Production of a unique cytotoxin by Campylobacter jejuni.

Campylobacter jejuni is an important diarrheal pathogen worldwide; the mechanisms by which it causes disease remain unclear. Because of its association with inflammatory diarrhea, we postulated that C. jejuni might produce a cytotoxin similar to that produced by Shigella sp., enterohemorrhagic Escherichia coli O157, or Clostridium difficile. Filtrates of 12 polymyxin-treated isolates of C. jejuni were placed on HeLa cells (sensitive to Shiga toxin cytotoxicity) and Chinese hamster ovary (CHO) cells. Of 12 isolates of C. jejuni tested, 5 killed 50% of the cells at greater than or equal to 1:4 dilutions of filtered suspensions of 10(9) bacteria per ml; killing was similar in HeLa and CHO cells (the CHO cells being insensitive to Shiga cytotoxin). One isolate produced a titer of 1:32 to 1:128. The relative potency in HeLa cells was comparable to that of E. coli strains that produce intermediate amounts of Shiga-like toxin. The other seven strains showed no cytotoxic effect, nor did the control diluents, polymyxin B, or supernatants of C. jejuni not treated with polymyxin B. Sonication also released active cytotoxin, but slightly less well than did polymyxin. The cytotoxic effect was dose dependent. Concentration of the C. jejuni in suspension by 10-fold before treatment with polymyxin B resulted in a 10-fold increase in the 50% cytotoxic dose. The cytotoxin effect was not neutralized by Shiga toxin immune serum against either Shiga-like toxin I or II or by anti-Clostridium difficile antiserum. The C jejuni cytotoxin was partially labile to trypsin (0.25%) and to heating to greater than or equal to 60 degrees C. Cytotoxicity was retained in Scientific Products dialysis tubing D1615-1 (Mr cutoff, 12,000 to 14,000). Some isolates of C. jejuni release a substance lethal to HeLa or CHO cells in vitro that is distinct from Shiga-like or Clostridium difficile toxin. This cytotoxin may contribute to the colonic mucosal invasive process that characterizes C. jejuni enteritis.     

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.     

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.     

Demonstration of a cytotoxin from Campylobacter jejuni

A 48-hour culture filtrate of Campylobacter jejuni was found to produce cytopathic effects on three human cell lines--that is, HeLa, MRC-5 and Hep-2. The cytopathic effects observed include cell rounding, loss of adherence and cell death after 24-48 h of incubation. Such morphological changes were observed with eight of the eleven strains of Campylobacter jejuni isolated from the blood/stools of patients who suffered from either acute gastroenteritis or septicaemia. The toxic factor did not retain its activity after treatment at 100 degrees C for 30 min. Trypsinisation of the filtrate totally abolished its toxic activity thus indicating that it was probably protein in nature. It is most probably an extracellular toxin as bacterial sonicates did not produce any toxic effect. This study reports the finding of toxic factor(s) in the culture filtrate of Campylobacter jejuni which is cytotoxic to human tissue culture cells.     

Production of a unique cytotoxin by Klebsiella oxytoca

Certain strains of Klebsiella oxytoca isolated from patients with hemorrhagic enterocolitis produced a unique cytotoxin. The cytotoxin induced rounding of tissue culture cells, such as HEp-2, Vero, CHO and HeLa cells. The induced morphologic changes were indistinguishable between cell types. Seventy to 80% of the rounded cells died in 48 h incubation. The cytotoxin was purified 1000-fold from culture supernatant by Sephadex G-25 and Bio-Gel P-2 gel filtration followed by reversed-phase high-performance liquid chromatography. The molecular weight of the purified cytotoxin was estimated to be less than or equal to 651 by mass spectrometry. The minimum concentrations of the purified cytotoxin required to cause 50% of rounding of cells were 0.6 micrograms/ml for HEp-2, 0.8 micrograms/ml for Vero, 0.8 micrograms/ml for CHO and 1.4 micrograms/ml for HeLa cells. The type strain of K. oxytoca, ATCC 13182, did not produce the cytotoxin and only the clinically isolated strains did, suggesting that the cytotoxin may play a role in the pathogenesis of the organisms.     

Production of enterotoxin and cytotoxin in Campylobacter jejuni strains isolated in Costa Rica.

