Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence

ABSTRACT Cardiolipin, an anionic phospholipid that resides at the poles of the inner and outer membranes, is synthesized primarily by the putative cardiolipin synthase ClsA in Shigella flexneri. An S. flexneri clsA mutant had no cardiolipin detected within its membrane, grew normally in vitro, and invaded cultured epithelial cells, but it failed to form plaques in epithelial cell monolayers, indicating that cardiolipin is required for virulence. The clsA mutant was initially motile within the host cell cytoplasm but formed filaments and lost motility during replication and failed to spread efficiently to neighboring cells. Mutation of pbgA, which encodes the transporter for cardiolipin from the inner membrane to the outer membrane, also resulted in loss of plaque formation. The S. flexneri pbgA mutant had normal levels of cardiolipin in the inner membrane, but no cardiolipin was detected in the outer membrane. The pbgA mutant invaded and replicated normally within cultured epithelial cells but failed to localize the actin polymerization protein IcsA properly on the bacterial surface and was unable to spread to neighboring cells. The clsA mutant, but not the pbgA mutant, had increased phosphatidylglycerol in the outer membrane. This appeared to compensate partially for the loss of cardiolipin in the outer membrane, allowing some IcsA localization in the outer membrane of the clsA mutant. We propose a dual function for cardiolipin in S. flexneri pathogenesis. In the inner membrane, cardiolipin is essential for proper cell division during intracellular growth. In the outer membrane, cardiolipin facilitates proper presentation of IcsA on the bacterial surface.

S. flexneri and by the inflammatory immune response of the host. Shigella spp. are closely related to Escherichia coli but encode a number of virulence determinants, many of which are encoded on a large plasmid (5). Virulence proteins include components of a Type III secretion system (T3SS) that injects effector proteins into the cytoplasm of the epithelial cells to initiate uptake of the bacteria and alter host cell responses (6). The subsequent spread to adjacent epithelial cells requires intracellular replication, movement mediated by cytoplasmic host actin (7), and reactivation of the T3SS (8)(9)(10).
S. flexneri, which is nonmotile, uses actin-based propulsion to move through the host cell cytoplasm and protrude into the neighboring cell. Actin-based motility requires proper localization of the virulence protein IcsA on the bacterial surface (11,12). IcsA mutants are invasive and replicate within epithelial cells but cannot spread to adjacent cells (13). IcsA is secreted through the inner membrane via the Sec system (14) and requires the periplasmic chaperone proteins DegP, Skp, and SurA for localization to the outer membrane (15,16). IcsA is a member of the autotransporter protein family (17); its carboxy-terminus domain forms a beta barrel channel to mediate the transfer of its amino-terminal passenger proteinase domain across the outer membrane (18). Once IcsA is properly oriented on the bacterial surface, the amino-terminal domain recruits phosphorylated neural Wiskott-Aldrich syndrome protein (N-WASP) (19,20), which in turn recruits the Arp 2/3 complex that activates actin polymerization to propel the bacteria (21). The localization of IcsA on the surface of S. flexneri is to the old pole of the bacterial cell (11); however, the exact mechanism for unipolar targeting of IcsA remains unknown (22). Previous studies have shown that mutations that alter the O-antigen chain length of S. flexneri lipopolysaccharide (LPS) are associated with disruption of IcsA localization (23). An increase in O-antigen length can shield IcsA to inhibit its function (24), whereas decreased O-antigen chain length results in a uniform distribution of IcsA on the surface (25).
S. flexneri, like other Gram-negative bacteria, has an asymmetric outer membrane in which the lipid A portion of the LPS forms the outer leaflet, while the inner leaflet is made up of phospholipids (26). Both leaflets of the inner membrane are phospholipid. In S. flexneri, mutation of vpsC, a component of the Mla pathway (27), causes an accumulation of phospholipids in the outer leaflet of the outer membrane and remodeling of the lipid A species from hexa-acylated to hepta-acylated and results in impaired intercellular spread (28). Active remodeling of lipid A species during infection has been reported for Gram-negative pathogens, and these alterations allow the pathogens to evade the host immune response and protect themselves from environmental stress (29). However, it is not known whether the effects of vpsC mutation on S. flexneri virulence are due to changes in lipid A or the phospholipids in the outer membrane. In general, relatively little is known of the effects of specific phospholipids on bacterial pathogenesis.
