Modulation of an Outer Membrane Protease Contributes to the Virulence Defect of Shigella flexneri Strains Carrying a Mutation in the virK Lo
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感染与免疫杂志 2005年第2期
Infectious Disease Division, Massachusetts General Hospital, Boston, Massachusetts
ABSTRACT
The Shigella actin assembly protein IcsA is removed from the bacterial surface by the protease IcsP. We show that decreased intracellular spreading of virK::Tn10 mutants is due in part to significant increases in IcsP and IcsP-mediated cleavage of IcsA and that IcsP expression is a critical determinant of Shigella virulence.
TEXT
Shigellae move through the cytoplasm of infected cells and into adjacent cells by assembly of a propulsive actin tail (1, 15, 16, 20, 22), mediated by the polar outer membrane protein IcsA (VirG) (1, 7, 8, 11, 15). The domain of IcsA that mediates actin assembly is removed from the bacterial surface by the outer membrane protease IcsP (SopA), releasing a truncated IcsA polypeptide into the culture supernatant (3, 23).
IcsP contributes to the intercellular spreading defect of virK transposon insertion mutants. Strains that carry a transposon insertion in the poorly characterized gene virK display decreased levels of IcsA in total cellular protein preparations and decreased intercellular spreading but wild-type levels of icsA mRNA (18). We postulated that these phenotypes might be due to increased cleavage of IcsA by IcsP. Levels of truncated IcsA in the culture supernatant prepared from mid-exponential-phase cultures as previously described (2, 26) were reproducibly increased threefold or more in a virK mutant compared to those in the wild-type strain (Fig. 1A), indicating increased cleavage of IcsA from the surface of the mutant. Furthermore, IcsP levels (26) were reproducibly increased fivefold or more in the mutant compared to those in the wild-type, consistent with the increased cleavage of IcsA in the virK mutants being mediated by increased levels of IcsP. As for wild-type strains (3, 7, 23), in the virK mutant, IcsP fractionated to the outer membrane and IcsA was localized to the pole (data not shown). The IcsA and IcsP phenotypes were indistinguishable for each of three virK mutants (V836, V956, and V1060) (18); we selected V956 for the studies described below.
Introduction of the icsP mutation into the virK::Tn10 mutant by P1L4 transduction (17) led to undetectable amounts of cleaved IcsA in the culture supernatant and a greater than 20-fold increase in full-length IcsA associated with the bacterial pellet (Fig. 1B). The increase in bacterium-associated IcsA of the double mutant was comparable to that of the icsP single mutant, indicating that IcsP mediates essentially all of the increase in IcsA cleavage in the virK mutant. As reported previously (3, 23), the level of full-length IcsA associated with the bacterial pellet was increased in the icsP mutant compared to that in the wild type.
To determine whether IcsP is responsible for the defect in intercellular spreading of the virK mutant (18), we tested whether introduction of a disruption of icsP into the virK::Tn10 mutant would rescue the spreading phenotype of the virK::Tn10 mutant. The virK::Tn10 icsP double mutant generated a mixture of plaques that were larger than or approximately equal in size to those of the virK::Tn10 mutant (Fig. 1C; Table 1), demonstrating partial rescue of intercellular spreading and indicating that the small-plaque phenotype of the virK::Tn10 mutant is due at least in part to IcsP. As reported previously (3, 18, 23), the virK::Tn10 mutant formed very small plaques, some of which were only visible microscopically, and the icsP mutant formed plaques approximately the size of those formed by the wild-type strain. The total number of plaques, including those seen only microscopically, was comparable for all of the strains tested. These data indicate that both the decrease in bacterium-associated IcsA and the defect in actin-based motility of the virK mutant are mediated at least partially by the effect of the virK::Tn10 mutation on expression of IcsP and the resultant increase in IcsP-mediated cleavage of IcsA at the bacterial surface.
