Isamu Kondo

Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli.

SummaryRestriction fragments of DNA from bacteriophage Sϕ-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of Sϕ-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.

An exfoliative toxin A-converting phage isolated from Staphylococcus aureus strain ZM.

Exfoliative toxin A (ETA) causes staphylococcal scalded‐skin syndrome in children. The gene for ETA was believed to be coded by the chromosomal DNA. We isolated temperate phages from an ETA‐producing strain, ZM, using a restriction minus strain, 1039, as an indicator. One of the prophages, designated φ‐ZM‐1 mediated lysogenic conversion of ETA. The polymerase chain reaction assay of the eta gene revealed that phage φ‐ZM‐1 carries the structural gene for ETA.

Mercury Resistance and R Plasmids in Escherichia coli Isolated from Clinical Lesions in Japan

The mercury and antibiotic resistance of 338 strains of Escherichia coli isolated from hospital patients was determined. Resistance to mercury was found in 58.6% of the isolates. The frequencies of resistance to streptomycin (Sm), tetracycline (Tc), chloramphenicol (Cm), kanamycin (Kan), cephaloridine (Cer), and gentamicin (Gm) were 66.3, 60.3, 56.5, 42.9, 32.1, and 1.5%, respectively. Among the above, 198 mercury- and antibiotic-resistant isolates were selected and tested for their ability to transfer the resistance to susceptible strains of E. coli K-12 and Klebsiella pneumoniae JK5. R plasmids carrying mercury resistance were demonstrated in 89.9% of the mercury-resistant strains of E. coli. Furthermore, R(Hg,Sm,Tc,Cm) plasmids were demonstrated most frequently, followed by R(Hg,Sm,Tc,Cm,Kan), R(Hg,Cm,Kan), and R(Hg,Sm,Tc,Cm,Kan,Cer) plasmids.

A Bacteriological Study on Children with Staphylococcal Toxic Epidermal Necrolysis in Japan

From 1973 to 1976, 21 patients with staphylococcal toxic epidermal necrolysis were admitted to the Jikei University Hospital in Tokyo. Of all of them were isolated coagulase-positive Staphylococcus aureus from various sites. In Japan, unlike in Europe and America, phage group II cocci were found in few cases. On the other hand, S. aureus belonging to other phage groups than group II (I, III mixed group and III group) were very commonly isolated. All the isolates produced exfoliatin which was capable of producing exfoliation in newborn mice. The type (A or B) of exfoliation which was detected by the antiserum agar method did not correlate to the severity of this disease.

DNA Sequencing of the eta Gene Coding for Staphylococcal Exfoliative Toxin Serotype A

We report the nucleotide sequence of a 1.45 kb segment containing the eta gene, coding for staphylococcal exfoliative toxin A (ETA), isolated from the recombinant plasmid pETA-J3. The coding region of 840 bp specified a polypeptide of 280 amino acid residues which included a putative 38 residue signal sequence. The amino acid composition deduced from the structural gene was in agreement with the results of peptide analysis of the ETA molecule reported by others. The sequence of the 35 N-terminal amino acid residues of ETA derived from Staphylococcus aureus strain ZM was also consistent with that deduced from the DNA sequencing.

Mechanism of the Spur Formation Observed between Two Forms of Extracellular Staphylococcal Protein A Produced by Mutants against Normal Canine Serum

Staphylococcal protein A (SpA) is now used as a tool for the purification of antigenic substances or as an immunofluorescent marker, since SpA reacts nonimmunologically with immunoglobulins from various mammalian species. The precise mechanism through which SpA reacts with immunoglobulins, however, has not been fully elucidated (1,4). In most staphylococcal strains SpA is present in the cell-bound form, which might make it difficult to purify SpA on a scale large enough to carry out sizable experiments to investigate the precise mechanism of the reaction between SpA and immunoglobulins. In order to overcome the difficulties in obtaining a sufficient amount of soluble SpA the authors have successfully isolated various mutants which release SpA into the culture medium (3). In the present investigation mutant V-I, which produces an extracellular SpA with a molecular weight of 13,000, and another mutant UV-2, which produces an extracellular SpA with a molecular weight of 41,000, were used as SpA producers (2). The two forms of soluble SpA produced by the mutant strains were purified by applying the culture supernatants of the mutants on human IgG-Sepharose columns as described in a previous paper (3). A precipitation line between UV-2 SpA and normal dog serum formed a spur projecting toward V-I SpA in the Ouchterlony immunodiffusion test. According to knowledge of the formation of a precipitation line in a gel due to an antigenantibody reaction, spur formation suggests partial identity in antigenicity between related antigen molecules. As a cross-reactive material for SpA produced by a mutant, however, previously failed to form a precipitation line with normal dog serum in the Ouchterlony immunodiffusion test, the spur observed between these forms of SpA and normal dog serum might be due to the actual reaction of SpA with canine immunoglobulins (5). For the isolation from whole canine serum of immunoglobulin components which might participate in the spur formation, a column of Sepharose CL 4B conjugated with V-I SpA was prepared (V-I Sepharose, column: 0.6 X 3 cm). Two ml of normal dog serum was applied on the column and it was extensively washed with Tris HCI/NaCl (0.05 MjO.l M) buffer, pH 8.0, and the absorbed materials were eluted by pH shift (McIlvaines buffer; pH 5.0,4.5,4.0,3.5, and 3.0).

The Effects of Intravenous Administration of Staphylococcal Protein A in Staphylococcus-Infected Mice

Staphylococcal protein A is endowed with the peculiar property of reacting with the Fc portion of immunoglobulins from various mammalian species (2).In vitro, protein A forms a precipitate with mammalian serum and activates the complement system (5). In vivo, the administration of protein A is found to cause an Arthus-like phenomenon in guinea pigs (3). Some species, however, manifest a less conspicuous reaction to protein A. For example normal rabbit serum gives no precipitation with protein A although a soluble complex is formed in the reaction mixture. The normal mouse reveals no demonstrable symptom when it receives an intravenous or intradermal injection of purified soluble protein A. The biological effects of the interaction between protein A and immunoglobulins are less extensively elucidated than the specific antigen-antibody reaction and subsequent activation of the complement system. During the course of our investigation on the pathogenicity of a protein Adeficient mutant, some interesting findings in which leukocyte and complement component are involved were observed after intravenous administration of protein A to mice previously infected with Staphylococcus aureus. Female ICR strain mice, about 6 weeks old and weighing 25 g,served as infected mice.Staphylococcus aureus βH strain and its derivative protein A-deficient mutant PN 1 were used as infecting agents (6). Mice were intravenously inoculated with one-tenth of the lethal dose of

Immunological properties in staphylococcal toxic epidermal necrolysis.

The analysis of host defenses in 4 patients with staphylococcal toxic epidermal necrolysis revealed normal serum immunoglobulin levels, but a deficiency in cell-mediated immunity as detected by the tuberculin reactions. We suspected that the transient deficiency of cell-mediated immunity may have contributed to the development of this disease.