Masanosuke Yoshikawa

A Genetic Determinant Required for Continuous Reinfection of Adjacent Cells on Large Plasmid in S. flexneri 2a

We have identified a region (virG) on the 230 kb virulence plasmid of S. flexneri that is required for cell-to-cell spread of the bacterium. Tn5 insertions into this region result in avirulent mutants that can initially invade and multiply in epithelial cells, but tend to lose active movement and tend to localize within the cytoplasm, where they are gradually extinguished without infecting adjacent cells. The virG region was localized to within 4 kb and may contain a single cistron. Sequences hybridizing to this region were found in all intact virulence plasmids of Shigellae and enteroinvasive E. coli.

A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence‐associated antigens of Shigella flexneri

The expression of plasmid‐encoded, invasion‐related antigens lpa b, c and d of Shigella flexneri was found to be positively regulated at transcriptional level by a 33kD protein produced by the previously defined, virulence‐associated Region 1 on the SalI fragment B of the 230 kb invasion plasmid. The gene (designated virB) was identified and its nucleotide sequence determined. No Ipa b or c was produced in the absence of an intact virB gene although lower levels of d were produced. The previously reported regulatory activity of the virF gene some 30 kb distance away was shown to act exclusively through virB. In contrast, the activation of the virG gene necessary for intercellular spread occurred directly by virF without the requirement for virB. This study thus ascribes a critical function to a previously recognized, but functionally undefined, virulence locus on the large invasion plasmid of S. flexneri. The virF gene appears to have a central role in activation of the 230kb plasmid‐encoded virulence genes.

Identification and Characterization of hsa, the Gene Encoding the Sialic Acid-Binding Adhesin of Streptococcus gordonii DL1

ABSTRACT Oral colonization by Streptococcus gordonii, an important cause of subacute bacterial endocarditis, involves bacterial recognition of sialic acid-containing host receptors. The sialic acid-binding activity of this microorganism was previously detected by bacterium-mediated hemagglutination and associated with a streptococcal surface component identified as the Hs antigen. The gene for this antigen (hsa) has now been cloned in Escherichia coli, and its expression has been detected by colony immunoblotting with anti-Hs serum. Mutants of S. gordonii containing hsa inactivated by the insertion of an erythromycin resistance gene or deletion from the chromosome were negative for Hs-immunoreactivity, bacterium-mediated hemagglutinating activity, and adhesion to α2-3-linked sialoglycoconjugates. The deletion in the latter mutants was complemented by plasmid-borne hsa, resulting in Hs antigen production and the restoration of cell surface sialic acid-binding activity. The hsa gene encodes a 203-kDa protein with two serine-rich repetitive regions in its 2,178-amino-acid sequence. The first serine-rich region occurs within the amino-terminal region of the molecule, between different nonrepetitive sequences that may be associated with sialic acid binding. The second serine-rich region, which is much longer than the first, is highly repetitive, containing 113 dodecapeptide repeats with a consensus sequence of SASTSASVSASE. This long repetitive region is followed by a typical gram-positive cell wall anchoring region at the carboxyl-terminal end. Thus, the predicted properties of Hsa, which suggest an amino-terminal receptor-binding domain attached to the cell surface by a molecular stalk, are consistent with the identification of this protein as the sialic acid-binding adhesin of S. gordonii DL1.

Expression of four virulence antigens of Shigella flexneri is positively regulated at the transcriptional level by the 30 kiloDalton virF protein.

On the virulence plasmid of Shigella flexneri the virG region required for cell‐to‐cell spread of the bacteriumencodes a 130 kiloDalton (kD) antigen and Region‐2essential for the bacterial invasion of epithelial cells encodes 57, 43 and 39 kD antigens. The expression of these four antigens is positively regulated by the 30 kD protein encoded by virF, whose nucleotide sequence had been determined and which was previously found to be essential for virulence. An approximately 3.8kilobase (kb) RNA transcript is found to be transcribed by the virG region and is positively regulated by the virF protein resulting in increased production of the 130 kD antigen. The virF sequence is conserved among all shigellae and enteroinvasive Escherichia coli.

Identity of molecular structure of Shiga-like toxin I (VT1) from Escherichia coli O157 : H7 with that of Shiga toxin

The primary structures of the A and B subunits of Shiga toxin and of Shiga-like toxin I (VT1), isolated from the culture supernatants of Shigella dysenteriae 1 and Escherichia coli O157:H7, respectively, were analyzed by Edman degradation of intact proteins and peptides in their digests with trypsin or Achromobacter protease I and also by fast atom bombardment mass spectrometry of the digests. The results indicated that the A and B subunits of Shiga toxin and Shiga-like toxin I have the same primary structures. The identity of their primary structures was confirmed by determining the nucleotide sequence of the gene encoding Shiga-like toxin I cloned from a Shiga-like toxin I converting phage. This nucleotide sequence was different from that reported by Jackson et al. (Microbial Pathogenesis 1987; 2: 147-153), by Calderwood et al. (Proc Natl Acad Sci USA 1987; 84: 4364-8) and by Grandis et al. (J Bacteriol 1987; 169: 4313-9) in one base at position 231, which was found to be adenine instead of thymine, which they reported. The amino acid residue at position 45 from the N-terminus of the A subunit of Shiga-like toxin I deduced from the nucleotide sequence determined in this study is threonine, which corresponds with that found by amino acid sequencing, whereas from previous reports by other investigators it is serine. Edman degradation of the intact A subunit of Shiga toxin indicated that the A subunit was nicked between Ala253 and Ser254 to form A1 and A2 fragments linked by a disulfide bond.