Overexpression of IcsP leads to decreased actin-based motility and decreased intercellular spreading. To directly examine the effect of overexpression of IcsP on intercellular spreading, we constructed a strain in which expression of a plasmid-borne icsP gene can be induced with isopropyl--D-thiogalactopyranoside (IPTG) (strain SSA9, Table 2). With increasing concentrations of IPTG, the amount of IcsP increased and the amount of IcsA associated with the bacteria decreased (Fig. 2A), with no effect on the growth rate (data not shown). Furthermore, with increasing IPTG concentrations, actin tail assembly was progressively less efficient (Fig. 2B). A small effect was seen even in the absence of added IPTG, consistent with the known leakiness of the promoter. In the presence of 0.01 or 0.025 mM IPTG, actin tails were infrequent and when present were stunted; in the presence of 0.05 mM IPTG, actin tails were almost completely absent and the bacteria formed tight clusters in the cell, a phenotype seen with icsA mutants (1) and consistent with a total absence of actin-based motility. Intercellular spreading, as measured by the presence and size of bacterial plaques on a cell monolayer (19), was impaired in a similar manner (Fig. 2C; Table 3), whereas bacterial entry was unaffected (data not shown). IPTG alone had no effect on the actin tail formation, intercellular spreading, or IcsP levels of the wild-type strain (Fig. 2; Table 3). Thus, artificially increasing the level of IcsP leads to marked defects in actin assembly, indicating that IcsP is an important determinant of Shigella virulence.
We speculate that the effect of the virK::Tn10 mutation on IcsP may reflect alteration of interactions in the outer membrane between lipopolysaccharide (LPS) and IcsP, which likely binds LPS. First, although the precise function of virK has not been determined, its genetic context within the locus shf-rfbU-virK-msbB2 suggests that it may be involved in LPS modification. Whereas the function of shf is unknown (29), rfbU and msbB2 each modify LPS (4, 9). Second, although complementation of the IcsA and intercellular spreading phenotypes of a virK::Tn10 mutant by virK without msbB2 (18) implicates virK in these phenotypes, it does not eliminate the possibility that both virK and msbB2 are involved.
Third, IcsP is a member of the omptin family of outer membrane proteases (25), which includes PgtE of Salmonella enterica serovar Typhimurium (30), Pla of Yersinia pestis (24), and OmpT (27) and OmpP (10) of Escherichia coli. Five of 11 residues involved in LPS binding by the outer membrane protein FhuA (5, 6) are conserved in OmpT, including 3 that interact with lipid A (28). Four of these five are also conserved in IcsP (M. B. Goldberg, unpublished data) and in other members of the omptin family (13). Moreover, the in vitro activity of OmpT is increased in the presence of LPS (12). Therefore, structural changes in lipid A that occur with mutation of msbB2 and perhaps virK::Tn10 may alter an interaction of lipid A with IcsP, which in turn may alter its stability or activity. These issues are the subject of ongoing investigation.
ACKNOWLEDGMENTS
We thank C. Sasakawa for generously providing strains YSH6000T, V836, V956, and V1060.
This work was supported by Public Health Service grants AI43562 and AI35817 from the National Institute of Allergy and Infectious Diseases (M.B.G.), a Charles H. Hood Foundation (Boston, Mass.) postdoctoral research fellowship from The Medical Foundation (H.J.W.), and a Massachusetts General Hospital Fund for Medical Discovery postdoctoral fellowship (H.J.W.).
H.J.W. and S.R.G. contributed equally to this work.
Present address: Department of Biological Sciences, University of Nevada-Las Vegas, Las Vegas.
REFERENCES
1. Bernardini, M. L., J. Mounier, H. d'Hauteville, M. Coquis-Rondon, and P. J. Sansonetti. 1989. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc. Natl. Acad. Sci. USA 86:3867-3871.
2. Caldwell, R. B., and C. T. Lattemann. 2004. Simple and reliable method to precipitate proteins from bacterial culture supernatant. Appl. Environ. Microbiol. 70:610-612.
