Mutagenesis of the Shigella flexneri autotransporter IcsA reveals novel functional regions involved in IcsA biogenesis and recruitment of host neural Wiscott-Aldrich syndrome protein

The IcsA (VirG) protein of Shigella flexneri is a polarly localized, outer membrane protein that is essential for virulence. Within host cells, IcsA activates the host actin regulatory protein, neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn recruits the Arp2/3 complex, which nucleates host actin to form F-actin comet tails and initiate bacterial motility. Linker insertion mutagenesis was undertaken to randomly introduce 5-amino-acid in-frame insertions within IcsA. Forty-seven linker insertion mutants were isolated and expressed in S. flexneri Delta icsA strains. Mutants were characterized for IcsA protein production, cell surface expression and localization, intercellular spreading, F-actin comet tail formation, and N-WASP recruitment. Using this approach, we have identified a putative autochaperone region required for IcsA biogenesis, and our data suggest an additional region, not previously identified, is required for N-WASP recruitment.     

Neural Wiskott-Aldrich syndrome protein is implicated in the actin-based motility of Shigella flexneri.

Shigella, the causative agent of bacillary dysentery, is capable of directing its own movement in the cytoplasm of infected epithelial cells. The bacterial surface protein VirG recruits host components mediating actin polymerization, which is thought to serve as the propulsive force. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP), which is a critical target for filopodium formation downstream of Cdc42, is required for assembly of the actin tail generated by intracellular S.flexneri. N-WASP accumulates at the front of the actin tail and is capable of interacting with VirG in vitro and in vivo, a phenomenon that is not observed in intracellular Listeria monocytogenes. The verprolin-homology region in N-WASP was required for binding to the glycine-rich repeats domain of VirG, an essential domain for recruitment of F-actin on intracellular S.flexneri. Overexpression of a dominant-negative N-WASP mutant greatly inhibited formation of the actin tail by intracellular S.flexneri. Furthermore, depletion of N-WASP from Xenopus egg extracts shut off Shigella actin tail assembly, and this was restored upon addition of N-WASP protein, suggesting that N-WASP is a critical host factor for the assembly of the actin tail by intracellular Shigella.     

Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.     

Quantification of Shigella IcsA required for bacterial actin polymerization

Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500-2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella-infected cells.     

Cdc42 facilitates invasion but not the actin-based motility of Shigella

The enteric pathogen Shigella utilizes host-encoded proteins to invade the gastrointestinal tract. Efficient invasion of host cells requires the stimulation of Rho-family GTPases and cytoskeletal alterations by Shigella-encoded IpaC. Following invasion and lysis of the phagosome, Shigella exploits the host's actin-based polymerization machinery to assemble an actin tail that serves as the propulsive force required for spreading within and between cells. The Shigella surface protein IcsA stimulates actin-tail formation by recruiting host-encoded N-WASP to drive Arp2/3-mediated actin assembly. N-WASP is absolutely required for Shigella motility, but not for Shigella invasion. Although Rho-family GTPases have been implicated in both the invasion and motility of Shigella, the role of Cdc42, an N-WASP activator, in this process has been controversial. In these studies, we have examined the role of Cdc42 in Shigella invasion and actin-based motility using Cdc42-deficient cells. We demonstrate that Cdc42 is required for efficient Shigella invasion but reveal a minor Cdc42-independent pathway that can permit Shigella invasion. However, the actin-based motility of Shigella, as well as vaccinia, proceeds unperturbed in the absence of Cdc42. These data further support the involvement of distinct host-encoded proteins in the steps regulating invasion and intercellular spread of Shigella.     

Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement.

Shigella flexneri uses elements of the host cell cytoskeleton to move within cells and from cell to cell. IcsA, an S. flexneri protein involved in this movement, was purified and studied in vitro. IcsA bound the radiolabelled ATP analog 3'(2')-O-(4-benzoyl)benzoyl-ATP and hydrolyzed ATP. In addition, the surface localization of IcsA on both extracellular and intracellular shigellae was unipolar. Further, in HeLa cells infected with shigellae, IcsA antiserum labelled the actin tail throughout its length, thereby suggesting that IcsA interacts with elements within the tail. Localization of IcsA within the tail at a distance from the bacterium would require its secretion; we demonstrate here that in vitro IcsA is secreted into the culture supernatant in a cleaved form.     

EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization

Extended Fer-CIP4 homology (EFC)/FCH-BAR (F-BAR) domains generate and bind to tubular membrane structures of defined diameters that are involved in the formation and fission of endocytotic vesicles. Formin-binding protein 17 (FBP17) and Toca-1 contain EFC/F-BAR domains and bind to neural Wiskott-Aldrich syndrome protein (N-WASP), which links phosphatidylinositol (4,5)-bisphosphate (PIP(2)) and the Rho family GTPase Cdc42 to the Arp2/3 complex. The N-WASP-WASP-interacting protein (WIP) complex, a predominant form of N-WASP in cells, is known to be activated by Toca-1 and Cdc42. Here, we show that N-WASP-WIP complex-mediated actin polymerization is activated by phosphatidylserine-containing membranes depending on membrane curvature in the presence of Toca-1 or FBP17 and in the absence of Cdc42 and PIP(2). Cdc42 further promoted the activation of actin polymerization by N-WASP-WIP. Toca-1 or FBP17 recruited N-WASP-WIP to the membrane. Conserved acidic residues near the SH3 domain of Toca-1 and FBP17 positioned the N-WASP-WIP to be spatially close to the membrane for activation of actin polymerization. Therefore, curvature-dependent actin polymerization is stimulated by spatially appropriate interactions of EFC/F-BAR proteins and the N-WASP-WIP complex with the membrane.     

Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex

An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, we describe the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome.     

IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed

Asymmetric localization of proteins is essential to many biological functions of bacteria. Shigella IcsA, an outer membrane protein, is localized to the old pole of the bacillus, where it mediates assembly of a polarized actin tail during infection of mammalian cells. Actin tail assembly provides the propulsive force for intracellular movement and intercellular dissemination. Localization of IcsA to the pole is independent of the amino-terminal signal peptide (Charles, M., Perez, M., Kobil, J.H., and Goldberg, M.B., 2001, Proc Natl Acad Sci USA 98: 9871-9876) suggesting that IcsA targeting occurs in the bacterial cytoplasm and that its secretion across the cytoplasmic membrane occurs only at the pole. Here, we characterize the mechanism by which IcsA is secreted across the cytoplasmic membrane. We present evidence that IcsA requires the SecA ATPase and the SecYEG membrane channel (translocon) for secretion. Our data suggest that YidC is not required for IcsA secretion. Furthermore, we show that polar localization of IcsA is independent of SecA. Finally, we demonstrate that while IcsA requires the SecYEG translocon for secretion, components of this apparatus are uniformly distributed within the membrane. Based on these data, we propose a model for coordinate polar targeting and secretion of IcsA at the bacterial pole.     

Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading

Shigella , the causative agent of bacillary dysentery, is capable of directing its movement within host cells by forming an actin comet tail. The VirG (IcsA) pro-tein expressed at one pole of the bacterium recruits neural Wiskott–Aldrich syndrome protein (N-WASP), a member of the WASP family, which in turn stimulates actin-related protein (Arp) 2/3 complex-mediated actin polymerization. As all the WASP family proteins induce actin polymerization by recruiting Arp2/3 complex, we investigated their involvement in Shigella motility. Here, we show that VirG binds to N-WASP but not to the other WASP family proteins. Using a series of chimeras obtained by swapping N-WASP and WASP domains, we demonstrated that the specificity of VirG to interact with N-WASP lies in the N-terminal region containing the pleckstrin homology (PH) domain and calmodulin-binding IQ motif of N-WASP. A conformational change in N-WASP was important for the VirG–N-WASP interaction, as elimination of the C-terminal acidic region, which is responsible for the intramolecular interaction with the central basic region of N-WASP, affected the specific binding to VirG. We observed that, in haematopoietic cells such as macrophages, polymorphonuclear leucocytes (PMNs) and platelets, WASP was predominantly expressed, whereas the expression of N-WASP was greatly suppressed. Indeed, unlike Listeria , Shigella was unable to move in macrophages at all, although the movement was restored as N-WASP was expressed ectopically. Thus, our findings demonstrate that N-WASP is a specific ligand of VirG, which determines the host cell type allowing actin-based spreading of Shigella .     

