic eae deletion mutant of that strain were challenged with the homologous wild-type strain. There was no significant effect of prior infection on the incidence of diarrhea in either study. However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain. Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide. These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge. Further studies may require the development of new model systems.

Susceptibility and serologic response of healthy adults to reinfection with Cryptosporidium parvum.

Okhuysen PC. Chappell CL. Sterling CR. Jakubowski W. DuPont HL.
Division of Infectious Diseases, The University of Texas Medical School and Center for Infectious Diseases, Houston 77030, USA. Okhuysen@heart.med.uth.tmc.edu
Healthy adults are susceptible to infection with small numbers of Cryptosporidium parvum oocysts, resulting in self-limited infection. We investigated if infection of humans with C. parvum is protective 1 year after primary exposure. At 1 year after a primary challenge with 30 to 10(6) oocysts, 19 healthy immunocompetent adults were rechallenged with 500 oocysts and monitored for the development of infection and/or illness. Oocyst excretion was quantitated by direct immunofluorescence with a C. parvum-specific monoclonal antibody, and anti-C. parvum antibodies in serum were detected by an enzyme-linked immunosorbent assay. Fewer subjects shed oocysts after the second exposure (3 of 19; 16%) than after the first exposure (12 of 19; 63%) (P < 0.005). Although the rates of diarrhea were comparable after each of the two exposures, the clinical severity as determined by the mean number of unformed stools passed was lower after reexposure (11.25 versus 8.62; P < 0.05). The number of anti-Cryptosporidium immunoglobulin G and A seroconversions increased after secondary exposure. However, the C. parvum serum antibody response did not correlate with the presence or absence of infection.

Diffusely adhering Escherichia coli strains induce attaching and effacing phenotypes and secrete homologs of Esp proteins.

Beinke C. Laarmann S. Wachter C. Karch H. Greune L. Schmidt MA.
Institut fur Infektiologie, Zentrum fur Molekularbiologie der Entzundung, Westfalische Wilhelms-Universitat, Munster, Germany.
Recent epidemiological studies indicate that Escherichia coli strains which exhibit the diffuse-adherence phenotype (DAEC strains) represent a potential cause of diarrhea in infants. We investigated the interaction of DAEC strains isolated from diarrhea patients in Brazil and in Germany with epithelial cells in tissue culture. The investigated strains were identified as DAEC strains by (i) their attachment pattern, (ii) presence of genes associated with the Dr family of adhesins, and (iii) lack of genetic markers for other diarrhea-associated E. coli categories. Several clinical DAEC isolates were shown to secrete similar patterns of proteins into tissue culture medium. Protein secretion was found to be regulated by environmental parameters, namely, medium, temperature, pH, and iron concentration. DAEC strains secreting these proteins induced accumulation of actin and tyrosine-phosphorylated proteins at sites of bacterial attachment, leading to the formation of pedestals and/or extended surface structures. These changes were phenotypically similar to the attaching and effacing (A/E) lesions observed with enteropathogenic and some enterohemorrhagic E. coli strains carrying the locus of enterocyte effacement (LEE) pathogenicity island. Proteins homologous to the EspA, EspB, and EspD proteins, necessary for signal transduction events inducing A/E lesions, were identified by sequence analysis and cross-reaction of specific antibodies. However, initially nonadhering strains secreting these proteins induced signal transduction events only after prolonged infection. These results indicate that secretion of the Esp proteins alone is not sufficient for efficient signal transduction. This study further shows that some DAEC strains are likely to contain a homolog(s) of the LEE locus which may contribute to the pathogenic potential of DAEC.

Secretory immune response to membrane antigens during Giardia lamblia infection in humans.

Rosales-Borjas DM. Diaz-Rivadeneyra J. Dona-Leyva A. Zambrano-Villa SA. Mascaro C. Osuna A. Ortiz-Ortiz L.
Grupo de Bioquimica y Parasitologia Molecular, Instituto de Biotecnologia, Universidad de Granada, Spain.
The secretory immune response in humans infected with Giardia lamblia was studied by using saliva samples and a membrane-rich protein fraction. The membrane fraction, studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed 24 antigen bands, ranging from 170 to 14 kDa. Saliva samples from giardiasis patients showed a heterogeneous response against the membrane fraction when they were assayed by immunoblotting. Among the antigens recognized by patient saliva samples, those of 170, 105, 92, 66, 32, 29, and 14 kDa stood out. These antigens were not recognized by saliva samples from healthy individuals. They may be of importance in future studies of protection from or diagnosis of G. lamblia infections.