The production of toxins by 79 strains of Campylobacter jejuni isolated in Costa Rica from children with campylobacter-induced diarrhoea (44 strains) and from chickens (35 strains) was studied. An enterotoxic effect giving a rounding of mouse adrenocortical tumour (Y1) cells, which could be neutralised with antitoxin against Escherichia coli heat-labile enterotoxin, was detected in supernates from 16 (62%) of 26 strains from children with watery diarrhoea, in 5 (28%) of 18 strains from children with bloody or inflammatory diarrhoea, and in 12 (34%) of the 35 strains from chickens. Cytotoxic effects in human lung fibroblasts (MRC-5), African Green monkey kidney (Vero) cells and human cervical carcinoma (HeLa) cells were observed in none of the 26 strains from children with watery diarrhoea, in 2 (11%) of the 18 strains from children with bloody or inflammatory diarrhoea, and in 6 (17%) of the 35 strains from chickens. The simultaneous production of enterotoxin and cytotoxin was detected in four strains. The cytotoxic effect, which was most prominent in cells freshly seeded at a low density, appeared as a lethal rounding of the cells. Fibroblasts were more sensitive than epithelial cells. The effects of the supernates were inactivated by heating at 100 degrees C for 10 min and decreased after 1 week at 4 degrees C. The production of toxins was lost after storage of the strains for one year at -70 degrees C.     

In-vitro and in-vivo studies of a cytotoxin from Campylobacter jejuni

Studies were performed on a cytotoxin (CT) from human strains of Campylobacter jejuni isolated in Malaysia. CT was detected by cytopathic effect (CPE) on HeLa cells at titres from 8 to 32, in culture filtrates from 14 (48%) of 29 human isolates. The CPE correlated well with a quantitative 51Cr-release assay where a specific release of 54-68% was noted. CT production was lost after 5-7 subcultures. CT activity was also detected in 5 (26%) of 19 faecal filtrates from which CT-producing isolates were subsequently obtained. The mol. wt of CT was estimated by Sephadex G-50 chromatography to be greater than 30,000. In a suckling-mouse assay, CT consistently failed to demonstrate fluid accumulation after intragastric inoculation of culture filtrate. The Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) assay was also used. Rabbits given CT-producing strains of C. jejuni developed bacteraemia and severe watery mucus-containing diarrhoea for the duration of the experiment with death of some animals. Rabbits given CT non-producing strains had less severe disease and none died. Rabbits given partially-purified CT had diarrhoea for 3 days but none died.     

Enterobacter cloacae producing a Shiga-like toxin II-related cytotoxin associated with a case of hemolytic-uremic syndrome.

Two Shiga-like toxin-producing organisms were isolated from the feces of an infant with hemolytic-uremic syndrome by PCR followed by colony blot hybridization. One strain was identified as Escherichia coli OR:H9, while the other was identified as Enterobacter cloacae. Both isolates were highly cytotoxic for Vero cells, and Southern hybridization analysis of chromosomal DNA indicated that both contained a single slt-II-related gene and that these genes were located on similarly sized restriction fragments. Nucleotide sequence analysis indicated that the toxin encoded by the E. cloacae slt-II-related gene was very similar to Shiga-like toxin II variants from E. coli, differing from the most closely related toxin by 3 residues in the A subunit.     

Production of an anti-tumour cytotoxin by human monocytes.

Human monocytes incubated in vitro for 20 hr at 37 degrees release a factor which is cytotoxic to a number of human and murine tumour cell lines: untransformed cells appear to be less susceptible. A similar factor is produced under comparable conditions by myelomonocytic leukaemic cells and by macrophages derived from monocytes by in vitro culture for 8 days. Maximum production of the factor occurred in the presence of foetal calf serum or autologous plasma and endotoxin. The factor is newly synthesized in culture as its production is reduced if the monocytes are treated with cycloheximide or actinomycin D or incubated at lower temperatures. Freshly isolated monocytes do not release the factor on freeze--thaw or hypotonic lysis. The monocyte cytotoxin has apparent molecular weights of 34,000 on Ultrogel AcA54 gel filtration and 140,000 on gradient polyacrylamide gel electrophoresis; it has beta 2--gamma 1 electrophoretic mobility in polyacrylamide gel and does not appear to be C3a or arginase.     

Isolation, characterization, and host-cell-binding properties of a cytotoxin from Campylobacter jejuni.

A 68,000-molecular-weight protein was isolated by polyacrylamide gel electrophoresis from the organism-free filtrate of a fully virulent clinical strain of Campylobacter jejuni. The eluted protein was heat labile, was inactivated at either pH 3.0 or 9.0, was sensitive to trypsin, and was lethal for fertile chicken eggs. It also had toxic effects on chicken embryo fibroblast, Chinese hamster ovary (CHO), and intestinal 407 (Int407) cells. A monoclonal antibody (CETPMAb4) raised to this eluted toxic protein (ETP) from C. jejuni abolished these toxic activities. Homology between C. jejuni ETP and Vibrio cholerae toxin was not observed in that specific antisera to each did not block their respective toxic activities. In enzyme-linked immunosorbent assays, ETP, unlike chlorea enterotoxin, did not bind to GM1 ganglioside. Furthermore, the C. jejuni toxin had cytotoxinlike properties and induced rounding of CHO cells. Binding of ETP to Int407 and primary chicken embryo fibroblast cells was maximal after 2 h as assessed by enzyme-linked immunosorbent assay, and this toxin adherence to host cell membranes was significantly reduced by prior treatment of the cells with proteolytic enzymes, neuraminidase, or glutaraldehyde but not by treatment with beta-galactosidase, lipase, Nonidet P-40, or sodium metaperiodate. In competitive binding assays, sugars, lectins, or GM1 ganglioside did not adversely influence uptake of ETP by these cells. These results suggest that the ETP produced by C. jejuni is a cytotoxin which binds to Int407 cells via a protein- or glycoproteinlike receptor on cell membranes and possesses properties dissimilar to those of V. cholerae toxin.     

