Osamu Matsushita

Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan.

The ixodid tick Ixodes persulcatus is the most important vector of Lyme disease in Japan. Most spirochete isolates obtained from I. persulcatus ticks have been classified as Borrelia burgdorferi sensu lato because of their genetic, biological, and immunological characteristics. However, we found that a small number of isolates obtained from I. persulcatus contained a smaller 38-kDa endoflagellar protein and single 23S-5S rRNA gene unit. Representative isolate HT31T (T = type strain) had the same 23S rRNA gene physical map as Borrelia turicatae. The DNA base composition of strain HT31T was 28.6 mol% G+C. DNA-DNA hybridization experiments revealed that strain HT31T exhibited moderate levels of DNA relatedness (24 to 51%) with Borrelia hermsii, B. turicatae, Borrelia parkeri, and Borrelia coriaceae. However, the levels of DNA reassociation with the previously described Lyme disease borreliae (B. burgdorferi, Borrelia garinii, and Borrelia afzelii) were only 8 to 13%. None of the previously described species examined exhibited a high level of DNA relatedness with strain HT31T. In addition, the 16S rRNA gene sequence (length, 1,368 nucleotides) of strain HT31T was determined and aligned with the 16S rRNA sequences of other Borrelia species. Distance matrix analyses were performed, and a phylogenetic tree was constructed. The results showed that isolate HT31T is only distantly related to both previously described Lyme disease borreliae and relapsing fever borreliae. Thus, the spirochetes isolated from I. persulcatus and closely related isolates should be classified as members of a new Borrelia species. We propose the name Borrelia miyamotoi sp. nov. for this spirochete; strain HT31 is the type strain.

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.

Substrate Recognition by the Collagen-binding Domain of Clostridium histolyticum Class I Collagenase

Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b. S3a and S3b bound to insoluble collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD). Because S3a+S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3a+S3b) CBDs bound to atelocollagen, which contains only the collagenous region. However, they did not bind to telopeptides immobilized on Sepharose beads. These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region. The CBD bound to immobilized collagenous peptides, (Pro-Hyp-Gly) n and (Pro-Pro-Gly) n , only whenn is large enough to allow the peptides to have a triple-helical conformation. They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triple-helical conformation. The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region. These observations suggested that the CBD specifically recognizes the triple-helical conformation made by three polypeptide chains in the collagenous region.

A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation

The crystal structure of a collagen‐binding domain (CBD) with an N‐terminal domain linker from Clostridium histolyticum class I collagenase was determined at 1.00 Å resolution in the absence of calcium (1NQJ) and at 1.65 Å resolution in the presence of calcium (1NQD). The mature enzyme is composed of four domains: a metalloprotease domain, a spacing domain and two CBDs. A 12‐residue‐long linker is found at the N‐terminus of each CBD. In the absence of calcium, the CBD reveals a β‐sheet sandwich fold with the linker adopting an α‐helix. The addition of calcium unwinds the linker and anchors it to the distal side of the sandwich as a new β‐strand. The conformational change of the linker upon calcium binding is confirmed by changes in the Stokes and hydrodynamic radii as measured by size exclusion chromatography and by dynamic light scattering with and without calcium. Furthermore, extensive mutagenesis of conserved surface residues and collagen‐binding studies allow us to identify the collagen‐binding surface of the protein and propose likely collagen–protein binding models.

Clostridial hydrolytic enzymes degrading extracellular components.

Bacteria belonging to the genus Clostridium, both glycolytic and proteolytic, and both pathogenic and non-pathogenic, produce a battery of hydrolytic enzymes to obtain nutrients from various biopolymers. The clostridial hydrolytic enzymes are diverse, and are used or are potentially useful for fundamental and applied research purposes. Among them, enzymes degrading the major components in the extracellular matrix or on the cell surface in vertebrates are herein reviewed with special emphasis on recent knowledge gained through molecular biology of clostridial collagenases, sialidases and hyaluronidases. This paper also reviews some literature on the biotechnological approach to the designing of new molecular tools and drug delivery systems involving clostridial hydrolytic enzymes.

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.

Accumulation of Clostridium perfringens Epsilon-Toxin in the Mouse Kidney and Its Possible Biological Significance

ABSTRACT In this paper we show that Clostridium perfringens epsilon-toxin accumulates predominantly in the mouse kidney, where it is distributed mainly in glomeruli, capillaries, and collecting ducts. Although some pycnotic and exfoliated epithelial cells were observed in distal tubuli and collecting ducts, there were no findings indicative of severe renal injury. Bilateral nephrectomy increased the mouse lethality of the toxin, suggesting that the kidney contributes to the host defense against the lethal toxicity of epsilon-toxin.

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.