3. Egile, C., H. d'Hauteville, C. Parsot, and P. J. Sansonetti. 1997. SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri. Mol. Microbiol. 23:1063-1073.
4. Fallarino, A., C. Mavrangelos, U. H. Stroeher, and P. A. Manning. 1997. Identification of additional genes required for O-antigen biosynthesis in Vibrio cholerae O1. J. Bacteriol. 179:2147-2153.
5. Ferguson, A. D., E. Hofmann, J. W. Coulton, K. Diederichs, and W. Welte. 1998. Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. Science 282:2215-2220.
6. Ferguson, A. D., W. Welte, E. Hofmann, B. Lindner, O. Holst, J. W. Coulton, and K. Diederichs. 2000. A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins. Struct. Fold Des. 8:585-592.
7. Goldberg, M. B., O. Barzu, C. Parsot, and P. J. Sansonetti. 1993. Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement. J. Bacteriol. 175:2189-2196.
8. Goldberg, M. B., and J. A. Theriot. 1995. Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility. Proc. Natl. Acad. Sci. USA 92:6572-6576.
9. Kaniuk, N. A., E. Vinogradov, J. Li, M. A. Monteiro, and C. Whitfield. 2004. Chromosomal and plasmid-encoded enzymes are required for assembly of the R3-type core oligosaccharide in the lipopolysaccharide of Escherichia coli O157:H7. J. Biol. Chem. 279:31237-31250.
10. Kaufmann, A., Y. D. Stierhof, and U. Henning. 1994. New outer membrane-associated protease of Escherichia coli K-12. J. Bacteriol. 176:359-367.
11. Kocks, C., J.-B. Marchand, E. Gouin, H. d'Hauteville, P. J. Sansonetti, M.-F. Carlier, and P. Cossart. 1995. The unrelated surface proteins ActA of Listeria monocytogenes and IcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocua and Escherichia coli, respectively. Mol. Microbiol. 18:413-423.
12. Kramer, R. A., K. Brandenburg, L. Vandeputte-Rutten, M. Werkhoven, P. Gros, N. Dekker, and M. R. Egmond. 2002. Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT. Eur. J. Biochem. 269:1746-1752.
13. Kukkonen, M., M. Suomalainen, P. Kyllonen, K. Lahteenmaki, H. Lang, R. Virkola, I. M. Helander, O. Holst, and T. K. Korhonen. 2004. Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. Mol. Microbiol. 51:215-225.
14. LaBrec, E. H., H. Schneider, T. J. Magnani, and S. B. Formal. 1964. Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysentery. J. Bacteriol. 88:1503-1518.
15. Lett, M.-C., C. Sasakawa, N. Okada, T. Sakai, S. Makino, M. Yamada, K. Komatsu, and M. Yoshikawa. 1989. virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence. J. Bacteriol. 171:353-359.
16. Makino, S., C. Sasakawa, K. Kamata, T. Kurata, and M. Yoshikawa. 1986. A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell 46:551-555.
17. Maurelli, A. T., and R. Curtiss, 3rd. 1984. Bacteriophage Mu d1(Apr lac) generates vir-lac operon fusions in Shigella flexneri 2a. Infect. Immun. 45:642-648.
18. Nakata, N., C. Sasakawa, N. Okada, T. Tobe, I. Fukuda, T. Suzuki, K. Komatsu, and M. Yoshikawa. 1992. Identification and characterization of virK, a virulence-associated large plasmid gene essential for intercellular spreading of Shigella flexneri. Mol. Microbiol. 6:2387-2395.
19. Oaks, E. V., M. E. Wingfield, and S. B. Formal. 1985. Plaque formation by virulent Shigella flexneri. Infect. Immun. 48:124-129.
20. Ogawa, H., A. Nakamura, and R. Nakaya. 1968. Cinemicrographic study of tissue cell cultures infected with Shigella flexneri. Jpn. J. Med. Sci. Biol. 21:259-273.