During growth in exponential phase, phosphoethanolamine (PE) is the major phospholipid in the membrane of E. coli, representing almost 80% of the total phospholipids (30). The anionic phospholipids phosphatidylglycerol (PG) and cardiolipin account for about 18% and 2.5%, respectively (30). In the family Enterobacteriaceae, cardiolipin is synthesized within the inner membrane by ClsA, ClsB, and ClsC (30)(31)(32), and a portion of the cardiolipin is transported to the outer membrane by PbgA (33)(34)(35). Cardiolipin is a large anionic glycerol phospholipid composed of four large acyl chains connected by a small glycerol head group (36), giving it a conical shape. This shape allows it to accumulate at membrane regions that have negative curvature (37), including the negative membrane curvature regions of the inner mitochondrial matrix where cardiolipin was first identified (38). In E. coli, cardiolipin appears to localize to the poles of the bacterial inner leaflets of both the inner and outer membranes (39). Cardiolipin has been shown to play roles in both the localization and activity of electron transport proteins both in the mitochondria (36) and in the inner membrane of E. coli (40). Cardiolipin has also been shown to be important for localization or activity of proteins required for cell division (41) and osmotic stress response (42) in E. coli. In the absence of cardiolipin, the anionic phospholipid PG, which shares the same glycerol head group as cardiolipin, will localize to the bacterial poles (37) and interact (43) with proteins in a manner similar to that of cardiolipin, which has complicated studying the role of cardiolipin in bacterial membranes.
In this study, we determined that clsA encodes the major cardiolipin synthase and pgbA encodes the phospholipid transporter in S. flexneri. Both of these genes were required for S. flexneri plaque formation but acted at different points in the virulence pathway. This study demonstrates a role for phospholipids, specifically cardiolipin, in S. flexneri pathogenesis, and provides a model for their contribution to IcsA localization to the bacterial surface.
gene, which encodes the PbgA protein that is responsible for transporting cardiolipin to the outer membrane, a requirement for maintaining outer membrane integrity during growth in the host cells (34,46). Complete deletion of pbgA in E. coli and S. Typhimurium is lethal; however, mutants with a deletion of the C-terminal periplasmic portion of PbgA were viable but lacked cardiolipin transport to the outer membrane (33). Therefore, we made a similar deletion of the C terminus of the S. flexneri pbgA homolog (yejM) to determine the effects of eliminating cardiolipin from the outer  (30 -32). The chemical structures of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL) were produced using ChemDraw (PerkinElmer). (B) TLC analysis of total S. flexneri membrane phospholipids of the wild type (WT) and cardiolipin synthesis mutants. Bacteria were grown to mid-log phase, and phospholipids were extracted and separated by TLC. The percentage of cardiolipin in the sample is indicated above each lane. pclsA, plasmid expressing clsA. (C) Bacteria were grown into stationary phase (OD 650 of~2.0); phospholipids were then extracted and separated by TLC. The percentage of cardiolipin in the sample is indicated above each lane. Phospholipids were visualized using molybdenum blue spray reagent (Sigma). membrane. Membrane fractionation and analysis of the phospholipids confirmed that the S. flexneri pbgA mutant lacked cardiolipin in the outer membrane ( Fig. 2A), while the phospholipid composition of the inner membrane of the pbgA mutant looked identical to that of the wild type ( Fig. 2A). The clsA mutant did not have cardiolipin in the outer membrane but had increased PG, maintaining the proportion of anionic lipids that make up the outer membrane similar to that of the wild type, approximately 32%. In contrast, because the pbgA mutant did not have increased PG levels in the outer membrane, the outer membrane anionic lipid percentage significantly decreased from wild type to only 18%. Together, these results suggest that S. flexneri PbgA, like the S. Typhimurium homolog, is responsible for transporting cardiolipin and some PG to the outer membrane and maintaining the normal levels of anionic phospholipids in the outer membrane.
