The unrelated surface proteins ActA of Listeria monocytogenes and lcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocua and Escherichia coli respectively

Listeria monocytogenes and Shigella flexneri are two unrelated facultative intracellular pathogens which spread from cell to cell by using a similar mode of intracellular movement based on continuous actin assembly at one pole of the bacterium. This process requires the asymmetrical expression of the ActA surface protein in L. monocytogenes and the lcsA (VirG) surface protein in S. flexneri . ActA and lcsA share no sequence homology. To assess the role of the two proteins in the generation of actin-based movement, we expressed them in the genetic context of two non-actin polymerizing, non-pathogenic bacterial species, Listeria innocua and Escherichia coli . In the absence of any additional bacterial pathogenicity determinants, both proteins induced actin assembly and propulsion of the bacteria in cytoplasmic extracts from Xenopus eggs, as visualized by the formation of characteristic actin comet tails. E. coli expressing lcsA moved about two times faster than Listeria and displayed longer actin tails. However, actin dynamics (actin filament distribution and filament half-lives) were similar in lcsA- and ActA-induced actin tails suggesting that by using unrelated surface molecules, L. monocytogenes and S. flexneri move intracellularly by interacting with the same host cytoskeleton components or by interfering with the same host cell signal transduction pathway.     

A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin.

The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.     

The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton.

Listeria monocytogenes, a facultative intracellular pathogen, employs actin and other microfilament-associated proteins to move through the host cell cytoplasm. Isogenic mutants of L. monocytogenes lacking the surface-bound ActA polypeptide no longer interact with cytoskeletal elements and are, as a consequence, non-motile (Domann et al., 1992, EMBO J., 11, 1981-1990; Kocks et al., 1992, Cell, 68, 521-531). To investigate the interaction of ActA with the microfilament system in the absence of other bacterial factors, the listerial actA gene was expressed in eukaryotic cells. Immunofluorescence studies revealed that the complete ActA, including its C-terminally located bacterial membrane anchor, colocalized with mitochondria in transfected cells. When targeted to mitochondria, the ActA polypeptide recruited actin and alpha-actinin to these cellular organelles with concomitant reorganization of the microfilament system. Removal of the internal proline-rich repeat region of ActA completely abrogated interaction with cytoskeletal components. Our results identify the ActA polypeptide as a nucleator of the actin cytoskeleton and provide the first insights into the molecular nature of such controlling elements in microfilament organization.     

LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes

The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogen es EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes .     

Asymmetric distribution of the Listeria monocytogenes ActA protein is required and sufficient to direct actin-based motility

Listeria monocytogenes is a Gram-positive facultative intracytoplasmic bacterial pathogen that exhibits rapid actin-based motility in eukaryotic cells and in cell-free cytoplasmic extracts. The protein product of the actA gene is required for bacterial movement and is normally expressed in a polarized fashion on the bacterial surface. Here we demonstrate that the ActA protein is sufficient to direct motility in the absence of other L. monocytogenes gene products, and that polarized localization of the protein is required for efficient unidirectional movement. We have engineered a fusion protein combining ActA with the C-terminal domain of the LytA protein of Streptococcus pneumoniae , which mediates high-affinity binding to DEAE-cellulose and to choline moieties present in the S. pneumoniae cell wall. DEAE-cellulose fragments or S. pneumoniae coated uniformly with the ActA/LytA fusion protein nucleate actin filament growth in cytoplasmic extracts, but do not move efficiently. However, when ActA/LytA-coated S. pneumoniae is grown to polarize the distribution of the fusion protein, the bacteria exhibit unidirectional actin-based movement similar to the normal movement of L. monocytogenes .     

Production, purification and characterization of hemolysins from Listeria ivanovii and Listeria monocytogenes Sv4b

In culture supernatants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as listeriolysin O (LLO). In the case of L. ivanovii a second major supernatant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supernatants of L. ivanovii a sphingomyelinase and a lecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-terminal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown.     