21. Okada, N., C. Sasakawa, T. Tobe, K. A. Talukder, K. Komatsu, and M. Yoshikawa. 1991. 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. Mol. Microbiol. 5:2171-2180.
22. Pal, T., J. W. Newland, B. D. Tall, S. B. Formal, and T. L. Hale. 1989. Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein. Infect. Immun. 57:477-486.
23. Shere, K. D., S. Sallustio, A. Manessis, T. G. D'Aversa, and M. B. Goldberg. 1997. Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility. Mol. Microbiol. 25:451-462.
24. Sodeinde, O. A., and J. D. Goguen. 1989. Nucleotide sequence of the plasminogen activator gene of Yersinia pestis: relationship to ompT of Escherichia coli and gene E of Salmonella typhimurium. Infect. Immun. 57:1517-1523.
25. Stathopoulos, C. 1998. Structural features, physiological roles, and biotechnological applications of the membrane proteases of the OmpT bacterial endopeptidase family: a micro-review. Membr. Cell Biol. 12:1-8.
26. Steinhauer, J., R. Agha, T. Pham, A. W. Varga, and M. B. Goldberg. 1999. The unipolar Shigella surface protein IcsA is directly targeted to the old pole; IcsP cleavage of IcsA occurs over the entire bacterial surface. Mol. Microbiol. 32:367-378.
27. Sugimura, K., and T. Nishihara. 1988. Purification, characterization, and primary structure of Escherichia coli protease VII with specificity for paired basic residues: identity of protease VII and OmpT. J. Bacteriol. 170:5625-5632.
28. Vandeputte-Rutten, L., R. A. Kramer, J. Kroon, N. Dekker, M. R. Egmond, and P. Gros. 2001. Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site. EMBO J. 20:5033-5039.
29. Venkatesan, M. M., M. B. Goldberg, D. J. Rose, E. J. Grotbeck, V. Burland, and F. R. Blattner. 2001. Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri. Infect. Immun. 69:3271-3285.
30. Yu, G. Q., and J. S. Hong. 1986. Identification and nucleotide sequence of the activator gene of the externally induced phosphoglycerate transport system of Salmonella typhimurium. Gene 45:51-57.(Helen J. Wing , Seth R. G)
ABSTRACT
The Shigella actin assembly protein IcsA is removed from the bacterial surface by the protease IcsP. We show that decreased intracellular spreading of virK::Tn10 mutants is due in part to significant increases in IcsP and IcsP-mediated cleavage of IcsA and that IcsP expression is a critical determinant of Shigella virulence.
TEXT
Shigellae move through the cytoplasm of infected cells and into adjacent cells by assembly of a propulsive actin tail (1, 15, 16, 20, 22), mediated by the polar outer membrane protein IcsA (VirG) (1, 7, 8, 11, 15). The domain of IcsA that mediates actin assembly is removed from the bacterial surface by the outer membrane protease IcsP (SopA), releasing a truncated IcsA polypeptide into the culture supernatant (3, 23).
IcsP contributes to the intercellular spreading defect of virK transposon insertion mutants. Strains that carry a transposon insertion in the poorly characterized gene virK display decreased levels of IcsA in total cellular protein preparations and decreased intercellular spreading but wild-type levels of icsA mRNA (18). We postulated that these phenotypes might be due to increased cleavage of IcsA by IcsP. Levels of truncated IcsA in the culture supernatant prepared from mid-exponential-phase cultures as previously described (2, 26) were reproducibly increased threefold or more in a virK mutant compared to those in the wild-type strain (Fig. 1A), indicating increased cleavage of IcsA from the surface of the mutant. Furthermore, IcsP levels (26) were reproducibly increased fivefold or more in the mutant compared to those in the wild-type, consistent with the increased cleavage of IcsA in the virK mutants being mediated by increased levels of IcsP. As for wild-type strains (3, 7, 23), in the virK mutant, IcsP fractionated to the outer membrane and IcsA was localized to the pole (data not shown). The IcsA and IcsP phenotypes were indistinguishable for each of three virK mutants (V836, V956, and V1060) (18); we selected V956 for the studies described below.
