FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2149254554> ?p ?o ?g. }

• W2149254554 endingPage "2903" @default.
• W2149254554 startingPage "2894" @default.
• W2149254554 abstract "Article15 May 1998free access Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins Brigitte Demers Corresponding Author Brigitte Demers Unité de Pathogénie Microbienne Moléculaire and Unité INSERM U389, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris, Cédex 15, France Search for more papers by this author Philippe J. Sansonetti Philippe J. Sansonetti Unité de Pathogénie Microbienne Moléculaire and Unité INSERM U389, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris, Cédex 15, France Search for more papers by this author Claude Parsot Claude Parsot Unité de Pathogénie Microbienne Moléculaire and Unité INSERM U389, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris, Cédex 15, France Search for more papers by this author Brigitte Demers Corresponding Author Brigitte Demers Unité de Pathogénie Microbienne Moléculaire and Unité INSERM U389, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris, Cédex 15, France Search for more papers by this author Philippe J. Sansonetti Philippe J. Sansonetti Unité de Pathogénie Microbienne Moléculaire and Unité INSERM U389, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris, Cédex 15, France Search for more papers by this author Claude Parsot Claude Parsot Unité de Pathogénie Microbienne Moléculaire and Unité INSERM U389, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris, Cédex 15, France Search for more papers by this author Author Information Brigitte Demers 1, Philippe J. Sansonetti1 and Claude Parsot1 1Unité de Pathogénie Microbienne Moléculaire and Unité INSERM U389, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris, Cédex 15, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2894-2903https://doi.org/10.1093/emboj/17.10.2894 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Shigella, the etiological agent of human bacillary dysentery, invades the colonic epithelium where it induces an intense inflammatory response. Entry of Shigella into epithelial cells involves a type III secretion machinery, encoded by the mxi and spa operons, and the IpaA–D secreted proteins. In this study, we have identified secreted proteins of 46 and 60 kDa as the products of virA and ipaH9.8, respectively, the latter being a member of the ipaH multigene family. Inactivation of virA did not affect entry into epithelial cells. Using lacZ transcriptional fusions, we found that transcription of virA and four ipaH genes, but not that of the ipaBCDA and mxi operons, was markedly increased during growth in the presence of Congo red and in an ipaD mutant, two conditions in which secretion through the Mxi–Spa machinery is enhanced. Transcription of the virA and ipaH genes was also transiently activated upon entry into epithelial cells. These results suggest that transcription of the virA and ipaH genes is regulated by the type III secretion machinery and that a regulatory cascade differentially controls transcription of genes encoding secreted proteins, some of which, like virA, are not required for entry. Introduction Numerous Gram-negative bacteria that are pathogens for humans, animals or plants use homologous protein secretion machineries to secrete their virulence factors. The Sec-independent type III secretion pathway is involved in secretion of Yersinia anti-host proteins, Salmonella and Shigella spp. effectors of entry into epithelial cells, EPEC signal transducing proteins, Pseudomonas aeruginosa toxins and virulence factors of many plant pathogens, as well as in flagellum assembly of bacteria such as Salmonella typhimurium and Bacillus subtilis (reviewed in Van Gijsegem et al., 1993; Mecsas and Strauss, 1996; Alfano and Collmer, 1997). Possible characteristic features of this secretion pathway include the fact that secretion is activated by contact of the bacterium with host cells (Ménard et al., 1994; Watarai et al., 1995; Zierler and Galán, 1995), that some of the secreted proteins are delivered into the cytoplasm of host cells (Rosqvist et al., 1994; Sory and Cornelis, 1994; Wood et al., 1996; Collazo and Galan, 1997) and that transcription of genes encoding secreted proteins