Siderophore production by cystic fibrosis isolates of Burkholderia cepacia.

Darling P. Chan M. Cox AD. Sokol PA.
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Alberta, Canada.
Sixty-one Burkholderia cepacia isolates from patients with cystic fibrosis (CF) and four plant isolates were screened for production of the siderophores salicylic acid (SA), pyochelin, cepabactin, and ornibactins and fingerprinted by a PCR-based randomly amplified polymorphic DNA (RAPD) method. Of the 24 RAPD types determined, 22 (92%) were associated with isolates that produced SA, 21 (87%) were associated with isolates that produced ornibactins, 15 (60%) were associated with isolates that produced pyochelin, and 3 (12%) were associated with isolates that produced cepabactin. Of the 24 RAPD types plus 2 phenotypic variants of types 1 and 9, 3 were associated with isolates that produced all four siderophores, 8 were associated with isolates that produced three siderophores, 12 were associated with isolates that produced two siderophores, and 3 were associated with isolates that produced only one siderophore. These results suggest that the numbers and types of siderophores produced by CF isolates of B. cepacia correlate with RAPD type and that SA and ornibactins are the most prevalent siderophores produced.

Signal transduction pathways involved in enterohemorrhagic Escherichia coli-induced alterations in T84 epithelial permeability.

Year 1998
Philpott DJ. McKay DM. Mak W. Perdue MH. Sherman PM.
Department of Pediatrics, University of Toronto, Ontario, Canada.
Enterohemorrhagic Escherichia coli (EHEC) infection is associated with watery diarrhea and can lead to complications, including hemorrhagic colitis and the hemolytic-uremic syndrome. The mechanisms by which these organisms produce diarrheal disease remain to be elucidated. Changes in T84 epithelial cell electrophysiology were examined following EHEC infection. T84 cell monolayers infected with EHEC O157:H7 displayed a time-dependent decrease in transepithelial resistance. Increases in the transepithelial flux of both [3H]mannitol and 51Cr-EDTA accompanied the EHEC-induced decreases in T84 resistance. Altered barrier function induced by EHEC occurred at the level of the tight junction since immunofluorescent staining of the tight-junction-associated protein ZO-1 was disrupted when examined by confocal microscopy. Decreased resistance induced by EHEC involved a protein kinase C (PKC)-dependent pathway as the highly specific PKC inhibitor, CGP41251, abrogated the EHEC-induced drop in resistance. PKC activity was also increased in T84 cells infected with EHEC. Calmodulin and myosin light chain kinase played a role in EHEC-induced resistance changes as inhibition of these effector molecules partially reversed the effects of EHEC on barrier function. These studies demonstrate that intracellular signal transduction pathways activated following EHEC infection link the increases in T84 epithelial permeability induced by this pathogen.

Divergent signal transduction responses to infection with attaching and effacing Escherichia coli.

Year 1998
Ismaili A. McWhirter E. Handelsman MY. Brunton JL. Sherman PM.
Research Institute, The Hospital for Sick Children, Department of Medical Genetics, University of Toronto, Ontario, Canada.
Shiga toxin-producing Escherichia coli (STEC) O157:H7 is an attaching and effacing pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. Although this organism causes adhesion pedestals, the cellular signals responsible for the formation of these lesions have not been clearly defined. We have shown previously that STEC O157:H7 does not induce detectable tyrosine phosphorylation of host cell proteins upon binding to eukaryotic cells and is not internalized into nonphagocytic epithelial cells. In the present study, tyrosine-phosphorylated proteins were detected under adherent STEC O157:H7 when coincubated with the non-intimately adhering, intimin-deficient, enteropathogenic E. coli (EPEC) strain CVD206. The ability to be internalized into epithelial cells was also conferred on STEC O157:H7 when coincubated with CVD206 ([158 +/- 21] % of control). Neither the ability to rearrange phosphotyrosine proteins nor that to be internalized into epithelial cells was evident following coincubation with another STEC O157:H7 strain or with the nonsignaling espB mutant of EPEC. E. coli JM101(pMH34/pSSS1C), which overproduces surface-localized O157 intimin, also rearranged tyrosine-phosphorylated and cytoskeletal proteins when coincubated with CVD206. In contrast, JM101 (pMH34/pSSS1C) demonstrated rearrangement of cytoskeletal proteins, but not tyrosine-phosphorylated proteins, when coincubated with intimin-deficient STEC (strains CL8KO1 and CL15). These findings indicate that STEC O157:H7 forms adhesion pedestals by mechanisms that are distinct from those in attaching and effacing EPEC. Taken together, these findings point to diverging signal transduction responses to infection with attaching and effacing bacterial enteropathogens.

Pseudomonas aeruginosa lasR transcription correlates with the transcription of lasA, lasB, and toxA in chronic lung infections associated with cystic fibrosis.

Year 1998
Storey DG. Ujack EE. Rabin HR. Mitchell I.
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4. storey@acs.ucalgary.ca
The role of Pseudomonas aeruginosa quorum-sensing systems in the lung infections associated with cystic fibrosis (CF) has not been examined. The purpose of this study was to determine if genes regulated by the LasR-LasI quorum-sensing system were coordinately regulated by the P. aeruginosa populations during the lung infections associated with CF. We also wanted to ascertain if there was a relationship between the expression of lasR, a transcriptional regulator, and some P. aeruginosa virulence factors during these infections. We extracted RNAs from the bacterial populations of 131 sputa taken from 23 CF patients. These RNAs were blotted and hybridized with probes to P. aeruginosa lasA, lasB, and toxA. The hybridization signals from each probe were ranked, and the rankings were analyzed by a Spearman rank correlation to determine if there was an association between the population transcript accumulations for the three genes. The correlations between the transcript accumulation patterns of pairs of the genes suggested that lasA, lasB, and toxA might be coordinately regulated during CF lung infections. To determine if this coordinate regulation might be due to regulation by LasR, we probed RNAs, extracted from 84 sputa, with the lasR, lasA, lasB, toxA, and algD probes. Statistical analysis indicated that lasR transcript accumulation correlated to lasA, lasB, toxA, and algD transcript accumulations. These results indicated that lasR may at least partially regulate or be coordinately regulated with lasA, lasB, toxA, and algD during the lung infections associated with CF. These results also suggested that the LasR-LasI quorum-sensing system may control the expression of at least some virulence factors in the lungs of patients with CF.

Antibody-secreting cells in the stomachs of symptomatic and asymptomatic Helicobacter pylori-infected subjects.

Year 1998
Mattsson A. Quiding-Jarbrink M. Lonroth H. Hamlet A. Ahlstedt I. Svennerholm A.
Department of Medical Microbiology, Goteborg University, Goteborg, Sweden.
In this study we analyzed whether infection with Helicobacter pylori gives rise to specific B-cell responses against a number of putative virulence factors of H. pylori, e.g., urease, flagellin, and different bacterial surface antigens, locally in the gastric mucosa. This was studied in antrum and corpus biopsies collected from 11 H. pylori-infected patients with duodenal ulcers, 11 asymptomatic H. pylori carriers, and 13 noninfected, healthy controls. Mononuclear cells were isolated from the biopsies and assayed for frequencies of total and H. pylori-specific antibody-secreting cells (ASCs) by means of the enzyme-linked immunospot technique. The H. pylori-infected subjects had remarkably higher frequencies of total immunoglobulin A (IgA)- and IgM-secreting cells than the noninfected subjects, while the frequencies of IgG-secreting cells were virtually the same in the different groups. In addition, most of the infected subjects had IgA ASCs reacting with H. pylori membrane proteins, flagellin, and urease, while none of the noninfected subjects had any detectable H. pylori-reactive ASCs. Furthermore, half of the infected subjects also had ASCs reacting with a Helicobacter-specific 26-kDa protein, while only a few of them had ASCs reacting with neutrophil-activating protein, the neuraminyllactose-binding hemagglutinin HpaA, or lipopolysaccharides purified from different H. pylori strains. The frequencies of H. pylori-specific ASCs in the antrum and corpus were almost identical, and no differences in either antigen specificity or magnitude of the B-cell response in the stomach could be detected between the ulcer patients and the asymptomatic H. pylori carriers. This study demonstrates that H. pylori infection induces strong antibody responses in the human gastric mucosa, both in asymptomatic carriers and in duodenal ulcer patients. However, the outcome of infection could not be explained by differences in the local B-cell response to any of the antigens used in this study.

Pseudomonas aeruginosa exoenzyme S is a mitogen but not a superantigen for human T lymphocytes.

