Junzaburo Minami

Lambda-Toxin of Clostridium perfringens Activates the Precursor of Epsilon-Toxin by Releasing Its N- and C-Terminal Peptides

The effect of γ‐toxin, a thermolysin‐like metalloprotease of Clostridium perfringens, on the inactive ε‐prototoxin produced by the same organism was examined. When the purified ε‐prototoxin was incubated with the purified γ‐toxin at 37 C for 2 hr, the 32.5‐kDa ε‐prototoxin was processed into a 30.5‐kDa polypeptide, as determined by SDS‐polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without γ‐toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by γ‐toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The ε‐toxin gene was cloned and sequenced. Determination of the N‐terminal amino acid sequence of each activated ε‐prototoxin revealed that γ‐toxin cleaved between the 10th and 11th amino acid residues from the N‐terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated ε‐prototoxin was also determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The C‐terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C‐terminus of the prototoxin, suggesting that removal of not only N‐terminal but also C‐terminal peptide is responsible for activation of the prototoxin.

Clostridium perfringens epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of MDCK cells and rat synaptosomes

Clostridium perfringens ε-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled ε-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-β-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When ε-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.

Collagen-Binding Domain of a Clostridium Histolyticum Collagenase Exhibits a Broad Substrate Spectrum Both in Vitro and in Vivo

The substrate spectrum of the tandem collagen-binding domain (CBD) of Clostridium histolyticum class I collagenase (Co1G) was examined both in vitro and in vivo. CBD bound to insoluble type I, II, III and IV collagens in vitro, and to skin, aorta, tendon, kidney. trachea and corneal tissues containing various types of collagen fibrils or sheets. CBD hound to all kinds of collagen fibrils regardless of their diameters and also bound to sheet-forming collagen in the glomerular basal lamina or Descemets membrane of the cornea. This wide substrate spectrum expands possible applications of the drug delivery system we proposed previously (PNAS 95:7018-7023, 1998). Therapeutic agents fused with CBD will hind not only to subcutaneous tissues, but also to other tissues containing non-type I collagen.

Comparison of the Virulence of Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus

The virulence of methicillin‐resistant Staphylococcus aureus (MRSA) was compared with that of methicillin‐sensitive S. aureus (MSSA), using 13 MRSA and 7 MSSA strains isolated from clinical specimens. The infectivity and lethality of the two groups were examined as to the inoculum required to infect 50% of guinea pigs (ID50) and to kill 50% of mice (LD50), respectively. The mean ID50 [log10 colony forming units (CFU)] for MRSA strains was 7.1 ± 0.60 standard deviation, which was 1.5 higher than that for MSSA strains (P < 0.001). The mean LD50 (log10 CFU) for MRSA strains was 9.0 ± 0.42, being 1.1 higher than that for MSSA strains (P = 0.001). Pretreatment of mice with cyclophosphamide decreased the mean LD50 for MRSA strains more than that for MSSA strains, resulting in the difference in the mean LD50 being insignificant (P = 0.502). These results indicate that MRSA is less virulent than MSSA in normal hosts, but that they are equally virulent in immunocompromised hosts. The growth of MRSA strains was much slower than that of MSSA strains in the lag phase, although their growth rates were almost the same in the exponential growth phase, suggesting that the difference in virulence between them may be at least partly due to such a difference in growth.

Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature‐dependent manner

The phospholipase C gene (plc) of Clostridium perfringens possesses three phased A‐tracts forming bent DNA upstream of the promoter. An in vitro transcription assay involving C.perfringens RNA polymerase (RNAP) showed that the phased A‐tracts have a stimulatory effect on the plc promoter, and that the effect is proportional to the number of A‐tracts, and more prominent at lower temperature. A gel retardation assay and hydroxyl radical footprinting revealed that the phased A‐tracts facilitate the formation of the RNAP–plc promoter complex through extension of the contact region. The upstream (UP) element of the Escherichia coli rrnB P1 promoter stimulated the downstream promoter activity temperature independently, differing from the phased A‐tracts. When the UP element was placed upstream of the plc promoter, low temperature‐dependent stimulation was observed, although this effect was less prominent than that of the phased A‐tracts. These results suggest that both the phased A‐tracts and UP element cause low temperature‐dependent activation of the plc promoter through a similar mechanism, and that the more efficient low temperature‐dependent activation by the phased A‐tracts may be due to an increase in the bending angle at a lower temperature.

