On the 230-kilobase-pair (kb) virulence plasmid of Shigella flexneri 2a strain YSH6000, at least seven separate genetic determinants have been identified. One of them, an approximately 4-kb region, virG, that is required for the Sereny reaction, was extensively studied to examine the role of the virG region. The phenotype of a VirG-mutant (M94) of YSH6000 in the cytoplasm of cultured MK cels was characterized by a kinetic study of the invading shigellae. The observed phenotype of M94 in the cytoplasm indicated that the virG locus is not required for multiplication of the invading shigellae, but is essential for their spread to adjacent cells. The DNA region necessary for the VirG function was localized to a 3.6-kb DNA sequence on the 230-kb plasmid. A 130-kilodalton polypeptide was confirmed to be the virG product. External labeling of bacteria with 1251 indicated that the 130-kilodalton virG protein is exposed on the bacterial surface. The nucleotide sequence of 4,472 bp, which contains the functional virG gene and its own regulatory sequence, was determined, and a large open reading frame encoding 1,102 amino acid residues was identified.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.The Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is an important human pathogen that causes a number of serious suppurative and inflammatory infections of the skin (impetigo, erysipelas, cellulitis, and necrotizing fasciitis) and of the throat (pharyngitis). The poststreptococcal sequelae that produce rheumatic fever and acute glomerulonephritis are of particular concern, as is the increasing incidence of invasive group A streptococcal infections, including the streptococcal toxic shock-like syndrome (1). Each streptococcal infection is initiated by the interaction of bacteria with host cells, and the consequence of this interaction induces a local inflammatory host reaction that finally forms suppurative lesions. This initial interaction between bacteria and host cells is mediated by the binding of structures on the bacterial surface (adhesins) to specific receptors on the surface of the host cells.

The pathogenic gram-positive bacterium Streptococcus pyogenes (group A streptococcus) causes numerous diseases of cutaneous tissue, each of which is initiated after the interaction of the bacterium with the cells of the epidermis. In this study, we show that different surface proteins of S. pyogenes play important roles in determining the cell-specific tropism of the bacterium in skin. Using streptococcal strains with defined mutations in the genes which encode surface proteins in combination with primary cultures of human skin and an in situ adherence assay which uses histological sections of human skin, we show that the M protein of S. pyogenes mediates the binding of the bacterium to keratinocytes, while a second streptococcal surface protein, protein F. directs the adherence of the organism to Langerhans' cells. Characterization of binding revealed that adherence was inhibited by purified streptococcal proteins and pretreatment of both host cells with the protease typsin. Adherence was only slightly affected by the state of keratinocyte differentiation in vitro, but was considerably modulated in response to environmental conditions known to regulate expression of M protein and protein F, suggesting that the interaction between these bacterial cell-surface structures/ adhesins and keratinocytes and Langerhans' cells may play an important role in streptococcal skin disease. (J. Clin. Invest. 1994. 94:965-977.)

By random transposon TnS insertions, we previously identified six virulence-associated SaIl fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of Shigellaflexneri 2a. In this study, we analyzed the sites of 134 independent TnS insertions on four contiguous Sall fragments, B, P, H, and D, of pMYSH6000 and identified five virulence-associated regions; four were associated with inducing a positive Sereny test (Ser), invasion into epithelial cells (Inv), binding to Congo red (Pcr), and inhibition of bacterial growth (Igr), and one was associated with the Ser and Inv but not with the Pcr or Igr phenotypes. Hybridization studies revealed that these virulence-associated DNA regions were highly conserved among 15 other virulence plasmids of four species of Shigella and enteroinvasive Escherichuz coli. These data indicate that at least seven separate genetic determinants on the virulence plasmid are required for fuDl expression of the virulence phenotype of shigeliae.Shigellae are enteroinvasive bacteria that cause bacillary dysentery in humans and monkeys. These organisms invade colonic epithelial cells, multiply intracellularly, and spread to adjacent cells (9). The genetic determinants required for these abilities are located on at least three separate sites of the chromosome (3-5, 18) and on a 100-to 140-megadalton (MDa) plasmid. Commonly, plasmids of shigellae and enteroinvasive Escherichia coli (EIEC) contain genetic regions that are required in the early steps of the invasion process (18, 19); loss of the plasmid or deletion of an essential region consistently leads to loss of virulence (13,21).Expression of virulence in shigellae is dependent on temperature (12). Shigellae grown at 37°C are fully virulent, whereas bacteria grown at 30°C neither invade epithelial cells (12) nor provoke keratoconjunctivitis in guinea pigs (24). Making use of this property, Hale et al. (6) identified at least seven plasmid-coded, virulence-associated peptides produced by Shigella flexneri 2a and 5 and EIEC strain 0143. By Western blot (immunoblot) analysis of extracts of whole cells, four peptides of 78, 62, 43, and 38 kDa were recognized by convalescent-phase monkey antisera. These workers proposed that these proteins function as components of the invasion phenotype and are expressed on the bacterial surface. Oaks et al. (15) identified an additional plasmid-encoded surface peptide of 140 kDa which was also specifically recognized by convalescent-phase human or monkey sera. To identify the genetic regions associated with invasion, Maurelli et al. (14) shotgun-cloned Sau3A digests of the plasmid DNA into a cosmid vector, which was subsequently introduced into plasmid-free S. flexneri 5. A clone containing a 37-kilobase (kb) minimum sequence necessary for invasion was isolated. This recombinant clone also produced the four virulence-associated peptides described by Hale et al. (6). A DNA fragment coding for three antigenic proteins of 57, 43, and 39 kDa was cloned into a A expression vector by Buysse et al. (1). T...