is controlled by secretion of regulatory proteins (Hughes et al., 1993; Pettersson et al., 1996). Members of the genus Shigella cause bacillary dysentery in humans by invading the colonic epithelial mucosa and inducing a strong inflammatory response (LaBrec et al., 1964). In vitro, cell invasion involves two steps: entry and intercellular dissemination. Genes involved in both steps are carried on a 200 kb virulence plasmid (reviewed by Hale, 1991; Parsot, 1994). A 31 kb fragment of this plasmid is necessary and apparently sufficient for entry into epithelial cells (Maurelli et al., 1985; Sasakawa et al., 1988). This fragment is organized in two divergently transcribed regions which schematically encode secreted proteins, the IpaA–D proteins and a type III secretion system, the Mxi–Spa secretion apparatus. The first region contains eight genes, including ipaBCDA, which are transcribed from a promoter located upstream from icsB. The second region contains 20 genes, designated ipg, mxi and spa, which are clustered in large operons. Inactivation of ipa, mxi and spa genes leads to a non-invasive phenotype, due to either loss of effector proteins (Sasakawa et al., 1989; Ménard et al., 1993) or failure to secrete them (Andrews and Maurelli, 1992; Venkatesan et al., 1992; Allaoui et al., 1993b; Sasakawa et al., 1993). Only a small proportion of IpaA–D proteins is secreted by wild-type Shigella growing in laboratory media. Inactivation of ipaD enhances secretion of IpaA, IpaB, IpaC and ∼15 other proteins (Ménard et al., 1994; Parsot et al., 1995). These latter proteins are absent or barely detectable in the medium of the wild-type strain unless Congo red, a dye that induces secretion (Bahrani et al., 1997), is present in the culture medium (Parsot et al., 1995). In this study, we have characterized secreted proteins of 46 and 60 kDa which are overproduced by a ΔipaBCDA mutant. The 46 kDa protein was identified as the product of virA, a gene which previously had been characterized in an Shigella flexneri 2a strain (Uchiya et al., 1995). The 60 kDa protein was identified as the product of ipaH9.8, a member of the ipaH multigene family that comprises five genes which are designated by the size of the HindIII fragment on which they are carried by the virulence plasmid (Hartman et al., 1990; Venkatesan et al., 1991). Using lacZ transcriptional fusions, we have investigated transcription of virA, of four members of the ipaH family and of the ipaBCDA and mxi operons. We present evidence that transcription of virA and of four ipaH genes, but not that of the ipaBCDA and mxi operons, is increased when secretion through the type III secretion machinery is enhanced in response to addition of Congo red to the growth medium and to inactivation of ipaD. In addition, transcription of virA–lacZ and ipaH–lacZ fusions was activated during entry of bacteria into epithelial cells. Characterization of a virA mutant indicated that VirA, in contrast to IpaB, IpaC and IpaD, is not required for entry into epithelial cells, which suggests that the differential expression of secreted proteins might reflect differences in the function of these proteins during infection. Results Some secreted proteins are overproduced by constitutively secreting strains Inactivation of either ipaB or ipaD and deletion of the ipaB, C, D and A genes lead to the secretion of ∼15 proteins that associate in the extracellular medium (Parsot et al., 1995). Aggregates containing proteins secreted by the ΔipaBCDA mutant (SF635) were used to immunize mice, and the resulting antiserum was tested by Western blotting on extracts of whole cultures, bacterial pellets and culture supernatants of M90T (wild-type), SF622 (ipaD), SF635 (ΔipaBCDA), SF634 (ipaD mxiD) and BS176 (a virulence plasmid-cured strain). The serum reacted most strongly with a 46 kDa protein; this protein was present in high amounts in extracts of ipaD and ΔipaBCDA strains, was present in low amounts in extracts of wild-type and ipaD mxiD strains, and was not present in extracts of the virulence plasmid-cured strain (Figure 1). SDS–PAGE analysis and Coomassie Blue staining also revealed that a protein, or possibly a mixture of proteins, of ∼60 kDa was present in higher amounts in extracts of the ipaD and ΔipaBCDA strains