Actin-based motility of pathogens: the Arp2/3 complex is a central player

Bacterial actin-based motility has provided cell biologists with tools that led to the recent discovery that, in many forms of actin-based motilities, a key player is a protein complex named the Arp2/3 complex. The Arp2/3 complex is evolutionally conserved and made up of seven polypeptides involved in both actin filament nucleation and organization. Interestingly, this complex is inactive by itself and recent work has highlighted the fact that its activation is achieved differently in the different types of actin-based motilities, including the well-known examples of Listeria and Shigella motilities. Proteins of the WASP family and small G-proteins are involved in most cases. It is interesting that bacteria bypass or mimic some of the events occurring in eukaryotic systems. The Shigella protein IcsA recruits N-WASP and activates it in a Cdc42-like fashion. This activation leads to Arp2/3 complex recruitment, activation of the complex and ultimately actin polymerization and movement. The Listeria ActA protein activates Arp2/3 directly and, thus, seems to mimic proteins of the WASP family. A breakthrough in the field is the recent reconstitution of the actin-based motilities of Listeria and N-WASP-coated E. coli (IcsA) using a restricted number of purified cellular proteins including F-actin, the Arp2/3 complex, actin depolymerizing factor (ADF or cofilin) and capping protein. The movement was more effective upon addition of profilin, alpha-actinin and VASP (for Listeria). Bacterial actin-based motility is now one of the best-documented examples of the exploitation of mammalian cell machineries by bacterial pathogens.     

Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination

Actin reorganization is important for regulation of neuronal morphology. Neural Wiskott-Aldrich syndrome protein (N-WASP) is an important regulator of actin polymerization and also known to be strongly expressed in brain. Recently, Toca-1 (transducer of Cdc42-dependent actin assembly) has been shown to be required for Cdc42 to activate N-WASP from biochemical experiments. Toca-1 has three functional domains: an F-BAR/EFC domain at the N terminus, an HR1 at the center, and an SH3 domain at the C terminus. The F-BAR/EFC domain induces tubular invagination of plasma membrane, while Toca-1 binds both N-WASP and Cdc42 through the SH3 domain and the HR1, respectively. However, the physiological role of Toca-1 is completely unknown. Here we have investigated the neural function of Toca-1. Toca-1 is strongly expressed in neurons including hippocampal neurons in developing brain at early times. Knockdown of Toca-1 in PC12 cells significantly enhances neurite elongation. Consistently, overexpression of Toca-1 suppresses neurite elongation through the F-BAR/EFC domain with a membrane invaginating property, suggesting an implication of membrane trafficking in the neural function of Toca-1. In addition, knockdown of N-WASP, to our surprise, also enhances neurite elongation in PC12 cells, which is in clear contrast to the previous report that dominant negative mutants of N-WASP suppress neurite extension in PC12 cells. On the other hand, knockdown of Toca-1 in cultured rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of N-WASP enhances both axon elongation and branching. These results suggest that a vesicle trafficking regulator Toca-1 regulates different aspects of neuronal morphology from N-WASP.     

Host and Bacterial Proteins That Repress Recruitment of LC3 to Shigella Early during Infection

Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min.), the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4–6 hr) by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG). Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.     

Functional Analysis of a Rickettsial OmpA Homology Domain of Shigella flexneri IcsA

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.     

Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteins

Shigella move through the cytosol of infected cells by assembly of a propulsive actin tail at one end of the bacterium. Vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins, is important in cellular actin dynamics and is present on intracellular Shigella. VASP binds both profilin, an actin monomer-binding protein, and vinculin, a component of intercellular contacts that also binds the Shigella actin assembly protein IcsA. It has been postulated that VASP might serve as a linker between vinculin and profilin on intracellular Shigella, thereby delivering profilin to the Shigella actin assembly machinery. We show that Shigella actin-based motility is unaltered in cells that are deficient for the Ena/VASP family of proteins. In these cells, Shigella form normal-appearing actin tails and move at rates that are comparable to the rates of bacterial movement in Ena/VASP-deficient cells complemented with the Ena/VASP family member Mena. Finally, whereas vinculin can bind the Arp2/3 complex, we show that Arp2/3 recruitment to Shigella is not correlated with vinculin recruitment, indicating that the role of vinculin in Shigella motility is not recruitment of Arp2/3. Thus, although VASP is recruited to the surface of intracellular Shigella, it is not essential for Shigella actin-based motility.     

A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization

Wiskott-Aldrich syndrome protein (WASP) and N-WASP have emerged as key proteins connecting signalling cascades to actin polymerization. Here we show that the amino-terminal WH1 domain, and not the polyproline-rich region, of N-WASP is responsible for its recruitment to sites of actin polymerization during Cdc42-independent, actin-based motility of vaccinia virus. Recruitment of N-WASP to vaccinia is mediated by WASP-interacting protein (WIP), whereas in Shigella WIP is recruited by N-WASP. Our observations show that vaccinia and Shigella activate the Arp2/3 complex to achieve actin-based motility, by mimicking either the SH2/SH3-containing adaptor or Cdc42 signalling pathways to recruit the N-WASP-WIP complex. We propose that the N-WASP-WIP complex has a pivotal function in integrating signalling cascades that lead to actin polymerization.     