Cardiolipin synthesis is required for plaque formation. To determine the role of cardiolipin in S. flexneri pathogenesis, we performed plaque assays (47) with each of the cardiolipin synthesis mutants. Plaque formation required invasion of the monolayer, intracellular replication, and spread to the adjacent cells. After 72 h, the clsA mutant formed pinpoint plaques compared to the WT, and plaque formation was complemented by clsA on a plasmid (Fig. 3). Because ClsC contributed to cardiolipin synthesis in stationary phase, it was possible that the ClsB or ClsC was expressed in the intracellular environment and contributed sufficient cardiolipin to support formation of very small plaques. Both clsB and clsC were induced approximately 10-fold in intracel- Bacteria were grown to mid-log phase, and bacterial membranes were separated by solubilization in Sarkosyl. The phospholipids were then extracted, spotted for separation by TLC, and visualized using molybdenum blue spray reagent (Sigma). The phospholipids include phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL). The percentage of anionic phospholipids (AL) in the sample is indicated above each lane, and means Ϯ standard deviations of three experiments are shown. The value that is significantly different (P Ͻ 0.05) from the value for the outer membrane of the wild type by Student's t test is indicated by an asterisk. (B) Confirmation of clean inner and outer membrane fractions. Bacteria were grown to mid-log phase, and the bacterial membranes were separated by solubilization using Sarkosyl. Proteins were resolved by (10%) SDS-PAGE and stained with Coomassie blue (CB) or immunoblotted using either polyclonal anti-SecA or polyclonal anti-OmpA. The molecular masses (in kilodaltons) of molecular mass markers are shown to the left of the gels. ppbgA, plasmid expressing pbgA; C, cytoplasm; IM, inner membrane; OM, outer membrane. lular bacteria (see Fig. S1 in the supplemental material). However, mutations in clsB and clsC did not affect S. flexneri plaque formation (Fig. 3), indicating that cardiolipin synthesis by ClsA is required for wild-type plaque formation and that ClsB and ClsC do not synthesize sufficient cardiolipin in intracellular bacteria to compensate fully for the loss of clsA.
PbgA, the transporter of cardiolipin to the outer membrane, is required for S. flexneri plaque formation. To determine whether cardiolipin is required in the outer membrane of S. flexneri for virulence, the pbgA mutant was tested for plaque formation. The pbgA mutant was unable to form plaques, and plaque formation was restored by full-length pbgA on a plasmid (Fig. 3). This indicates that transport of cardiolipin to the outer membrane is critical for plaque formation. The clsA and pbgA mutants were further characterized to determine more precisely the roles of cardiolipin in S. flexneri virulence.
Cardiolipin synthesis and transport do not contribute to S. flexneri membrane integrity. Previous studies have shown that a disruption in outer membrane integrity inhibits S. flexneri plaque formation (28). To determine whether the defect in plaque formation by the clsA and pbgA mutants is the result of reduced outer membrane integrity due to lack of cardiolipin, we assessed the mutant for increased sensitivity to sodium deoxycholate (DOC), an ionic bile acid that disrupts the membrane. The clsA and pbgA mutants grew similarly to the wild type, with an average doubling time of In contrast, the vpsC mutant, which has compromised outer membrane stability (28), was inhibited in the presence of DOC. This suggests that cardiolipin within the outer membrane of S. flexneri is not playing a structural role in the integrity of the outer membrane of S. flexneri.
Cardiolipin synthesis and transport are not required for S. flexneri cellular invasion and intracellular replication. The initial stage of S. flexneri virulence requires the bacteria to invade colonic epithelial cells (2) followed by intracellular replication (48). A requirement for cardiolipin for either of these processes would result in reduced plaque formation. Therefore, we compared the clsA and pbgA mutants to the wild type and found that neither mutant had a significant defect in invasion compared to the wild-type parental strain (Table S1). Infection rates were determined for cells grown in the presence and absence of DOC, since previous work (49) had shown that DOC increased virulence protein secretion and infectivity of wild-type Shigella. To assess intracellular replication, we isolated intracellular bacteria at 60 and 180 min postinfection and determined their doubling time. We found that compared to the WT, neither the clsA nor pbgA mutant had a significant defect in intracellular replication (Table S1). Together, these data indicate that cardiolipin is not required for Henle cell invasion and intracellular replication during the first 3 h of infection.