than in extracts of the wild-type and ipaD mxiD strains (Figure 1). These results suggested that production of 46 and 60 kDa secreted proteins was increased in the constitutively secreting ipaD and ΔipaBCDA strains compared with the wild-type and secretion-deficient ipaD mxiD strains. Figure 1.Secretion of proteins by various Shigella strains. Cultures of M90T (wild-type), BS176 (the virulence plasmid-cured strain) and the ipaD (SF622), ΔipaBCDA (SF635) and ipaD mxiD (SF634) mutants were used to prepare either whole culture extracts, by adding Laemmli sample buffer directly to the cultures, or bacterial pellets and culture supernatants, by centrifugation of the cultures. Proteins present in culture supernatants were concentrated 10 times by TCA precipitation. Samples were separated by SDS–PAGE and analyzed by either Coomassie Blue staining or immunoblotting using an antiserum raised against aggregates recovered from the medium of the ΔipaBCDA mutant. Numbers indicate the position and the size (in kDa) of standard proteins and arrows indicate the position of the 60 and 46 kDa proteins. Download figure Download PowerPoint Characterization of the gene encoding the 46 kDa secreted protein The 46 kDa protein secreted by the ΔipaBCDA mutant was transferred onto a PVDF membrane and subjected to Edman degradation and proteolysis by endolysin. The N-terminal sequence of the protein was identified as M-Q-T-S-N-I-T-N-H-E and those of two internal peptides as I-I-T-F-G-I-Y-S-P-H-E-T-L-A and V-H-T-I-T-A-P-V-S-G-N. Oligonucleotides based on the N-terminal sequence and one internal peptide were used to screen, by Southern blotting, a set of overlapping cosmids representing the entire virulence plasmid. Both probes hybridized to a 6.4 kb HindIII fragment of cosmid pCos3 (data not shown) which was then cloned into pUC19 to give rise to pBD3 (Figure 2). Escherichia coli derivatives harboring this plasmid produced a 46 kDa protein that was recognized by the serum raised against the mixture of secreted proteins (data not shown), thereby indicating that the entire gene had been cloned. Figure 2.Structure of plasmids carrying virA and ipaH9.8. A schematic genetic map of a portion of the virulence plasmid pWR100 is shown in the center, along with the position of some relevant restriction sites. Symbols used for restriction sites are: B, BspEI; C, HincII; E, EcoRI; H, HindIII; N, NdeI; P, HpaI; S, Sau3AI; T, StuI; V, BbvI; X, XbaI. The DNA corresponding to virA and ipaH9.8 is shown by shaded bars and the lacZ gene by a solid bar. Arrows indicate the orientation of transcription of the genes. Restriction sites of the virulence plasmid that were used for cloning are indicated in brackets. Download figure Download PowerPoint Subcloning experiments and Southern blot analysis of recombinant plasmids using oligonucleotides as probes allowed us to localize the gene encoding the 46 kDa protein on a 3.2 kb HincII–HindIII fragment located upstream from icsA (Bernardini et al., 1989; Lett et al., 1989). Sequence analysis revealed an open reading frame (ORF) starting 487 bp upstream from the icsA translation start codon and oriented in the opposite direction. Amino acid sequences deduced from positions 43–71, 159–200 and 442–473 of the ORF were identical to those determined for the N-terminal end and the two internal peptides of the secreted protein. These sequence data have been submitted to the DDBJ/EMBL/GenBank databases under the accession number AF047364. The deduced sequence of the 46 kDa protein was identical to that of VirA, a secreted protein encoded by the virulence plasmid of an S.flexneri strain of serotype 2a (Uchiya et al., 1995) and, therefore, the corresponding gene of S.flexneri 5 was designated virA. No other ORFs were detected immediatly upstream or downstream from virA. Restriction analysis of overlapping cosmids indicated that virA was located ∼10 kb downstream from the spa operon (Venkatesan et al., 1992; Sasakawa et al., 1993) on the virulence plasmid pWR100. Characterization of the gene encoding a 60 kDa secreted protein The 60 kDa proteins which were secreted in high amount by the ΔipaBCDA strain were transferred onto a PVDF membrane and the lower part of the band was used for N-terminal sequence determination and proteolysis by