Nck-independent actin assembly is mediated by two phosphorylated tyrosines within enteropathogenic Escherichia coli Tir

Enteropathogenic Escherichia coli (EPEC) stimulates tyrosine-kinase signalling cascades to trigger localized actin assembly within mammalian cells. During actin 'pedestal' formation, the EPEC effector protein Tir is translocated into the plasma membrane, becomes phosphorylated on tyrosine-474 (Y474) and promotes recruitment of the mammalian adaptor protein Nck to efficiently activate N-WASP-Arp2/3-mediated actin polymerization. Tir also triggers localized actin assembly in the absence of Nck, but the Tir sequences involved in this signalling cascade have not been defined. To identify and characterize the phosphotyrosines that contribute to Nck-independent pedestal formation, we investigated the regulation of Tir tyrosine phosphorylation and found that phosphorylation is stimulated by Tir clustering. In addition to Y474, residue Y454 is also phosphorylated, although at lower efficiency. These tyrosines differentially contribute to actin polymerization in a fashion reminiscent of actin 'tail' formation mediated by the vaccinia virus envelope protein A36R, which utilizes two similarly spaced phosphotyrosines to recruit the adaptors Nck and Grb2, respectively, in order to stimulate N-WASP. Neither phosphorylated Y454 nor Y474 directly bind Grb2, but Tir derivatives harbouring these residues ultimately recruit N-WASP and Arp2/3 independently of Nck, suggesting that EPEC exploits additional phosphotyrosine-binding adaptors capable of initiating actin assembly.     

The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface

Shigella flexneri is an intracellular pathogen that is able to move within the cytoplasm of infected cells by the continual assembly of actin onto one pole of the bacterium. IcsA, an outer membrane protein, is localized to the old pole of the bacterium and is both necessary and sufficient for actin assembly. IcsA is slowly cleaved from the bacterial surface by the protease IcsP (SopA). Absence of IcsP leads to an alteration in the distribution of surface IcsA, such that the polar cap is maintained and some IcsA is distributed along the lateral walls of the bacillus. The mechanism of unipolar localization of IcsA and the role of IcsP in its unipolar localization are incompletely understood. Here, we demonstrate that cleavage of IcsA occurs exclusively in the outer membrane and that IcsP is localized to the outer membrane. In addition, we show that IcsA at the old pole is susceptible to cleavage by IcsP and that native IcsP is active at the pole. Taken together, these data indicate that IcsP cleaves IcsA over the entire bacterial surface. Finally, we show that, immediately after induction from a tightly regulated promoter, IcsA is expressed exclusively at the old pole in both the icsP- icsA- and the icsA- background. These data demonstrate that unipolar localization of IcsA results from its direct targeting to the pole, followed by its diffusion laterally in the outer membrane.     

SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri

The spreading ability of Shigella flexneri , a facultative intracellular Gram-negative bacterium, within the host-cell cytoplasm is the result of directional assembly and accumulation of actin filaments at one pole of the bacterium. IcsA/VirG, the 120 kDa outer membrane protein that is required for intracellular motility, is located at the pole of the bacterium where actin polymerization occurs. Bacteria growing in laboratory media and within infected cells release a certain proportion of the surface-exposed IcsA after proteolytic cleavage. In this study, we report the characterization of the sopA gene which is located on the virulence plasmid and encodes the protein responsible for the cleavage of IcsA. The deduced amino acid sequence of SopA exhibits 60% identity with those of the OmpT and OmpP outer membrane proteases of Escherichia coli . The construction and phenotypic characterization of a sopA mutant demonstrated that SopA is required for exclusive polar localization of IcsA on the bacterial surface and proper expression of the motility phenotype in infected cells.     

Multicopy icsA is able to suppress the virulence defect caused by the wzz(SF) mutation in Shigella flexneri

The lipopolysaccharides (LPS) of Shigella flexneri are important for virulence and their O antigen (Oag) polysaccharide chains affect IcsA (VirG)-mediated actin-based motility (ABM) within mammalian cells. S. flexneri 2a 2457T has smooth LPS whose Oag chains have two modal lengths (short (S)-type and very long (VL)-type), and has IcsA predominantly located at one pole on its cell surface. A S. flexneri 2457T wzz(SF) mutant (RMA696) has VL-type Oag but not S-type Oag chains, less IcsA detectable by immunofluorescence on its cell surface, reduced virulence and defective ABM. Introduction of a plasmid encoding IcsA into S. flexneri wzz(SF) showed that multicopy icsA could suppress the virulence defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation) caused by the wzz(SF) mutation suggesting that the VL-type Oag chains were masking IcsA and limiting the amount available to initiate ABM.