Localization Of IcsA Research Articles

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

ABSTRACTCardiolipin, 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.

Biogenesis of YidC Cytoplasmic Membrane Substrates Is Required for Positioning of Autotransporter IcsA at Future Poles

Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved across multiple Gram-negative bacterial species and has thus served as an important and informative model for studying polar localization. We present evidence that in Escherichia coli, the establishment of polar positional information recognized by IcsA requires the activity of the cytoplasmic membrane protein insertase YidC. We show that the role of YidC in IcsA localization is independent of the cell septation and cytokinesis proteins FtsQ and FtsEX. FtsQ is required for polar localization of IcsA and, based on cross-linking studies, is inserted in the vicinity of YidC, but, we find, is not dependent on YidC for membrane insertion. FtsEX is a YidC substrate, but we find that it is not required for polar localization of IcsA. These findings indicate that polar positional information recognized by IcsA depends on one or more membrane proteins that require YidC for proper membrane insertion.

Genetic Reporter System for Positioning of Proteins at the Bacterial Pole

ABSTRACTSpatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells.

Evaluation by Real-Time PCR of the Expression of S. Flexneri Virulence-Associated Genes ospB and phoN2 under Different Genetical Backgrounds

Under conditions of activated type III secretion Shigella flexneri up-regulates the expression of numerous genes, including the virulence plasmid (pINV)-encoded ospB and phoN2 genes. ospB and phoN2 are virulence-associated genes which are part of a bicistronic transcriptional unit encoding OspB, a protein (effector) of unknown function secreted by the type III secretion (TTS) apparatus, and PhoN2 (apyrase or ATP-diphosphohydrolase), a periplasmic protein involved in polar IcsA localization on the surface of S. flexneri. In this work we used real-time PCR to measure transcription of ospB and phoN2 of wild-type S. flexneri strain M90T as well as of derivative mutants impaired in definite virulence traits. The results obtained confirmed and extended previous reports indicating that the expression of ospB and phoN2 genes is modulated in a virB-dependent, mxiE-independent manner under conditions of non-activated secretion, while their expression is considerably induced in a mxiE-dependent manner under conditions of activated secretion. That the expression of the ospB-phoN2 operon is up-regulated in condition of activated secretion, indicates that probably the expression of these two genes might be important, especially during the later stages of infection of S. flexneri.

IcsA Surface Presentation in Shigella flexneri Requires the Periplasmic Chaperones DegP, Skp, and SurA

A Shigella flexneri degP mutant, which was defective for plaque formation in Henle cell monolayers, had a reduced amount of IcsA detectable on the bacterial surface with antibody. However, the mutant secreted IcsA to the outer membrane at wild-type levels. This suggests that IcsA adopts an altered conformation in the outer membrane of the degP mutant with reduced exposure on the cell surface. IcsA is, therefore, unlikely to be accessible to actin-nucleating proteins within the eukaryotic cell cytoplasm, which is required for bacterial movement within the host cell and cell-to-cell spread. The degP mutant was somewhat more sensitive to detergents, antibiotics, and the antimicrobial peptide magainin, indicating that the degP phenotype was not limited to IcsA surface presentation. The plaque defect of the degP mutant, which is independent of DegP protease activity, was suppressed by overexpression of the periplasmic chaperone Skp but not by SurA. S. flexneri skp and surA mutants failed to form plaques in Henle cell monolayers and were defective in cell surface presentation and polar localization of IcsA. Therefore, the three periplasmic folding factors DegP, Skp, and SurA were all required for IcsA localization and plaque formation by S. flexneri.

Polar positional information in Escherichia coli spherical cells

Shigella surface protein IcsA and its cytoplasmic derivatives are localized to the old pole of rod-shaped cells when expressed in Escherichia coli. In spherical mreB cells, IcsA is targeted to ectopic sites and close to one extremity of actin-like MamK filament. To gain insight into the properties of the sites containing polar material, we studied the IcsA localization in spherical cells. GFP was exported into the periplasm via the Tat pathway and used as a periplasmic space marker. GFP displayed zonal fluorescence in both mreB and rodA-pbpA spherical E. coli cells, indicating an uneven periplasmic space. Deconvolution images revealed that the cytoplasmic IcsA fused to mCherry was localized outside or at the edge of the GFP zones. These observations strongly suggest that polar material is restricted to the positions where the periplasm possesses particular structural or biochemical properties.