endolysin. Analysis of the N-terminal sequence indicated that the sample contained two proteins; the sequence of the major species was determined as M-L-P-I-N-N-N-F-S-L-P-Q. The sequence of an internal peptide was determined as Y-E-M-L-E-N-E-Y-P-Q-R-V-A-D-R, which was almost identical to a fragment of the constant region of members of the IpaH family. IpaH proteins are characterized by a constant C-terminal region of ∼300 residues which is preceded by a variable N-terminal region composed of repetitive motifs (Hartman et al., 1990; Venkatesan et al., 1991). The N-terminal sequence of the 60 kDa secreted protein was different from those deduced from the 5′ end of ipaH7.8, ipaH4.5, ipaH2.5 and ipaH1.4 (Hartman et al., 1990; Venkatesan et al., 1991), which suggested that this protein might correspond to the fifth IpaH protein, IpaH9.8, whose gene had not been sequenced yet. Southern blot analysis using a probe derived from the constant region of ipaH genes indicated that ipaH9.8 was present in cosmid pCos87 (data not shown). Deletion derivatives of pCos87 were constructed to give rise to pBD4 (Figure 2), whose 2.4 kb insert was entirely sequenced. The amino acid sequences deduced from positions 40–75 and 1477–1521 of the ORF identified by sequence analysis were identical to those of the N-terminal end and of the internal peptide of the 60 kDa secreted protein. These sequence data have been submitted to the DDBJ/EMBL/GenBank databases under the accession number AF047365. The ipaH9.8 gene encodes a 545 residue protein with a deduced Mr of 61 886. No ORFs were identified upstream or downstream from ipaH9.8. Restriction analysis of overlapping cosmids indicated that ipaH9.8 was located 45 kb downstream from the spa operon. Inactivation of ipaD increases transcription of virA and ipaH genes Western blot analysis indicated that a higher amount of VirA was produced by the ipaD mutant than by the wild-type strain (Figure 1). To investigate virA transcription, we constructed a virA–lacZ transcriptional fusion by cloning the icsA–virA intergenic region and the 5′ part of virA upstream from the lacZ gene in a suicide vector. The recombinant plasmid pLAC4 (Figure 2) was integrated at the virA locus of the virulence plasmid harbored by the wild-type and ipaD strains to construct SF1001 (virA–lacZ ipaD+) and SF1002 (virA–lacZ ipaD−) (Table I). Expression of the virA–lacZ fusion was 17 times higher in the ipaD− strain as compared with the ipaD+ strain (Table II), indicating that the increased production of VirA by the ipaD mutant was due to an increased transcription of virA. To determine whether VirA was involved in the regulation of the virA promoter, a DNA fragment internal to virA was cloned upstream from the lacZ gene in a suicide vector, and the recombinant plasmid pLAC5 (Figure 2) was integrated at the virA locus of the wild-type and ipaD strains to construct strains SF1003 (virA–lacZ ipaD+ virA−) and SF1004 (virA–lacZ ipaD− virA−). Inactivation of virA had no effect on transcription of the virA–lacZ fusion in either the ipaD+ or ipaD− backgrounds (data not shown), indicating that virA was not autoregulated. Table 1. Shigella strains Strain Genotype Reference M90T Wild type Sansonetti et al. (1985) M90T-Sm spontaneous SmR derivative of M90T Allaoui et al. (1992) BS176 plasmidless derivative of M90T Sansonetti et al. (1985) SF132 icsB–lacZ in M90T-Sm Allaoui et al. (1992) SF134 ipgD–lacZ in M90T-Sm Allaoui et al. (1993a) SF401 mxiD Allaoui et al. (1993b) SF403 mxiD–lacZ in M90T-Sm Allaoui et al. (1993b) SF622 ipaD Ménard et al. (1993) SF623 ipaA–lacZ in M90T-Sm Ménard et al. (1993) SF624 ipaA–lacZ in SF622 (ipaD) Ménard et al. (1993) SF634 ipaD mxiD Ménard et al. (1994) SF635 ΔipaBCDA Parsot et al. (1995) SF803 icsB–lacZ in SF622 (ipaD) this work SF806 ipgD–lacZ in SF622 (ipaD) this work SF808 mxiD–lacZ in SF622 (ipaD) this work SF1001 virA–lacZ in M90T-Sm (virA+) this work SF1002 virA–lacZ in SF622 (virA+) this work SF1003 virA–lacZ in M90T-Sm (virA−) this work SF1004 virA–lacZ in SF622 (virA−) this work SF1005 ipaH9.8–lacZ in M90T-Sm this work SF1006 ipaH9.8–lacZ in SF622 (ipaD) this work SF1007 ipaH7.8–lacZ in M90T-Sm this work SF1008 ipaH4.5–lacZ in M90T-Sm this work SF1009 ipaH4.5–lacZ in SF622 (ipaD) this work SF1010 ipaH1.4–lacZ in M90T-Sm this work SF1011 ipaH1.4–lacZ in SF622 (ipaD) this work SF1012 virA–lacZ in SF401 (mxiD) this work Table 2. Expression of lacZ transcriptional fusions by bacteria growing in vitro Fusion β-Galactosidase activity (Miller units)a ipaD+ ipaD− Ratio Ib ipaD++CR Ratio IIc virA–lacZ 16 280 17 280 17 virA–lacZ mxiD 17 NA NA 18 1.1 ipaH9.8–lacZ 28 580 21 325 12 ipaH7.8–lacZ 20 NA NA 235 12 ipaH4.5–lacZ 31 360 12 110 3.5 ipaH1.4–lacZ 53 280 5.3 270 5.1 ipaA–lacZ 485 510 1.1 390 0.8 icsB–lacZ 290 305 1.1 285 1.0 mxiD–lacZ 275 260 0.9 320 1.2 ipgD–lacZ 450 400 0.9 475 1.1 a Activities are the means of at least three independent experiments. Standard deviations are within 25% of the reported values. b Activity present in ipaD− strains versus activity present in ipaD+ strains. c Activity present in derivatives of the ipaD+ strain grown in the presence of Congo red versus activity present in the same strains grown in the absence of Congo red. NA: not applicable. To analyze transcription of the various ipaH genes, the constant region of ipaH9.8 was cloned upstream from the lacZ gene in a suicide plasmid, and the recombinant plasmid pLAC6 (Figure 2) was transferred by conjugation into the wild-type and ipaD strains. Transconjugants were screened by Southern blotting to identify the ipaH gene into which the plasmid was integrated. This allowed us to construct ipaH9.8–lacZ, ipaH4.5–lacZ and ipaH1.4–lacZ fusions in both the wild-type and ipaD strains, as well as an ipaH7.8–lacZ fusion in the wild-type strain. Expression of ipaH9.8–lacZ, ipaH4.5–lacZ and ipaH1.4–lacZ fusions was low in derivatives of the wild-type strain and was increased 5–20 times in derivatives of the ipaD mutant (Table II). To investigate transcription of genes of the entry region, we used lacZ transcriptional fusions in icsB and ipaA, which are the first and last genes of the ipaBCDA operon, respectively, and in ipgD and mxiD, which are the first and twelfth genes of the mxi operon, respectively (Figure 2). These fusions were constructed in both the wild-type and ipaD strains. Integration of the suicide plasmids used to construct these fusions did not affect the secretion phenotype of recombinant strains (data not shown). For each fusion, similar amounts of β-galactosidase were present in derivatives of the wild-type and ipaD strains, indicating that transcription of these genes was not affected by inactivation of ipaD (Table II), which was consistent with the observation that production of IpaB and IpaC was not affected in an ipaD mutant (Ménard et al., 1993, 1994). Congo red increases transcription of virA and ipaH genes Secretion of IpaB and IpaC is enhanced when bacteria grow in the presence of Congo red (Parsot et al., 1995). To investigate the effect of Congo red on virA transcription, we assayed the β-galactosidase activity in strain SF1001 (virA–lacZ ipaD+) after growth in the presence of various concentrations of Congo red. Transcription of the virA–lacZ fusion was low at concentrations of dye up to 20 μg/ml and then increased with the concentration of the dye to reach a plateau at ∼100 μg/ml of Congo red (Table II; data not shown). Likewise, ∼3–12 times more β-galactosidase activity was present in strains carrying ipaH9.8–, ipaH7.8–, ipaH4.5– and ipaH1.4–lacZ fusions after growth in the presence of 100 μg/ml of Congo red (Table II). In contrast, transcription of icsB–, ipaA–, ipgD– and mxiD–lacZ fusions was not affected by the presence of Congo red in the growth medium (Table II), which was consistent with the observation that the amount of IpaB and IpaC was not affected by the presence of Congo red in the growth medium (Parsot et al., 1995). Secretion is required for activation of virA transcription To determine whether regulation of virA transcription was dependent on the type III secretion machinery, we compared the β-galactosidase activities produced by the virA–lacZ fusion in derivatives of wild-type and mxiD strains during growth in the presence of Congo red and the production of VirA in ipaD and mxiD ipaD strains. The presence of Congo red in the growth medium of the derivative of the mxiD strain carrying the virA–lacZ fusion did not lead to an increase in β-galactosidase activity (Table II), and lesser amounts of VirA were present in the