Introduction of the icsP mutation into the virK::Tn10 mutant by P1L4 transduction (17) led to undetectable amounts of cleaved IcsA in the culture supernatant and a greater than 20-fold increase in full-length IcsA associated with the bacterial pellet (Fig. 1B). The increase in bacterium-associated IcsA of the double mutant was comparable to that of the icsP single mutant, indicating that IcsP mediates essentially all of the increase in IcsA cleavage in the virK mutant. As reported previously (3, 23), the level of full-length IcsA associated with the bacterial pellet was increased in the icsP mutant compared to that in the wild type.
To determine whether IcsP is responsible for the defect in intercellular spreading of the virK mutant (18), we tested whether introduction of a disruption of icsP into the virK::Tn10 mutant would rescue the spreading phenotype of the virK::Tn10 mutant. The virK::Tn10 icsP double mutant generated a mixture of plaques that were larger than or approximately equal in size to those of the virK::Tn10 mutant (Fig. 1C; Table 1), demonstrating partial rescue of intercellular spreading and indicating that the small-plaque phenotype of the virK::Tn10 mutant is due at least in part to IcsP. As reported previously (3, 18, 23), the virK::Tn10 mutant formed very small plaques, some of which were only visible microscopically, and the icsP mutant formed plaques approximately the size of those formed by the wild-type strain. The total number of plaques, including those seen only microscopically, was comparable for all of the strains tested. These data indicate that both the decrease in bacterium-associated IcsA and the defect in actin-based motility of the virK mutant are mediated at least partially by the effect of the virK::Tn10 mutation on expression of IcsP and the resultant increase in IcsP-mediated cleavage of IcsA at the bacterial surface.
Overexpression of IcsP leads to decreased actin-based motility and decreased intercellular spreading. To directly examine the effect of overexpression of IcsP on intercellular spreading, we constructed a strain in which expression of a plasmid-borne icsP gene can be induced with isopropyl--D-thiogalactopyranoside (IPTG) (strain SSA9, Table 2). With increasing concentrations of IPTG, the amount of IcsP increased and the amount of IcsA associated with the bacteria decreased (Fig. 2A), with no effect on the growth rate (data not shown). Furthermore, with increasing IPTG concentrations, actin tail assembly was progressively less efficient (Fig. 2B). A small effect was seen even in the absence of added IPTG, consistent with the known leakiness of the promoter. In the presence of 0.01 or 0.025 mM IPTG, actin tails were infrequent and when present were stunted; in the presence of 0.05 mM IPTG, actin tails were almost completely absent and the bacteria formed tight clusters in the cell, a phenotype seen with icsA mutants (1) and consistent with a total absence of actin-based motility. Intercellular spreading, as measured by the presence and size of bacterial plaques on a cell monolayer (19), was impaired in a similar manner (Fig. 2C; Table 3), whereas bacterial entry was unaffected (data not shown). IPTG alone had no effect on the actin tail formation, intercellular spreading, or IcsP levels of the wild-type strain (Fig. 2; Table 3). Thus, artificially increasing the level of IcsP leads to marked defects in actin assembly, indicating that IcsP is an important determinant of Shigella virulence.
We speculate that the effect of the virK::Tn10 mutation on IcsP may reflect alteration of interactions in the outer membrane between lipopolysaccharide (LPS) and IcsP, which likely binds LPS. First, although the precise function of virK has not been determined, its genetic context within the locus shf-rfbU-virK-msbB2 suggests that it may be involved in LPS modification. Whereas the function of shf is unknown (29), rfbU and msbB2 each modify LPS (4, 9). Second, although complementation of the IcsA and intercellular spreading phenotypes of a virK::Tn10 mutant by virK without msbB2 (18) implicates virK in these phenotypes, it does not eliminate the possibility that both virK and msbB2 are involved.