Production and partial characterization of a Vibrio fluvialis cytotoxin.

Conditions are described for the production of an extracellular cytotoxin or CHO cell-killing factor by Vibrio fluvialis, a recently recognized enteric pathogen. The cell-killing factor was ammonium sulfate precipitable, heat labile, and inactivated by proteases, and had an isoelectric point (estimated by sucrose density gradient electrofocusing) and an apparent molecular weight (estimated by gel filtration) of ca. 4.8 and 12,200, respectively. The culture supernatant fluids also possessed hemolytic and phospholipase A2 activities; however, they were separable from cell-killing factor activity by gel filtration. The substance(s) possessing the hemolytic and phospholipase activities coeluted when fractionated by gel filtration with Sephacryl S-200 (apparent molecular weight of ca. 34,500) and had isoelectric points of ca. 4.4 and 4.5, respectively.     

Production of dengue virus-induced macrophage cytotoxin in vivo

We have observed that dengue virus-induced cytotoxic factor (CF) induces peritoneal and splenic macrophages in vitro to produce a cytotoxin (CF2). This study demonstrates also production of CF2 in vivo in DV-infected mice and following inoculation with CF. The cell-type responsible for CF2 production in vivo is the macrophage (M phi) as M phi-depleted mice failed to produce CF2. CF2 activity could not be observed in the serum or peritoneal fluid though it is produced in peritoneal M phi. Once stimulated, CF2 is present for 4 h in M phi. M phi can be restimulated to produce CF2 only after a refractory period of 48 h.     

Production of dengue virus-induced macrophage cytotoxin in vivo.

We have observed that dengue virus-induced cytotoxic factor (CF) induces peritoneal and splenic macrophages in vitro to produce a cytotoxin (CF2). This study demonstrates also production of CF2 in vivo in DV-infected mice and following inoculation with CF. The cell-type responsible for CF2 production in vivo is the macrophage (M phi) as M phi-depleted mice failed to produce CF2. CF2 activity could not be observed in the serum or peritoneal fluid though it is produced in peritoneal M phi. Once stimulated, CF2 is present for 4 h in M phi. M phi can be restimulated to produce CF2 only after a refractory period of 48 h.     

Identification of a cytotoxin produced by Legionella pneumophila.

Culture filtrates of Legionella pneumophila were cytotoxic for Chinese hamster ovary cells. The cytotoxin was found to be methanol soluble, heat stable, and stable from pH 5 through 8.     

Production of an extracellular toxin by the oral pathogen Campylobacter rectus.

The ATCC type strain and six clinical isolates of Campylobacter rectus were tested for toxicity against HL-60 cells and human polymorphonuclear neutrophils (PMNs). After challenge with bacterial cell suspensions and media supernatants for up to 4 h, eukaryotic cell viability was assayed by trypan blue dye exclusion and lactate dehydrogenase release. Cells of the C. rectus type strain were not toxic. However, ethanol and (NH4)2SO4 extracts of culture media supernatants killed HL-60 cells in a time and dose dependent manner with 700 micrograms of supernatant protein killing 100% of HL-60 cells in 4 h. Concentrated media supernatants from clinical isolates also killed 100% of HL-60 cells in 30 to 60 min. The bacterial culture supernatants were toxic to PMNs with clinical isolates killing 70 to 90% of PMNs in 2 to 4 h. SDS-PAGE and immunoblot analysis of the toxic media supernatants revealed C. rectus specific proteins and lipopolysaccharide (LPS). The toxic activity was inhibited by protease, indicating that the toxin was protein. Non-toxic and toxic media supernatants were obtained by altering hemin and fumarate in the growth media. SDS-PAGE analysis of these revealed that all toxic supernatants contained a 104 kDa protein.     

Anti-tumour cytotoxin from macrophages: no correlation between cytotoxin adsorption by tumour cell lines and their cytotoxin susceptibility.