Year 1998
Bruno TF. Buser DE. Syme RM. Woods DE. Mody CH.
Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.
Virtually all cystic fibrosis (CF) patients become infected with Pseudomonas aeruginosa, and once the infection is established, the organism is rarely cleared. One of the P. aeruginosa virulence factors, exoenzyme S, has been shown to correlate with increased morbidity and mortality both in rat models of chronic pulmonary inflammation and in human CF patients. It has previously been shown that exoenzyme S is a potent stimulus for the proliferation of T cells in greater than 95% of adults, which could contribute to the pathogenesis of CF. The goal of this study was to determine the mechanism of T-cell stimulation by exoenzyme S in an effort to shed light on the immune response and contribute to understanding its role in P. aeruginosa pathogenesis. The current studies demonstrate that exoenzyme S stimulates naive T cells, since fetal blood lymphocytes proliferated and adult lymphocytes that expressed CD45RA proliferated. The percentage of T cells activated by exoenzyme S after a 4-h culture (as measured by CD69 surface expression) was intermediate in magnitude compared to levels induced by a panel of superantigens and mitogens. To determine the mechanism of activation, the requirement for accessory cells was investigated. The proliferative response to exoenzyme S was dependent on the presence of accessory cells but was not blocked by an anti-DR antibody. Exoenzyme S activated both CD4(+) and CD8(+) T cells, but CD4(+) T cells were preferentially activated. The Vbeta repertoire of donor T cells showed no preferential activation or preferential expansion after stimulation by exoenzyme S, suggesting that it is not a superantigen. Taken together, our data suggest that exoenzyme S is a T-cell mitogen but not a superantigen. Activation of a large percentage of T lymphocytes by exoenzyme S may produce a lymphocyte-mediated inflammatory response that should be considered in the pathogenesis of CF.

Burkholderia cepacia produces a hemolysin that is capable of inducing apoptosis and degranulation of mammalian phagocytes.

Year 1998
Hutchison ML. Poxton IR. Govan JR.
Department of Medical Microbiology, University of Edinburgh Medical School, Scotland. Mikeh@srv1.med.ed.ac.uk
Burkholderia cepacia is an opportunistic pathogen that has become a major threat to individuals with cystic fibrosis (CF). In approximately 20% of patients, pulmonary colonization with B. cepacia leads to cepacia syndrome, a fatal fulminating pneumonia sometimes associated with septicemia. It has been reported that culture filtrates of clinically derived strains of B. cepacia are hemolytic. In this study, we have characterized a factor which contributes to this hemolytic activity and is secreted from B. cepacia J2315, a representative of the virulent and highly transmissible strain belonging to the recently described genomovar III grouping. Biochemical data from the described purification method for this hemolysin allows us to hypothesize that the toxin is a lipopeptide. As demonstrated for other lipopeptide toxins, the hemolysin from B. cepacia was surface active and lowered the surface tension of high-pressure liquid chromatography-grade water from 72.96 to 29.8 mN m(-1). Similar to reports for other pore-forming cytotoxins, low concentrations of the hemolysin were able to induce nucleosomal degradation consistent with apoptosis in human neutrophils and the mouse-derived macrophage-type cell line J774.2. Exposure of human neutrophils to higher concentrations of toxin resulted in increased activities of the neutrophil degranulation markers cathepsin G and elastase. Based on the results obtained in this study, we suggest a role that allows B. cepacia to thwart the immune response and a model of the events that may contribute to the severe inflammatory response in the lungs of CF patients.

Characterization of the roles of hemolysin and other toxins in enteropathy caused by alpha-hemolytic Escherichia coli linked to human diarrhea.

Year 1998
Elliott SJ. Srinivas S. Albert MJ. Alam K. Robins-Browne RM. Gunzburg ST. Mee BJ. Chang BJ.
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore 21201, USA. selliot@umaryland.edu
Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.

Cryptosporidium parvum infection of human intestinal xenografts in SCID mice induces production of human tumor necrosis factor alpha and interleukin-8.

Year 1998
Seydel KB. Zhang T. Champion GA. Fichtenbaum C. Swanson PE. Tzipori S. Griffiths JK. Stanley SL Jr.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
The protozoan parasite Cryptosporidium parvum invades intestinal epithelial cells and can cause life-threatening diarrhea in immunocompromised individuals. Despite the clinical importance of this organism, much remains to be learned about the pathogenesis of C. parvum-induced diarrhea. To explore the role of the intestinal inflammatory response in C. parvum disease, using C. parvum oocysts we infected human intestinal xenografts in severe combined immunodeficient (SCID) mice. Seven days after infection, we found levels of human tumor necrosis factor alpha and interleukin-8 in C. parvum-infected human intestinal xenografts that were significantly higher than those seen in uninfected control xenografts. These results demonstrate that human intestinal cells produce proinflammatory cytokines in response to C. parvum infection and establish SCID-HU-INT mice as a model system to study the interactions of C. parvum with the human intestine.