ipaD mxiD strain as compared with the ipaD strain (Figure 1). This indicated that activation of the virA promoter in response to Congo red and inactivation of ipaD required the integrity of the secretion machinery. To investigate kinetics of activation of the virA promoter, Congo red (100 μg/ml) was added to the growth medium during the exponential phase of growth of derivatives of the wild-type and mxiD strains carrying the virA–lacZ fusion. Samples were then collected at 5 min intervals and assayed for β-galactosidase activity. An increase in the β-galactosidase specific activity was detected 10 min after addition of the dye to the medium of the derivative of the wild-type strain, whereas no transcriptional activation of the virA–lacZ fusion was detected in the derivative of the mxiD mutant (Figure 3). The 10 min lag time observed between addition of Congo red and activation of virA transcription in the derivative of the wild-type strain was similar to that observed for induction of IpaB and IpaC secretion by Congo red (Bahrani et al., 1997). Figure 3.Transcription of the virA–lacZ fusion upon addition of Congo red to the growth medium. Congo red (100 μg/ml) was added to the growth medium during the exponential phase of growth of derivatives of the wild-type (open symbols) and mixD (closed symbols) strains carrying the virA–lacZ fusion. Samples were then collected at 5 min intervals and assayed for β-galactosidase activity. For both strains, no increase in β-galactosidase activity was detected in the absence of Congo red. Download figure Download PowerPoint These results differentiated the virA and ipaH genes, the transcription of which was increased after growth in the presence of Congo red or by inactivation of ipaD, from the genes of the entry region, the transcription of which apparently was constitutive with respect to these parameters. Moreover, this suggested that transcription of the virA and ipaH genes was regulated by the Mxi–Spa secretion machinery, since (i) conditions leading to an enhanced transcription of these genes were the same as those known to increase secretion through the Mxi–Spa secretion machinery, and (ii) in these conditions, the secretion machinery was required for the enhanced transcription of the virA–lacZ fusion and for the enhanced production of the VirA protein. Transcription of virA– and ipaH–lacZ fusions upon entry and during intracellular multiplication To investigate virA and ipaH transcription during infection of epithelial cells, we measured the β-galactosidase activity that was present in bacteria shortly after entry into epithelial cells. Cells were infected for 30 min to allow entry and then treated with gentamicin for 30 min to kill extracellular bacteria. Infected cells were then washed to remove killed bacteria and lysed, and intracellular bacteria were recovered by centrifugation. The number of intracellular bacteria was determined by plating, and the β-galactosidase activity present in these bacteria was assayed by using 4-methyl-umbelliferyl-β-D-galactoside (MUG) as a substrate. The specific activity was first expressed in units of fluorescence per bacterium and then converted into Miller units. For the strain carrying the ipgD–lacZ fusion, chosen as a representative of genes which were expressed constitutively in vitro, the β-galactosidase activity present within intracellular bacteria recovered after 60 min of infection was similar to that found after growth in laboratory medium (Table III). This confirmed that, following gentamicin treatment, washes of infected cells were sufficient to remove killed extracellular bacteria which, otherwise, could have contributed to the total β-galactosidase activity without being numbered by plating. For strains carrying the virA– and ipaH–lacZ fusions, the β-galactosidase activity was 6–30 times higher in intracellular bacteria than in bacteria grown in vitro (Table III). This indicated that transcription of virA, ipaH9.8, ipaH7.8, ipaH4.5 and ipaH1.4 had been induced upon entry or shortly thereafter. Table 3. Expression of lacZ transcriptional fusions by intracellular bacteria Fusion β-Galactosidase activity (Miller units)a In vitro 60 min of infection Ratio Ib 150 min of infection Ratio IIc ipgD–lacZ 450 490 1.1 465 1.1 virA–lacZ 16 