Mononuclear phagocytes can synthesize a cytotoxin, similar to that in tumour necrosis serum, which is cytotoxic to certain tumour cell lines in vitro. This study has investigated whether susceptibility and resistance to the cytotoxin can be explained in terms of the amount of cytotoxin receptor expressed on the tumour cell surface. Binding of cytotoxin has been quantitated both by direct adsorption and competitive inhibition assays with cytotoxin-resistant or -susceptible tumour lines and with sublines of susceptible lines, selected for resistance to the cytotoxin. For both rabbit and human cytotoxin, there was no correlation between cytotoxin adsorption by tumour cell lines and their cytotoxin susceptibility, suggesting that resistance to the cytotoxin is expressed at a post-receptor stage. Preliminary studies on the cytotoxin receptor of K562 cells have shown that it is probably not the transferrin receptor, and that protein but not carbohydrate is essential for its function.     

Hybridization of Escherichia coli producing Shiga-like toxin I, Shiga-like toxin II, and a variant of Shiga-like toxin II with synthetic oligonucleotide probes.

Synthetic oligonucleotides, constructed from the nucleotide sequences of genes coding for the A subunit of Shiga-like toxin (SLT) I and the B subunit of SLT-II, were used as probes at different degrees of stringency to identify Escherichia coli producing different types of SLTs. At 45 degrees C, the A-I oligonucleotide probe hybridized with E. coli producing SLT-I, SLT-II, and variant of SLT-II (SLT-IIv). At 53 degrees C, only SLT-I-producing E. coli hybridized with this probe. At 45 degrees C, the B-II oligonucleotide probe hybridized with SLT-II- and SLT-IIv-producing E. coli. At 53 degrees C, this probe hybridized with only SLT-II-producing E. coli. The A-I and B-II oligonucleotide probes were subsequently tested for hybridization with 73 SLT-producing E. coli and 49 non-SLT-producing E. coli isolated in Asia and Canada. At 45 degrees C, the A-I oligomer had a sensitivity of 97% and a specificity of 100% in identifying SLT-producing E. coli. At 53 degrees C, the A-I oligonucleotide probe had a sensitivity of 92% and a specificity of 91% in identifying E. coli containing genes encoding SLT-I. At 45 degrees C, the B-II oligonucleotide had a 100% sensitivity and 97% specificity in identifying E. coli that hybridized with the SLT-II probe. Of 17 E. coli that hybridized only with the SLT-II probe, 10 did not hybridize with the B-II oligonucleotide at 53 degrees C. All 10 isolates were cytotoxic to Vero cells but not to HeLa cells, confirming that the B-II oligonucleotide probe used at 53 degrees C will differentiate isolates producing SLT-II and SLT-IIv.     

Demonstration of a cytotoxin fromCampylobacter pylori

In order to determine ifCampylobacter pylori produces a cytotoxin, a study was performed using bacterial lysates from three clinical isolates of the organism. The lysates were cytotoxic for Chinese hamster ovary cells, as determined by a microtiter assay. The lysates were also lethal for mice after intraperitoneal injection. Loss of toxicity and lethality followed trypsinization, heating and acidification of cell-free lysates. It is concluded that the toxic factor(s) inCampylobacter pylori may be a protein.     

Rapid Identification of Thermotolerant Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis from Various Geographic Locations by a GTPase-Based PCR-Reverse Hybridization Assay

Recently, a gene from Campylobacter jejuni encoding a putative GTPase was identified. Based on two semiconserved GTP-binding sites encoded within this gene, PCR primers were selected that allow amplification of a 153-bp fragment from C. jejuni, C. coli, C. lari, and C. upsaliensis. Sequence analysis of these PCR products revealed consistent interspecies variation, which allowed the definition of species-specific probes for each of the four thermotolerant Campylobacter species. Multiple probes were used to develop a line probe assay (LiPA) that permits analysis of PCR products by a single reverse hybridization step. A total of 320 reference strains and clinical isolates from various geographic origins were tested by the GTP-based PCR-LiPA. The PCR-LiPA is highly specific in comparison with conventional identification methods, including biochemical and whole-cell protein analyses. In conclusion, a simple method has been developed for rapid and highly specific identification of thermotolerant Campylobacter species.     

Identification of Campylobacter jejuni by using a biotinylated probe

A DNA biotinylated probe for the rapid identification of Campylobacter jejuni in culture (Enzo Biochem, New York) has been evaluated. The hybridized biotinylated DNA probe is detected by its interaction with streptavidin linked to horseradish peroxidase. Sixteen strains of C. jejuni, including type strain. 24 strains of other Campylobacter and Helicobacter species, and 59 strains of other general have been studied. The probe was highly sensitive (100%) and specific (100% inside the genera Campylobacter and Helicobacter). All Campylobacter strains gave strong signals, and only three weak signals have been observed with non-Campylobacter strains. Our results indicate that specific recombinant DNA probe should offer a reliable and rapid method for routine diagnosis of Campylobacter jejuni enteritis.