Functional organization and nucleotide sequence of virulence Region‐2 on the large virulence plasmid in Shigella flexneri 2a

The 7kb virulence Region‐2 of the large (virulence) plasmid in Shigella flexneri 2a encodes several proteins required for invasion of intestinal epithelial cells. Insertion and deletion mutagenesis, DNA subcloning and SDS‐polyacrylamide gel electro‐phoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (lpaB), 41 (lpaC) and 37 (lpaD)‐kiloDalton (kD) proteins. Complementation of Tn5‐induced mutations in Region‐2 with the above plasmid constructs indicated that Region‐2 consists of two operons and that the three lpa proteins are essential for the virulence phenotype. The transcriptional organization determined by Northern blotting, S1 nuclease protection and the effect of Tn5 insertions on expression of the lpa proteins revealed that Region‐2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, lpaB and C proteins and the 4.5 kb RNA for the ipsD gene. In addition, the full‐length RNA of 7.5 kb which covers Region‐2 supplemented full expression of the lpa proteins. The 7663 nucleotides of Region‐2 were determined to confirm the five open reading frames encoding 23655, 17755, 62168, 41077 and 36660 Dalton proteins, respectively, and their regulatory sequences.

Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading

A novel virulence gene (virA) was identified upstream of the virG gene on the large plasmid of Shigella flexneri 2a YSH6000. Characterization of virA mutants infecting MK2 epithelial cell monolayers revealed that their invasive capacity was decreased to less than one fifth of the wild‐type level. Nevertheless, the bacteria were capable of expressing and secreting IpaB, IpaC and IpaD proteins. The virA mutants were also impaired in their ability to spread intercellularly, since the bacteria gave rise to a small number of foci in a focus‐plaque‐forming test with MK2 cells. Although virG expression was slightly decreased in the virA mutants, introduction of a cloned virG gene into a virA mutant, N1945, failed to restore spreading ability. Although, introduction of a cloned virA gene into N1945 restored invasiveness and spreading ability, the reduced virG transcription level was not affected, indicating that the reduced virG expression in virA mutants does not play a major role in defective intercellular spreading. The nucleotide sequence of the virA region revealed that the virA gene was located 528 bp upstream of the virG gene, in the opposite orientation. The deduced amino acid sequence of the VirA protein indicated a 44.7 kDa protein with no homology to known proteins. The VirA protein was secreted into the culture supernatant, a process that required the Mxi and Spa loci. The expression of virA was under the control of the virB gene, the positive regulator of the ipa, mxi and spa operons. These results indicate that virA is a new member of the invasion regulon directed by virB and that the VirA function is involved in invasion and intercellular spreading.

Enzymatic Properties of Dipeptidyl Aminopeptidase IV Produced by the Periodontal Pathogen Porphyromonas gingivalis and Its Participation in Virulence

ABSTRACT Porphyromonas gingivalis is a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) fromP. gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. An Escherichia coli strain overproducing P. gingivalis DPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated fromP. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P. gingivalis DPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P. gingivalis DPPIV are involved in the catalytic reaction. DPPIV-deficient mutants of P. gingivalis were constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P. gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism.

p53-Independent Expression of p21CIP1/WAF1 in Plasmacytic Cells during G2 Cell Cycle Arrest Induced by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

ABSTRACT The cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans has been shown to induce cell cycle arrest in the G2/M phase in HeLa cells. In the present study, the mechanism of CDT-induced cell cycle arrest was investigated by using HS-72 cells, a murine B-cell hybridoma cell line. Using flow cytometric analysis, we found that the recombinant CDT (rCDT) from A. actinomycetemcomitans induced G2 cell cycle arrest in HS-72 cells and that rCDT upregulated expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 and the tumor suppressor protein p53. HS-72 cells transfected with the E6/E7 gene of human papillomavirus type 16, which lacked rCDT-induced accumulation of p53, exhibited expression of p21CIP1/WAF1 or G2 cell cycle arrest upon exposure to rCDT. Furthermore, ectopic expression of a dominant negative p53 mutant did not inhibit rCDT-mediated p21CIP1/WAF1 expression or G2 cell cycle arrest in HS-72 cells. These results suggest that the CDT from A. actinomycetemcomitans induces p21CIP1/WAF1 expression and G2 cell cycle arrest in B-lineage cells by p53-independent pathways. Together with additional observations made with HeLa cells and COS-1 cells cultured with the rCDT from A. actinomycetemcomitans, the results of this study indicate that CDT-induced p53 accumulation may not be required for G2 cell cycle arrest and that an increased level of p21CIP1/WAF1 may be important for sustaining G2 cell cycle arrest in several mammalian cells.

Reconstitution and Purification of Cytolethal Distending Toxin of Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (ΔcdtA, ΔcdtB, ΔcdtC, ΔcdtBC, ΔcdtAC, and ΔcdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre‐mixing and pre‐incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB‐intein‐CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted‐CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.