280 18 49 5.7 ipaH9.8–lacZ 28 350 13 49 7.1 ipaH7.8–lacZ 20 590 30 150 3.9 ipaH4.5–lacZ 31 280 9.0 21 13.3 ipaH1.4–lacZ 53 300 5.7 49 6.1 a Activities are the means of at least three independent experiments. Standard deviations are within 25% of the reported values. b Activity present after 60 min of infection versus activity present in bacteria grown in vitro. c Activity present after 60 min of infection versus activity present after 150 min of infection. To investigate virA transcription during growth in the intracellular compartment, infected cells were lysed after various periods of incubation in the presence of gentamicin, and intracellular bacteria were counted by plating and assayed for β-galactosidase activity. The number of intracellular bacteria carrying the virA–lacZ fusion increased with the time of incubation, which was consistent with their intracellular multiplication (Figure 4). In contrast, the specific β-galactosidase activity present in these bacteria decreased steadily; the slope of the decrease in β-galactosidase specific activity was similar to that of the increase in the number of intracellular bacteria, suggesting that the decrease in specific activity was due to bacterial multiplication. Similarly, the β-galactosidase activity present in bacteria carrying the various ipaH–lacZ fusions was 6–13 times lower after 150 min of infection as compared with the activity present after 60 min of infection (Table III). These results suggested that the virA– and ipaH–lacZ fusions had not been transcribed between 60 and 150 min of infection. In contrast, for the strain carrying the ipgD–lacZ fusion, similar amounts of β-galactosidase were present after 60 and 150 min of infection (Table III), suggesting that the intracellular compartment had no effect on ipgD transcription. Figure 4.Transcription of the virA–lacZ fusion by intracellular bacteria. Intracellular bacteria recovered after various times of infection of HeLa cells by SF1001 (virA–lacZ) were counted by plating (open symbols) and used to assay β-galactosidase activity (closed symbols). Download figure Download PowerPoint Phenotypic characterization of a virA mutant The presence of virA on the virulence plasmid, the regulation of its transcription by the type III secretion machinery, and previous results obtained with a virA mutant of S.flexneri 2a (Uchiya et al., 1995) suggested that VirA might be involved in Shigella virulence. To investigate the role of VirA, the virA gene of the wild-type strain was inactivated by integration of a suicide plasmid containing a virA internal fragment (Figure 2). Phenotypic characterization of the virA mutant was performed using both animal models of infection and cultured cell lines. Infection of rabbit ligated ileal loops revealed no difference between the mutant and the wild-type strains using such criteria as the volume of exudate, the intensities of ulceration and destruction of the villi, and the number of polymorphonuclear neutrophils accumulating in the mucosa (data not shown). In contrast, inoculation of guinea pig conjunctival sac (Sereny test) revealed an attenuation of the virulence of the virA mutant; whereas the wild-type strain provoked a frank keratoconjunctivitis within 48 h of infection, the virA mutant elicited a mild keratoconjuntivitis that was detectable only after 72 h of infection. The phenotype of the virA mutant harboring the plasmid pKvirA (Figure 2) was similar to that of the wild-type strain, which indicated that the atten" @default.
• W2149254554 created "2016-06-24" @default.
• W2149254554 creator A5069666784 @default.
• W2149254554 creator A5076149321 @default.
• W2149254554 creator A5077793315 @default.
• W2149254554 date "1998-05-15" @default.
• W2149254554 modified "2023-09-27" @default.
• W2149254554 title "Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins" @default.
• W2149254554 cites W1495526149 @default.
• W2149254554 cites W1504748286 @default.
• W2149254554 cites W1575061534 @default.
• W2149254554 cites W158273916 @default.
• W2149254554 cites W1732000839 @default.
• W2149254554 cites W180581491 @default.
• W2149254554 cites W1844896946 @default.
• W2149254554 cites W1852213005 @default.
• W2149254554 cites W1876016322 @default.
• W2149254554 cites W1900053682 @default.
