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Infection and Immunity, February 2005, p. 1217-1220, Vol. 73, No. 2
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.2.1217-1220.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Modulation of an Outer Membrane Protease Contributes to the Virulence Defect of Shigella flexneri Strains Carrying a Mutation in the virK Locus

Helen J. Wing ,{dagger},{ddagger} Seth R. Goldman,{dagger} Shabeen Ally, and Marcia B. Goldberg*

Infectious Disease Division, Massachusetts General Hospital, Boston, Massachusetts

Received 9 July 2004/ Returned for modification 16 September 2004/ Accepted 20 October 2004


   ABSTRACT
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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.


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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.



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  		FIG. 1. Role of IcsP in IcsA expression and the intercellular spreading phenotype of the virK::Tn10 mutant. (A and B) Western blot analysis with antiserum to IcsA or IcsP (7, 26). The amount of protein relative to that of the wild-type (WT) strain, as determined by band densitometry, is indicated below the lanes. IcsA or IcsP, full-length mature IcsA or IcsP in the bacterial pellet; IcsA', truncated IcsA in the culture supernatant. The strain genotype is indicated at the top, and the apparent molecular weight (103) is indicated at the left. Loading was normalized to the optical density of the bacterial culture at 600 nm. (C) Formation of plaques by intracellular bacteria. The strain genotype is adjacent to each panel. Images were taken at 72 h. Strain designations are as follows: WT, YSH6000T; virK:Tn10, V956; icsA, SRG10; icsP, HJW9; virK::Tn10 icsP, SRG12A (Table 2). For each panel, the images are representative of those obtained from three or more independent experiments.

 
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.


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  		TABLE 1. Partial rescue of plaque size by introduction of the icsP mutation into the virK::Tn10 mutant

 
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-ß-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.


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  		TABLE 2. Strains and plasmids used in this study

 


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  		FIG. 2. Defects in actin-based motility and intercellular spreading associated with increased levels of IcsP. The phenotype upon addition of IPTG to the wild-type (WT) strain (YSH6000T) carrying (+) or not carrying (–) an IPTG-inducible icsP expression construct (PIPTG-icsP) is shown. (A) IcsA and IcsP associated with the bacterial pellet. A Western blot analysis with antiserum to IcsA or IcsP is shown. The apparent molecular weight (103) is indicated at the left. Millimolar concentrations of IPTG are indicated above the lanes. (B) Actin tail formation by intracellular bacteria. Actin staining by phalloidin (top) and bacterial and cellular DNA staining by 4',6'-diamidino-2-phenylindole (DAPI) (bottom) are shown. Arrows, actin tails associated with intracellular bacteria; arrowheads, cluster of intracellular bacteria. (C) Formation of plaques by intracellular bacteria. Images were taken at 72 h. For each panel, the images are representative of those obtained from three or more independent experiments.

 

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  		TABLE 3. Sizes of plaques formed by bacteria expressing increased levels of icsP

 
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.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Bacterial Pathogenesis Laboratories, University Park, 65 Landsdowne St., Cambridge, MA 02139. Phone: (617) 768-8740. Fax: (617) 768-8738. E-mail: mgoldberg1{at}partners.org. Back

Editor: J. N. Weiser

{dagger} H.J.W. and S.R.G. contributed equally to this work. Back

{ddagger} Present address: Department of Biological Sciences, University of Nevada-Las Vegas, Las Vegas. Back


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Infection and Immunity, February 2005, p. 1217-1220, Vol. 73, No. 2
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.2.1217-1220.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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