S. flexneri intercellular spread requires cardiolipin synthesis and transport to the outer membrane. The lack of an effect of cardiolipin on invasion and intracellular replication suggested that the plaque defect was due to lack of cell-to-cell spread of the bacteria (50). To determine the role of cardiolipin in S. flexneri intercellular spread, we determined the percentage of primary Henle cell infections resulting in spread to neighboring cells after 4 h, using a cell-to-cell spread assay (28). Primary infected Henle cells were identified as cells containing large numbers of bacteria, indicating intracellular replication (Fig. 4A). Infective centers were scored positive for spread if one or more neighboring cells had three or more intracellular bacteria. After 4 h, wild-type S. flexneri had spread from Ͼ80% of the initially infected cells to neighboring cells (Fig. 4B). The clsA and pbgA mutants, however, had significant defects in intercellular spread with spread rates of only 20 and 24%, respectively. These rates mimicked the rate of an icsA mutant, which cannot spread (13). These results suggest that the defect in plaque formation of both the clsA and pbgA mutants is their inability to spread intercellularly.
Synthesis and transport of cardiolipin to the outer membrane of S. flexneri are required for intercellular dissemination in HT-29 cells. Intercellular spread of S. flexneri is a dynamic, multistep process (22); it requires the bacteria to polymerize host actin to move and penetrate neighboring cells (4), reactivate their T3SS to eject effector proteins into neighboring cell cytoplasm (51), and resolve the membrane protrusions into neighboring cells (9). To determine specifically at which point during intercellular spread the clsA and pbgA mutants have a defect, we used infection of HT-29 intestinal cells, which allowed us to employ time-lapse confocal microscopy to monitor cell-tocell spread over an extended period of time (52). The bacteria are easily visualized in HT-29 cells, but they grow and spread more slowly in this cell line than in HeLa cells. Thus, longer time periods were used for the spread analysis. First, cell-to-cell spread was quantified by using computer-assisted image analysis to measure the areas of the infected foci in cellular monolayers (Fig. 5A). After 8 h, the pbgA mutant, but not the complemented strain, showed a significant decrease in the area of intercellular spread compared to the WT (Fig. 5B), indicating that outer membrane cardiolipin is required for S. flexneri intercellular spread early in infection. The clsA mutant also showed a defect in spread, although a significant decrease in the focus area in HT-29 cells was not detected until 8 to 16 h of infection ( Fig. 5C and D). The difference in the rate of spread of the clsA mutant compared to that of the pbgA mutant may be due to increased PG in the outer membrane of the clsA mutant that partially compensates for the loss of cardiolipin, which does not occur in the strain lacking PbgA.
Cardiolipin synthesis is required for proper intracellular division of S. flexneri. Using the HT-29 intestinal cell line model, we analyzed the timing of cell-to-cell spread of the clsA mutant by time-lapse confocal microscopy (9). HT-29 cell monolayers expressing plasma membrane-targeted yellow fluorescent protein (YFP) were infected with S. flexneri expressing isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible cyan fluorescent protein (CFP), and individual bacteria were tracked. The wild-type strain showed rapid cell division, motility and spread to adjacent cells (Movie S1). The behaviour of the clsA mutant (Movie S2) was similar to that of the wild type at the early time points. It was motile in the initially infected cell and had normal intracellular growth (Fig. 6A). However, after the initial rounds of replication, the clsA mutant began to form filaments (Fig. 6), and there was a loss of motility (Movie S2). Some of the clsA mutant cells formed protrusions that were unable to form vacuoles and retracted back into the cell (Movie S2). Thus, cardiolipin is required in the membrane of S. flexneri for proper cell division after extended intracellular growth. Cardiolipin and Virulence of Shigella ® The transport of cardiolipin to S. flexneri's outer membrane is not required for intracellular growth. To define more precisely whether the defect in intracellular replication in the cardiolipin synthesis mutant (clsA) was due to lack of cardiolipin in the inner or outer membrane, we also performed time-lapse microscopy on the pbgA mutant, which has cardiolipin in the inner membrane, but not in the outer membrane (Movie S3). Unlike the clsA mutant, the pbgA mutant grew normally within the cytoplasm of HT-29 cells and showed no filamentation, indicating that cardiolipin is needed in the inner membrane for maintaining wild-type replication in the intracellular environment. However, the pbgA mutant was nonmotile inside the eukaryotic cell and did not spread intercellularly. Thus, cardiolipin or increased PG in the outer membrane promotes intracellular motility. For comparison, the vpsC mutant, which has altered phospholipids and lipid A modifications in the outer membrane (28), was analyzed (Movie S4). The vpsC mutant had an intracellular infection phenotype that was distinct from either of the cardiolipin mutants. It replicated normally and was motile, but its motility was less than the wild-type motility, and there was no spread to adjacent cells.