Listeria monocytogenes ActA protein interacts with phosphatidylinositol 4,5-bisphosphate in vitro

The N-terminal region of the Listeria monocytogenes ActA protein, in conjunction with host cell factors, is sufficient for actin polymerization at the bacterial surface. Previous data suggested that ActA could protect barbed ends from capping proteins. We tested this hypothesis by actin polymerization experiments in the presence of the ActA N-terminal fragment and capping protein. ActA does not protect barbed ends from capping protein. In contrast, this polypeptide prevents PIP(2) from inhibiting the capping activity of capping protein. Gel filtration and tryptophan fluorescence experiments showed that the purified ActA N-terminal fragment binds to PIP(2) and PIP, defining phosphoinositides as novels ligands for this functional domain of ActA. Phosphoinositide binding to the N-terminal region of ActA may induce conformational changes in ActA and/or facilitate binding of other cell components, important for ActA-induced actin polymerization.     

Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility

The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes.     

Listeria and autophagy escape: involvement of InlK, an internalin-like protein

Autophagy is a cell-autonomous mechanism of innate immunity that protects the cytosol against bacterial infection. Invasive bacteria, including Listeria monocytogenes, have thus evolved strategies to counteract a process that limits their intracellular growth. ActA is a surface protein produced by L. monocytogenes to polymerize actin and mediate intra- and intercellular movements, which plays a critical role in autophagy escape. We have recently investigated the role of another L. monocytogenes surface protein, the internalin InlK, in the infection process. We showed that in the cytosol of infected cells, InlK interacts with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoprotein particles named vaults. Although MVP has been implicated in a variety of key cellular process, its role remains elusive. We demonstrated that L. monocytogenes is able, via InlK, to decorate its surface with MVP in order to escape autophagic recognition. Strikingly, this new strategy used by L. monocytogenes to avoid autophagy is independent of ActA, suggesting that InlK-MVP interactions and actin polymerization are two processes that favor in the same manner the infection process. Understanding the role of MVP may provide new insights into bacterial infection and autophagy.     

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.     

Close packing of Listeria monocytogenes ActA, a natively unfolded protein, enhances F-actin assembly without dimerization

Studies of the biochemistry of Listeria monocytogenes virulence protein ActA have typically focused on the behavior of bacteria in complex systems or on the characterization of the protein after expression and purification. Although prior in vivo work has proposed that ActA forms dimers on the surface of L. monocytogenes, dimerization has not been demonstrated in vitro, and little consideration has been given to the surface environment where ActA performs its pivotal role in bacterial actin-based motility. We have synthesized and characterized an ActA dimer and provide evidence that the two ActA molecules do not interact with each other even when tethered together. However, we also demonstrate that artificial dimers provide superior activation of actin nucleation by the Arp2/3 complex compared with monomers and that increased activation of the Arp2/3 complex by dimers may be a general property of Arp2/3 activators. It appears that the close packing ( approximately 19 nm) of ActA molecules on the surface of L. monocytogenes is so dense that the kinetics of actin nucleation mimic that of synthetic ActA dimers. We also present observations indicating that ActA is a natively unfolded protein, largely random coil that is responsible for many of the unique physical properties of ActA including its extended structure, aberrant mobility during SDS-PAGE, and ability to resist irreversible denaturation upon heating.     

Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex

ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp2 and Arp3.     

Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway

Upon infection of mammalian cells, Listeria monocytogenes lyses the phagosome and enters the cytosol, where it secretes proteins necessary for its intracellular growth cycle. Consequently, bacterial proteins exposed to the cytosol are potential targets for degradation by host cytosolic proteases. One pathway for degradation of host cytosolic proteins, the N-end rule pathway, involves recognition of the N-terminal amino acid and is mediated by the proteasome. However, very few natural N-end rule substrates have been identified. We have examined the L. monocytogenes ActA protein as a potential target for this pathway. ActA is an essential determinant of L. monocytogenes pathogenesis that is required to induce actin-based motility and cell-to-cell spread. We show that the half-life of a secreted form of ActA can be altered in the mammalian cytosol by changing the N-terminal amino acid. Moreover, the introduction of a destabilizing N-terminus into the functional, surface-bound form of ActA results in a small-plaque phenotype in L2 cells, which is partially reversible by an inhibitor of the proteasome. These results indicate that the L. monocytogenes ActA protein is a natural N-end rule substrate, and that optimal function of ActA in mediating cell-to-cell spread is dependent upon its intracellular turnover rate.     