Apyrase, the Product of the Virulence Plasmid-Encoded phoN2 ( apy ) Gene of Shigella flexneri , Is Necessary for Proper Unipolar IcsA Localization and for Efficient Intercellular Spread

The role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.

The role of the Shigella flexneri yihE gene in LPS synthesis and virulence.

Previously, the authors have shown that inactivation of Shigella flexneri yihE, a gene of unknown function upstream of dsbA, which encodes a periplasmic disulphide catalyst, results in a global change of gene expression. Among the severely down-regulated genes are galETKM, suggesting that the yihE mutant, Sh54, may inefficiently produce the UDP-glucose and UDP-galactose required for LPS synthesis. This paper demonstrates that LPS synthesis in Sh54 is impaired. As a result, Sh54 is unable to polymerize host cell actin, due to aberrant localization of IcsA, or to cause keratoconjunctivitis in guinea pigs. Furthermore, Sh54 is more sensitive to some antimicrobial agents, and exhibits epithelial cytotoxicity characteristic of neither wild-type nor dsbA mutants. Supplying galETK in trans restores LPS synthesis and corrects all the defects. Hence, it is clear that the Shigella yihE gene is important not only in regulating global gene expression, as shown previously, but also in virulence through LPS synthesis via regulating the expression of the galETK operon.

Evidence for polar positional information independent of cell division and nucleoid occlusion.

We present evidence that, in Escherichia coli, polar positional information is present at midcell independent of known cell division factors. In filamented cells, IcsA, which is normally polar, localizes at or near potential cell division sites. Because the cell pole is derived from the septum, the sites to which IcsA localizes in filaments correspond to future poles. IcsA localization to these sites is independent of FtsZ, MinCDE, septation, and nucleoid occlusion, indicating that positional information for the future pole is independent of cell division and chromosome positioning. Upon IcsA localization to these sites, septation is inhibited, suggesting that IcsA recognition of this polar positional information may influence cell division.

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.

Shigella flexneri DegP facilitates IcsA surface expression and is required for efficient intercellular spread.

A degP mutant of Shigella flexneri was identified in a screen for insertion mutants that invaded cultured cells but did not form wild-type plaques in monolayers. The degP mutant SM1100 invaded Henle cells at wild-type levels and induced apoptosis in macrophages but formed smaller plaques than those formed by wild-type S. flexneri in confluent monolayers of Henle and Caco-2 cells. The proportion of SM1100 bacteria with IcsA localized to the bacterial pole, a process required for actin polymerization into actin "tails," was reduced compared to results with wild-type bacteria. The reduction in proper IcsA localization may account for the reduced plaque size of the degP mutant. Although DegP is a protease, the protease activity of S. flexneri DegP was not required for IcsA localization or the formation of plaques in Henle cell monolayers. DegP was also required for efficient polar IcsA localization in E. coli expressing icsA. In addition, the growth or survival of SM1100 was compromised compared to that of the wild type at elevated temperatures and in acidic conditions.

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.

Establishment of unipolar localization of IcsA in Shigella flexneri 2a is not dependent on virulence plasmid determinants.

Unipolar localization of IcsA on the surface of Shigella flexneri is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-type S. flexneri 2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic Escherichia coli strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the E. coli background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in Shigella and E. coli.

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.

Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in attenuation of the bacteria in both in vitro and in vivo models of virulence, although the precise block in pathogenesis is not known. We isolated defined mutations in two genes, galU and rfe, which directly affect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the membrane protrusions (fireworks) characteristic of intracellular motility displayed by wild-type shigellae. Furthermore, the galU mutant was unable to form plaques on a confluent monolayer of eucaryotic cells and the rfe mutant generated only tiny plaques. These observations indicated that the mutants were blocked in their ability to spread from cell to cell. Western immunoblot analysis of expression of IcsA, the protein essential for intracellular motility and intercellular spread, demonstrated that both mutants synthesized IcsA, although they secreted less of the protein to the extracellular medium than did the wild-type parent. More strikingly, the LPS mutants showed aberrant surface localization of IcsA. Unlike the unipolar localization of IcsA seen in the wild-type parent, the galU mutant expressed the protein in a circumferential fashion. The rfe mutant had an intermediate phenotype in that it displayed some localization of IcsA at one pole while also showing diffuse localization around the bacterium. Given the known structures of the LPS of wild-type S. flexneri 2a, the rfe mutant, and the galU mutant, we hypothesize that the core and O-antigen components of LPS are critical elements in the correct unipolar localization of IcsA. These observations indicate a more precise role for LPS in Shigella pathogenesis.

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.