• W2149254554 cites W1905967313 @default.
• W2149254554 cites W1923742340 @default.
• W2149254554 cites W1938013375 @default.
• W2149254554 cites W1941444893 @default.
• W2149254554 cites W1955204755 @default.
• W2149254554 cites W1960007027 @default.
• W2149254554 cites W1963528456 @default.
• W2149254554 cites W1966792663 @default.
• W2149254554 cites W1988282769 @default.
• W2149254554 cites W1996151076 @default.
• W2149254554 cites W2008850485 @default.
• W2149254554 cites W2018289835 @default.
• W2149254554 cites W2020196783 @default.
• W2149254554 cites W2034674930 @default.
• W2149254554 cites W2041653604 @default.
• W2149254554 cites W2054936556 @default.
• W2149254554 cites W2056922902 @default.
• W2149254554 cites W2080171465 @default.
• W2149254554 cites W2081966923 @default.
• W2149254554 cites W2100837269 @default.
• W2149254554 cites W2109460911 @default.
• W2149254554 cites W2110210117 @default.
• W2149254554 cites W2114285279 @default.
• W2149254554 cites W2115606209 @default.
• W2149254554 cites W2116548822 @default.
• W2149254554 cites W2117411464 @default.
• W2149254554 cites W2124537881 @default.
• W2149254554 cites W2126941629 @default.
• W2149254554 cites W2127135538 @default.
• W2149254554 cites W2128616172 @default.
• W2149254554 cites W2132247441 @default.
• W2149254554 cites W2137575812 @default.
• W2149254554 cites W2139161175 @default.
• W2149254554 cites W2150322683 @default.
• W2149254554 cites W2151837951 @default.
• W2149254554 cites W2152025494 @default.
• W2149254554 cites W2154460798 @default.
• W2149254554 cites W2155196333 @default.
• W2149254554 cites W2161767156 @default.
• W2149254554 cites W2166447817 @default.
• W2149254554 cites W2167032426 @default.
• W2149254554 cites W2168134728 @default.
• W2149254554 cites W2258671826 @default.
• W2149254554 cites W44949694 @default.
• W2149254554 doi "https://doi.org/10.1093/emboj/17.10.2894" @default.
• W2149254554 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1170630" @default.
• W2149254554 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9582283" @default.
• W2149254554 hasPublicationYear "1998" @default.
• W2149254554 type Work @default.
• W2149254554 sameAs 2149254554 @default.
• W2149254554 citedByCount "106" @default.
• W2149254554 countsByYear W21492545542012 @default.
• W2149254554 countsByYear W21492545542013 @default.
• W2149254554 countsByYear W21492545542014 @default.
• W2149254554 countsByYear W21492545542015 @default.
• W2149254554 countsByYear W21492545542016 @default.
• W2149254554 countsByYear W21492545542017 @default.
• W2149254554 countsByYear W21492545542018 @default.
• W2149254554 countsByYear W21492545542019 @default.
• W2149254554 countsByYear W21492545542020 @default.
• W2149254554 countsByYear W21492545542022 @default.
• W2149254554 countsByYear W21492545542023 @default.
• W2149254554 crossrefType "journal-article" @default.
• W2149254554 hasAuthorship W2149254554A5069666784 @default.
• W2149254554 hasAuthorship W2149254554A5076149321 @default.
• W2149254554 hasAuthorship W2149254554A5077793315 @default.
• W2149254554 hasBestOaLocation W21492545541 @default.
• W2149254554 hasConcept C104317684 @default.
• W2149254554 hasConcept C138885662 @default.
• W2149254554 hasConcept C156399914 @default.
• W2149254554 hasConcept C179926584 @default.
• W2149254554 hasConcept C2781217973 @default.
• W2149254554 hasConcept C41895202 @default.
• W2149254554 hasConcept C49039625 @default.
• W2149254554 hasConcept C54355233 @default.
• W2149254554 hasConcept C547475151 @default.
• W2149254554 hasConcept C55493867 @default.
• W2149254554 hasConcept C86339819 @default.
• W2149254554 hasConcept C86803240 @default.
• W2149254554 hasConcept C89423630 @default.

• next