Unipolar IcsA localization requires the cardiolipin transporter PbgA. Because the pbgA mutant was nonmotile within the intracellular environment, it was likely that the defect was in the export or localization of IcsA. To determine whether PbgA was required for IcsA localization, we used indirect immunofluorescence directed against IcsA to visualize surface IcsA localization of S. flexneri grown in vitro (16). Compared to the WT, the pbgA mutant displayed very low levels of surface IcsA recognized by the antibody (Fig. 7A). Wild-type, unipolar localization of IcsA was restored with full-length pbgA on a plasmid (Fig. 7A). This suggested that the lack of cardiolipin within the outer membrane, the only known defect of the pbgA mutant, reduced localization of IcsA on the surface of the bacteria.
The clsA mutant, which also lacks cardiolipin in the outer membrane, retained motility during the initial stage of infection of HT-29 cells, and IcsA localization at the pole was detected in the clsA mutant, although it was reduced compared to the wild  Fig. 2B), and immunoblotted using either polyclonal anti-IcsA antisera. C, cytoplasm; IM, inner membrane; OM, outer membrane. (C) LPS structure of clsA and pbgA mutants. Bacteria were grown to mid-log phase. LPS was extracted, resolved by (4 to 12%) SDS-PAGE, and visualized by silver staining. type (Fig. 7A). Therefore, if cardiolipin normally plays a role in IcsA localization in wild-type cells, the increase in outer membrane PG that occurs in the clsA mutant, but not the pbgA mutant, may be able to partially compensate for the loss of cardiolipin.
Although the anionic lipids cardiolipin and PG are known to be capable of localizing membrane proteins, the disruption in localization of IcsA could be indirect. Export of IcsA to the outer membrane (15) may be inefficient in the pbgA mutant, or mislocalization of IcsA could be due to disruption in LPS chain length (23) if the mutations affect LPS structure. To determine whether cardiolipin plays a role in efficient transport of IcsA to the outer membrane, we fractionated the inner and outer membranes of S. flexneri and examined the IcsA levels in each membrane using immunoblot analysis. We found that both the clsA and pbgA mutants had WT levels of IcsA within their outer membrane (Fig. 7B), indicating that cardiolipin synthesis or transport is not necessary for IcsA stability in the membrane or its export to the S. flexneri outer membrane. To determine whether the absence of cardiolipin disrupts the LPS of S. flexneri, we compared the LPS profiles of clsA and pbgA mutants to the wild-type LPS and to a previously characterized rol LPS mutant (23) by gel electrophoresis. The clsA and pbgA mutants did not have any detected differences in LPS chain lengths compared to the WT (Fig. 7C). Together, these data indicate that cardiolipin or compensatory levels of PG in the outer membrane are directly involved in the localization of IcsA on the surface.

DISCUSSION
To cause disease, S. flexneri must efficiently invade colonic epithelial cells and spread intercellularly to neighboring cells. Penetration of the adjacent cellular membranes requires S. flexneri to move within the cytoplasm, and this movement is a direct result of IcsA-mediated actin polymerization (13). We have previously shown that outer membrane integrity, mediated by asymmetric distribution of phospholipids and lipid A structure, is required by S. flexneri during intercellular spread (28); however, the roles of specific phospholipids in S. flexneri pathogenesis have not been determined. In this study, we show that the phospholipid cardiolipin is required in the inner membrane of S. flexneri for proper cell division during intercellular spread (Fig. 6B), while outer membrane cardiolipin is associated with proper localization of IcsA and intercellular spread (Fig. 7A).