The genetic determinants required for invasion of epithelial cells by Shigellaflexneri and for the subsequent bacterial spreading are encoded by the large virulence plasmid. Expression of the virulence genes is under the control of various genes on the large plasmid as well as on the chromosome. We previously identified one of the virulence-associated loci near phoBR in the NotI-C fragment of the chromosome of S. flexneri 2a YSH6000 and designated the locus vacC. The vacC mutant showed decreased levels of IpaB, IpaC, and IpaD proteins as well as transcription of ipa, an operon essential for bacterial invasion (N.

The formation of disulfide is essential for the folding, activity, and stability of many proteins secreted by Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into proteins exported from the cytoplasm to periplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we examined the role of the Dsb enzymes in Salmonella pathogenesis, and we demonstrated that DsbA, but not DsbC, is required for the full expression of virulence in a mouse infection model of Salmonella enterica serovar Typhimurium. Salmonella strains carrying a dsbA mutation showed reduced function mediated by type III secretion systems (TTSSs) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). To obtain a more detailed understanding of the contribution of DsbA to both SPI-1 and SPI-2 TTSS function, we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate protein for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Site-directed mutagenesis of the two cysteine residues present in the SpiA protein resulted in the loss of SPI-2 function in vitro and in vivo. Furthermore, we provided evidence that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA. Analysis of in vivo mixed infections demonstrated that a Salmonella dsbA srgA double mutant strain was more attenuated than either single mutant, suggesting that DsbA acts in concert with SrgA in vivo.

Transcription of the antiphagocytic M protein in the group A streptococcus (Streptococcus pyogenes) is environmentally regulated in response to CO2 and requires Mry, a trans-acting positive regulatory protein. We have examined the role of Mry in environmental regulation by analysing the factors that regulate expression of the gene that encodes Mry (mry). By employing a strategy that utilizes integrational plasmids, it was found that expression of mry requires the participation of DNA sequences that extend 473 base pairs upstream of the Mry coding region. Transcription of mry, as analysed in S1 nuclease protection assays, is initiated from two separate promoters located within this extended regulatory region. Construction and analysis of transcriptional fusions between the mry promoters and a promoterless chloramphenicol acetyltransferase gene demonstrated that mry is autoregulated and environmentally regulated in response to the level of CO2. These data suggest a model for the regulation of virulence in S. pyogenes where positive transcriptional control of mry in response to environmental stimuli regulates the expression of the M protein.

The 7 kb 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 electrophoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (IpaB), 41 (IpaC) and 37 (IpaD)-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 Ipa 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 Ipa proteins revealed that Region-2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5 kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, IpaB and C proteins and the 4.5 kb RNA for the ipaD gene. In addition, the full-length RNA of 7.5 kb which covers Region-2 supplemented full expression of the Ipa proteins. The 7663 nucleotides of Region-2 were determined to confirm the five open reading frames encoding 23,655, 17,755, 62,168, 41,077 and 36,660 Dalton proteins, respectively, and their regulatory sequences.