Third, IcsP is a member of the omptin family of outer membrane proteases (25), which includes PgtE of Salmonella enterica serovar Typhimurium (30), Pla of Yersinia pestis (24), and OmpT (27) and OmpP (10) of Escherichia coli. Five of 11 residues involved in LPS binding by the outer membrane protein FhuA (5, 6) are conserved in OmpT, including 3 that interact with lipid A (28). Four of these five are also conserved in IcsP (M. B. Goldberg, unpublished data) and in other members of the omptin family (13). Moreover, the in vitro activity of OmpT is increased in the presence of LPS (12). Therefore, structural changes in lipid A that occur with mutation of msbB2 and perhaps virK::Tn10 may alter an interaction of lipid A with IcsP, which in turn may alter its stability or activity. These issues are the subject of ongoing investigation.
ACKNOWLEDGMENTS
We thank C. Sasakawa for generously providing strains YSH6000T, V836, V956, and V1060.
This work was supported by Public Health Service grants AI43562 and AI35817 from the National Institute of Allergy and Infectious Diseases (M.B.G.), a Charles H. Hood Foundation (Boston, Mass.) postdoctoral research fellowship from The Medical Foundation (H.J.W.), and a Massachusetts General Hospital Fund for Medical Discovery postdoctoral fellowship (H.J.W.).
H.J.W. and S.R.G. contributed equally to this work.
Present address: Department of Biological Sciences, University of Nevada-Las Vegas, Las Vegas.
REFERENCES
1. Bernardini, M. L., J. Mounier, H. d'Hauteville, M. Coquis-Rondon, and P. J. Sansonetti. 1989. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc. Natl. Acad. Sci. USA 86:3867-3871.
2. Caldwell, R. B., and C. T. Lattemann. 2004. Simple and reliable method to precipitate proteins from bacterial culture supernatant. Appl. Environ. Microbiol. 70:610-612.
3. Egile, C., H. d'Hauteville, C. Parsot, and P. J. Sansonetti. 1997. SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri. Mol. Microbiol. 23:1063-1073.
4. Fallarino, A., C. Mavrangelos, U. H. Stroeher, and P. A. Manning. 1997. Identification of additional genes required for O-antigen biosynthesis in Vibrio cholerae O1. J. Bacteriol. 179:2147-2153.
5. Ferguson, A. D., E. Hofmann, J. W. Coulton, K. Diederichs, and W. Welte. 1998. Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. Science 282:2215-2220.
6. Ferguson, A. D., W. Welte, E. Hofmann, B. Lindner, O. Holst, J. W. Coulton, and K. Diederichs. 2000. A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins. Struct. Fold Des. 8:585-592.
7. Goldberg, M. B., O. Barzu, C. Parsot, and P. J. Sansonetti. 1993. Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement. J. Bacteriol. 175:2189-2196.
8. Goldberg, M. B., and J. A. Theriot. 1995. Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility. Proc. Natl. Acad. Sci. USA 92:6572-6576.
9. Kaniuk, N. A., E. Vinogradov, J. Li, M. A. Monteiro, and C. Whitfield. 2004. Chromosomal and plasmid-encoded enzymes are required for assembly of the R3-type core oligosaccharide in the lipopolysaccharide of Escherichia coli O157:H7. J. Biol. Chem. 279:31237-31250.
10. Kaufmann, A., Y. D. Stierhof, and U. Henning. 1994. New outer membrane-associated protease of Escherichia coli K-12. J. Bacteriol. 176:359-367.
11. Kocks, C., J.-B. Marchand, E. Gouin, H. d'Hauteville, P. J. Sansonetti, M.-F. Carlier, and P. Cossart. 1995. The unrelated surface proteins ActA of Listeria monocytogenes and IcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocua and Escherichia coli, respectively. Mol. Microbiol. 18:413-423.