Cardiolipin is a large anionic phospholipid that makes up only 5 to 10% of S. flexneri's membrane phospholipids (Fig. 1B). Much of what is currently known about cardiolipin's role in biological membranes is the result of studies of the eukaryotic mitochondria, where cardiolipin makes up a large portion of the inner mitochondrial matrix to promote the localization and activity of electron transport proteins (36). Cardiolipin function is largely conserved in E. coli, where it is required for localization of high-energy electron transport (40) and osmotic proteins (42) within the bacterial inner membrane. Still, much remains unknown regarding cardiolipin's role in bacterial membranes, and this is likely for two reasons. First, most enteric bacteria have three cardiolipin synthases, and all three must be inactivated in order to eliminate cardiolipin from the bacterial membrane (32); most studies thus far have been performed only on bacteria with reduced cardiolipin levels. Second, PG, which is similar to cardiolipin in that it also has a glycerol head group and is an anionic phospholipid, can interact with proteins in a similar manner and compensate for the lack of cardiolipin within the bacterial membrane (43). Thus, bacteria lacking cardiolipin do not display an in vitro growth phenotype.
To date, the roles of cardiolipin in bacterial pathogenesis have been identified in two pathogens, S. Typhimurium and Moraxella catarrhalis. In S. Typhimurium, cardiolipin is required in the outer membrane to provide membrane integrity during infection. Increased expression of pbgA and remodeling of the outer membrane occur in response to PhoPQ signaling during Salmonella infection (34). Salmonella, like Shigella, is an intracellular pathogen; however, Salmonella resides within lysosomes of macrophages, which may represent a more stressful environment than the cytoplasm. Since S. flexneri lacking cardiolipin does not have reduced membrane integrity when grown in the presence of DOC, and does not have an in vivo growth defect, it is unlikely that the role of cardiolipin in S. flexneri pathogenesis is maintenance of S. flexneri membrane integrity.
In M. catarrhalis, cardiolipin is required for proper bacterial attachment to human epithelial cells (53). It is hypothesized that cardiolipin is required for the localization or display of adhesion proteins. This may represent a similar function to cardiolipin's role in S. flexneri, where cardiolipin in the outer membrane is important for the localization of the IcsA (Fig. 7A).
In E. coli, cardiolipin specifically localizes to the inner leaflet of the bacterial poles (39). This is because cardiolipin has a small glycerol head group and a large acyl region with four chains, giving the overall structure of cardiolipin a conical shape. Bacteria lacking cardiolipin do not have altered cell morphology, supporting the model that the conical shape of cardiolipin does not dictate the negative curvature of the poles. Rather, its localization at the poles is a consequence of its shape. Cardiolipin localizes to bacterial poles via diffusion, because of its natural tendency to destabilize planar membranes. In the absence of cardiolipin, PG localizes to the bacterial pole and can interact with polar proteins in the same manner but less efficiently than cardiolipin (43). We predict that in cardiolipin's absence in S. flexneri, increased PG in the outer membrane can help localize IcsA to the pole; however, this localization does not appear to be as specific or as efficient as when cardiolipin is present. Polar IcsA localization is directed also by the outer leaflet LPS structure (23)(24)(25). Cardiolipin does not affect LPS structure, but it is not known whether the LPS structure affects outer membrane cardiolipin localization. It is possible that changes in the LPS structure disrupt polar localization of cardiolipin, indirectly causing the mislocalization of IcsA.
Cardiolipin may directly interact with IcsA, as has been shown for some proteins, to concentrate the protein at the pole. In the inner mitochondrial matrix, lysine residues on the surface of Drp1 interact with the glycerol head group of cardiolipin, helping it localize with cardiolipin (54). It is possible that positively charged residues on the surface of the IcsA beta barrel or the polar targeting (PT) domain identified in the N-terminal region of IcsA (55,56) help IcsA localize with cardiolipin. Alternatively, cardiolipin could indirectly localize IcsA by interacting with the positively charged residues of other proteins known to aid in its localization and activity (57). For example, IcsA chaperone (15,16,58,59) or secretion proteins (60) may interact with cardiolipin to direct insertion of IcsA into the outer membrane at the poles. These interactions would promote polar localization of IcsA to allow directed movement when actin polymerizes at the bacterial surface.