Detection of a gene encoding a phosphatidylinositol-specific phospholipase C that is co-ordinately expressed with listeriolysin in Listeria monocytogenes

A phosphatidylinositol-specific phospholipase C (PI-PLC) that is unique to the pathogenic Listeria species L. monocytogenes and L. ivanovii has been detected. Deletion analysis performed with Escherichia coli recombinants expressing PI-PLC activity together with maxicell analysis showed that a 34 kDa polypeptide was responsible for this activity. Nucleotide sequencing revealed that the gene encoding this polypeptide comprises 317 amino acid residues with a 22-amino-acid signal peptide. This gene, designated pic for phosphatidylinositol-specific phospholipase C, is located back to back with the listeriolysin gene on the chromosome of L. monocytogenes where these genes are transcribed by divergent non-overlapping promoters. Expression of the pic gene is dependent on the product of the prfA gene, which also regulates expression of the listeriolysin gene in L. monocytogenes.     

Identification and characterisation of regions in the cellular protein LaXp180 and the<Emphasis Type="Italic"> Listeria monocytogenes</Emphasis> surface protein ActA necessary for the interaction of the two proteins

The Listeria monocytogenes surface protein ActA is an important virulence factor that plays an essential role in intracellular movement of Listeria cells by inducing actin polymerisation. The ActA protein is known to interact with several mammalian proteins including the phosphoprotein VASP, actin and the Arp2/3 complex. In a search for additional ActA-binding proteins we recently employed the yeast two-hybrid system to search for proteins that interact with ActA, and identified, among others, the mammalian protein LaXp180 as a binding partner. In the present study the interaction of the two proteins was investigated in more detail. A number of variants were tested in the yeast two-hybrid system for their ability to interact. On the basis of these assays, the 14 C-terminal amino acids of LaXp180 were identified as being necessary for the interaction with ActA. The proline-rich repeat (PRR) region of ActA was found to be necessary for the interaction with LaXp180, but upstream or downstream sequences are also required to enhance the specificity of the interaction. The second and third repeats in ActA are especially important, and the minimal sequence of ActA capable of interacting with LaXp180 was a proline- and glutamate-rich stretch of PRR3 fused to part of the N-terminal sequence of ActA. Further analysis using site-specific mutations located in either the C-terminal region of LaXp180 or the proline-rich motif of PRR3 of ActA showed that three positively charged amino acids in LaXp180 and two negatively charged amino acids in ActA are critical for the interaction of the two proteins.     

Evolutionary history of the genus Listeria and its virulence genes

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9 kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence of the virulence gene cluster locus and its flanking regions was either determined in this study or assembled from public databases. Furthermore, some virulence-associated internalin loci were compared among the six species. Phylogenetic analyses were performed on a data set containing the sequences of prs, ldh, vclA, and vclB (all directly flanking the virulence gene cluster), as well as the iap gene and the 16S and 23S-rRNA coding genes which are located at different sites in the listerial chromosomes. L. grayi represents the deepest branch within the genus. The remaining five species form two groupings which have a high bootstrap support and which are consistently found by using different treeing methods. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch. Based on this perception, we tried to reconstruct the evolution of the virulence gene cluster. Since no traces of lateral gene transfer events could be detected the most parsimonious scenario is that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri and that the pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. This hypothesis is also supported by the location of the putative deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri.     

Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions

Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure-function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed.     

ActA from Listeria monocytogenes can interact with up to four Ena/VASP homology 1 domains simultaneously

The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP(4)). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultracentrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP(4) sites have roughly equivalent affinities with dissociation constants of about 4 microm. Mutational analysis of the FP(4) motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.