12. Kramer, R. A., K. Brandenburg, L. Vandeputte-Rutten, M. Werkhoven, P. Gros, N. Dekker, and M. R. Egmond. 2002. Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT. Eur. J. Biochem. 269:1746-1752.
13. Kukkonen, M., M. Suomalainen, P. Kyllonen, K. Lahteenmaki, H. Lang, R. Virkola, I. M. Helander, O. Holst, and T. K. Korhonen. 2004. Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. Mol. Microbiol. 51:215-225.
14. LaBrec, E. H., H. Schneider, T. J. Magnani, and S. B. Formal. 1964. Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysentery. J. Bacteriol. 88:1503-1518.
15. Lett, M.-C., C. Sasakawa, N. Okada, T. Sakai, S. Makino, M. Yamada, K. Komatsu, and M. Yoshikawa. 1989. virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence. J. Bacteriol. 171:353-359.
16. Makino, S., C. Sasakawa, K. Kamata, T. Kurata, and M. Yoshikawa. 1986. A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell 46:551-555.
17. Maurelli, A. T., and R. Curtiss, 3rd. 1984. Bacteriophage Mu d1(Apr lac) generates vir-lac operon fusions in Shigella flexneri 2a. Infect. Immun. 45:642-648.
18. Nakata, N., C. Sasakawa, N. Okada, T. Tobe, I. Fukuda, T. Suzuki, K. Komatsu, and M. Yoshikawa. 1992. Identification and characterization of virK, a virulence-associated large plasmid gene essential for intercellular spreading of Shigella flexneri. Mol. Microbiol. 6:2387-2395.
19. Oaks, E. V., M. E. Wingfield, and S. B. Formal. 1985. Plaque formation by virulent Shigella flexneri. Infect. Immun. 48:124-129.
20. Ogawa, H., A. Nakamura, and R. Nakaya. 1968. Cinemicrographic study of tissue cell cultures infected with Shigella flexneri. Jpn. J. Med. Sci. Biol. 21:259-273.
21. Okada, N., C. Sasakawa, T. Tobe, K. A. Talukder, K. Komatsu, and M. Yoshikawa. 1991. 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. Mol. Microbiol. 5:2171-2180.
22. Pal, T., J. W. Newland, B. D. Tall, S. B. Formal, and T. L. Hale. 1989. Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein. Infect. Immun. 57:477-486.
23. Shere, K. D., S. Sallustio, A. Manessis, T. G. D'Aversa, and M. B. Goldberg. 1997. Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility. Mol. Microbiol. 25:451-462.
24. Sodeinde, O. A., and J. D. Goguen. 1989. Nucleotide sequence of the plasminogen activator gene of Yersinia pestis: relationship to ompT of Escherichia coli and gene E of Salmonella typhimurium. Infect. Immun. 57:1517-1523.
25. Stathopoulos, C. 1998. Structural features, physiological roles, and biotechnological applications of the membrane proteases of the OmpT bacterial endopeptidase family: a micro-review. Membr. Cell Biol. 12:1-8.
26. Steinhauer, J., R. Agha, T. Pham, A. W. Varga, and M. B. Goldberg. 1999. The unipolar Shigella surface protein IcsA is directly targeted to the old pole; IcsP cleavage of IcsA occurs over the entire bacterial surface. Mol. Microbiol. 32:367-378.
27. Sugimura, K., and T. Nishihara. 1988. Purification, characterization, and primary structure of Escherichia coli protease VII with specificity for paired basic residues: identity of protease VII and OmpT. J. Bacteriol. 170:5625-5632.
28. Vandeputte-Rutten, L., R. A. Kramer, J. Kroon, N. Dekker, M. R. Egmond, and P. Gros. 2001. Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site. EMBO J. 20:5033-5039.
29. Venkatesan, M. M., M. B. Goldberg, D. J. Rose, E. J. Grotbeck, V. Burland, and F. R. Blattner. 2001. Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri. Infect. Immun. 69:3271-3285.
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