The loss of cardiolipin from the outer membrane without compensation by increased PG affects plaque formation by preventing IcsA localization. The effects of loss of cardiolipin from the inner membrane are less clear. The clsA mutant replicates normally in vitro and has no obvious defect early in infection of epithelial cells. However, longer exposure to the intracellular environment results in aberrant cell division and loss of motility. Studies of E. coli have shown that the cell division protein MinD and osmotic stress proteins associate with cardiolipin in the inner membrane (42,43). Similar effects of cardiolipin on localization of inner membrane proteins in S. flexneri could cause the growth defects seen in the host cell cytoplasm.

MATERIALS AND METHODS
Bacterial strains and growth conditions. Bacterial strains and plasmids used in this study are found in Table S2 in the supplemental material. All strains were maintained at Ϫ80°C in tryptic soy broth (TSB) containing 20% (vol/vol) glycerol. E. coli strains were grown on Luria-Bertani (LB) agar (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar [wt/vol]) at 37°C, and single colonies were selected and grown in LB broth at 37°C. S. flexneri strains were grown on TSB agar (TSB, 1.5% agar [wt/vol]) containing Congo red dye (0.01% [wt/vol]; Sigma) at 37°C. Congo red binding colonies (61), indicating a functional T3SS, were selected and grown in LB broth at 30°C for maintenance and were then subcultured 1:100 and grown at 37°C for assays. The following antibiotics were used at the indicated concentrations: kanamycin, 50 g/ml; ampicillin, 25 g/ml.
Construction of S. flexneri mutants. The S. flexneri clsA, clsB, clsC, and vpsC mutants were created by bacteriophage P1 transduction of the ΔclsA::kan, ΔclsB::kan, ΔclsC::kan, and ΔmlaD::kan alleles from IcsA localization was monitored as previously described (16). Briefly, bacteria were grown to midlogarithmic phase and fixed in 4% (vol/vol) paraformaldehyde in PBS. The cells were then labeled by indirect immunofluorescence, using rabbit polyclonal antibody against IcsA (rabbit 35) diluted 1:100, provided by Edwin Oaks (Walter Reed Army Institute of Research), and a fluorescent isothiocyanateconjugated goat anti-rabbit secondary antibody diluted 1:100 (15). Images were acquired using a DP73 digital microscope camera (Olympus) and processed using cellSens software (Olympus). Images were postprocessed with Lightroom (Adobe) to increase contrast. All images were processed using the same settings.
SDS-PAGE and immunoblotting. Inner and outer membranes were isolated by membrane fractionation as described above and resuspended in Laemmli SDS sample buffer (5% ␤-mercaptoethanol, 3% [wt/vol] SDS, 10% glycerol, 0.02% bromophenol blue, 63 mM Tris-Cl [pH 6.8]) (69), and boiled for 5 min. Samples were electrophoresed in quadruplicate (10%) SDS-polyacrylamide gels for separation. Proteins from three gels were transferred to a 0.45-m-pore-size nitrocellulose membrane (GE Healthcare) and incubated with either rabbit polyclonal anti-IcsA antibody (Edwin Oaks, Walter Reed Army Institute of Research) diluted 1:10,000, rabbit polyclonal anti-SecA antibody (Donald Oliver, Wesleyan University) diluted 1:10,000, or rabbit polyclonal anti-OmpA antibody (Donald Oliver, Wesleyan University) diluted 1:5,000. Proteins were detected using horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:5,000). Signal was detected by developing the blot with Pierce ECL detection kit (Thermo Fisher). Proteins from the fourth gel were visualized by Coomassie brilliant blue staining and used to assess equal loading of samples for immunoblotting.
Analysis of lipopolysaccharides. LPS was isolated and analyzed as previously described (70). Briefly, bacteria were grown at 37°C to an OD 650 of~0.5, and the equivalent of OD 650 of 1 was pelleted and resuspended in Laemmli SDS-PAGE sample buffer (69). Samples were then boiled for 10 min, cooled to room temperature, and treated with 25 g proteinase K for 1 h at 55°C. LPS was visualized by (4 to 12%) SDS-PAGE (Bolt Bis-Tris Plus; Invitrogen) and subsequent staining of the LPS with silver stain as follows. The gels were fixed in 40% isopropanol and 5% acetic acid, oxidized with 0.7% periodic acid, stained with 20% silver nitrate, and developed using 50 g/ml citric acid.