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.

An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter.

The plc gene, which encodes phospholipase C (alpha-toxin) of Clostridium perfringens, possesses three poly(A) tracts forming an intrinsically curved DNA region immediately upstream of the promoter. The in vivo transcriptional activity of the plasmid-borne plc gene was stimulated by this curved-DNA-containing sequence, depending on its proper linear and rotational orientation. The in vitro transcriptional activity of the plc gene was also stimulated by the upstream sequence. In addition, the stimulatory effect of the sequence and the degree of DNA bending were greater at lower temperature, as was demonstrated by both in vitro and in vivo transcription assays, and a gel-mobility assay, respectively. A similar temperature effect was also observed with the chromosomal plc gene. These observations suggest that the upstream DNA curvature per se stimulates the initiation of transcription of the plc gene, possibly through direct contact with RNA polymerase.

Role of the Upstream Region Containing an Intrinsic DNA Curvature in the Negative Regulation of the Phospholipase C Gene of Clostridium perfringens

The phospholipase C (α‐toxin) gene (plc) of Clostridium perfringens was cloned into pUC19 and the effects of the upstream regions on expression of the plc gene were examined in Escherichia coli JM109. When the 0.7‐kb region just upstream of the putative — 35 site of the gene was deleted, production of phospholipase C increased approximately 10‐fold. Northern blot hybridization analysis of the plc transcript showed that the upstream region inhibited transcription from the plc promoter. Nucleotide sequencing of this upstream region revealed that there are three periodically repeated (dA)5–6 tracts between positions −66 and −40 of the plc gene. A fragment containing this sequence showed anomalously slow electrophoretic mobility at low temperature, indicating that the region immediately upstream of the plc promoter is a locus of sequence directed DNA‐bending. Nested deletions of the upstream region were created from its 5′ end by exonuclease III and the effects of deletions on the expression of the plc gene were examined. When the 77‐bp fragment containing the two (dA)5–6 tracts was deleted, phospholipase C production increased markedly. These results indicate that the intrinsic DNA curvature upstream of the plc promoter is involved in the negative regulation of the plc gene transcription.

Biological activities and chemical composition of a cytotoxin of Klebsiella oxytoca

A low-molecular-mass cytotoxin produced by Klebsiella oxytoca isolated previously from patients with antibiotic-associated haemorrhagic enterocolitis was purified, and its biological and chemical properties were elucidated. The toxin inhibited the syntheses of DNA and RNA by HEp-2 cells dose-dependently, whereas protein synthesis was only slightly inhibited, as measured by the incorporation of radioactive precursors. When synchronously cultured HEp-2 cells were examined in the presence of cytotoxin, inhibition of DNA synthesis occurred promptly within 5 h, but cell-rounding, the earliest visible morphological change, was not observed until 6 h after exposure. The intracellular levels of ATP decreased with an approximately similar time course. These results suggest that cytotoxicity toward HEp-2 cells is primarily due to the inhibitory effect of the cytotoxin on nucleic acid synthesis, possibly on DNA synthesis. Cell rounding and cell death were induced even in the absence of the cytotoxin after incubation with the cytotoxin for 6 h. The cytotoxin was heat-labile, cytotoxic activity decreasing to 50% of the initial level on heating at 70 degrees C for 20 min. Plasmids were extracted from three strains of K. oxytoca producing the cytotoxin and analysed by agarose gel electrophoresis. Two strains possessed plasmids of different sizes, but one strain possessed no plasmid, indicating that the cytotoxin is probably chromosomally encoded. Analysis by NMR and FAB-mass-spectrometry revealed that the molecular mass of the cytotoxin should be 217.1062 Da (exact mass), its molecular formula being C8H15O4N3.

Expression of the colH Gene Encoding Clostridium histolyticum Collagenase in Bacillus subtilis and Its Application to Enzyme Purification

The colH gene encoding 116‐kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli‐Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colH gene was introduced into B. subtilis DB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion‐exchange chromatography. The yield of rColH from an 800‐ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type‐I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N‐terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N‐terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colH gene. Therefore, the recombinant B. subtilis culture is capable of serving as a useful source for enzyme purification.