N. Okada

Temperature‐regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid

The invasion phenotype of shigellae is subject to thermoregulation that is known to be expressed through activation of some invasion (inv) genes such as ipaB, ipaC, and ipaD encoded by the large virulence plasmid of Shigeila flexneri. The expression of ipa genes is regulated positively by virF through the activation of virB on the plasmid. To identify the mediator for the thermoregulation of the large plasmid, we have studied the effect of temperature on the transcription of virF and virB genes and ipa and the other two inv operons. The results showed that transcription of VirB was affected by temperature more strictly than that of virF. Analysis of the mRNA level of virB at different levels of virF transcription indicated that virB transcription depended upon both temperature and virF. On the other hand, transcriptions of ipa and the other two inv operons depended on the activation of virB transcription but not on temperature. By inducing wr8 transcription from a tac promoter fused with the virB region, invasion ability was restored to a WrF‐deletion mutant at 30°C as well as at 37°C. By using conditions in which the temperature‐dependent expression of the invasion phenotype was circumvented by the induction of virB transcription, intercellular spreading ability in a virF+, virB::Tn5 strain was shown to be expressed even at 30°C. These results suggest that the virB transcription stage is the main target for the thermoregulation.

Identification and characterization of virK, a virulence‐associated large plasmid gene essential for intercellular spreading of Shigella flexneri

Seven virulence loci have been identified by Tn5 insertion mutagenesis on the large 230 kb plasmid (pMYSH6000) of Shigella flexneri 2a. In this study, we used Tn10 insertion mutagenesis and identified a novel virulence locus on pMYSH6000 responsible for bacterial spread. Characterization of the invading bacteria of the Tn10 insertion mutants in the epithelial cells revealed that the bacteria were capable of at least some intracellular spreading but not intercellular spreading. Immunoblot analysis of lysates of the Tn10 insertion mutants with a VirG‐specific antipeptide antibody revealed diminished levels of the 116 kDa VirG protein. The virG mRNA in the mutants, however, was expressed at the same level as that in the wild type. The DNA region required for the virulence phenotype was localized to a 1.6 kb DNA sequence in the Sal I‐K fragment on the plasmid, and thus the locus was designated virK. Expression of virK in Escherichia coli using a T7 RNA polymerase‐dependent promoter system yielded a 36 kDa protein. The nucleotide sequence of 1642 bp encoding VirK function was determined, and an open reading frame encoding 316 amino acid residues was shown to encode the VirK protein. The virK region was highly conserved among the large virulence plasmids of shigellae and enteroinvasive Escherichia coli. These results suggest that VirK function is an essential virulence determinant for shigellae Involved in the expression of virG gene product at post‐transcriptional level.

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.

Construction of a physical map of the chromosome of Shigella flexneri 2a and the direct assignment of nine virulence‐associated loci identified by Tn5 insertions

To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl‐linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K‐12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K‐12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci Identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K‐12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5′ and 3’flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison of genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K‐12.

Molecular cloning and characterization of chromosomal virulence region kcpA of Shigella flexneri

In Shigella flexneri, in addition to several well‐recognized plasmid‐borne virulence loci, at least three genetic loci implicated in pathogenesis have been recognized on the chromosome. To understand more about the pathogenesis of bacillary dysentery at a molecular level, the genetically recognized but previously unidentified KcpA region (one of the chromosomal regions near purE) was cloned and sequenced. A single translatable open reading frame encoding a 12310 Dalton protein corresponding to the minicell product was found. Immunofluorescence microscopy, as well as optical and electron microscopic comparison of tissue‐cultured cells and guinea‐pigs’eyes infected with wild‐type or kcpA mutant bacteria, revealed that the kcpA product is required by invading bacteria for spread into adjacent cells.

Loss of virulence in Shigella strains preserved in culture collections due to molecular alteration of the invasion plasmid

Fifty-two Shigella strains long preserved by three Japanese culture collections were examined for virulence. All of them were avirulent when judged by the focus-plaque assay and the ability to bind Congo red. Fifteen strains had a plasmid comparable in size to that responsible for epithelial invasiveness and were positive in hybridization tests with a probe derived from a plasmid cistron, virG. Twenty-four strains had a similar plasmid but were negative in hybridization tests. The remaining 13 strains were negative in all the five criteria for virulence. Similar studies made on one hundred Shigella strains isolated from 1967 to 1985 clearly demonstrated loss of virulence with prolonged time of storage.

Thermoregulation of Invasion Genes in Shigella Flexneri Through the Transcriptional Activation of the Virb Gene on the Large Plasmid

Expression of several invasion (inv) genes on the large virulence plasmid of Shigella flexneri is subject to thermoregulation. The ipaBCD genes are expressed at 37 C but not at 30 C and this expression is regulated by virF through the activation of virB on the plasmid. To identify the mediator gene for the thermoregulation of the large plasmid, the transcription of virF, virB, ipa and the other two inv operons was examined by Northern dot hybridization and S1 nuclease assay methods. The results indicated that transcription of virF and virB was affected by temperature but the level of virB transcription was more strongly controlled by temperature than that of virF. On the other hand, transcription of ipa and the other two inv operons was not dependent on temerature but on virB transcription. By overexpressing the virB gene with a tac promoter, deregulation of invasion phenotype was achieved in the absence of VirF function, suggesting that the transcriptional activation of virB mediates the thermoregulation phenomenon.

Molecular Genetic Analysis of Virulence Factors of Shigella Flexneri

Labrec et al.7 established in 1964 that invasion into epithelial cells was the essential early step in the pathogenesis of bacillary dysentery. The virulent translucent strain of Shigella flexneri invaded the intestinal epithelium, while the avirulent opaque variant did not, in spite of the finding, that both had the same LD50 upon intraperitoneal injection. Ogawa et al.15 performed similar experiments and obtained essentially the same results. However, they made one important additional observation; within 24 hours of infection, the bacteria existed exclusively in the epithelial cells without direct penetration into the lamina propria and bacterial multiplication therein. Thus, virulent shigellae invade the colonic epithelium and spread successively to adjacent epithelial cells resulting in a severe inflammatory reaction, further spread of the bacteria to deeper tissue and, ultimately, to ulceration and bloody diarrhea.