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.     

Actin‐based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol

Listeria monocytogenes grows in the host cytosol and uses the surface protein ActA to promote actin polymerisation and mediate actin‐based motility. ActA, along with two secreted bacterial phospholipases C, also mediates avoidance from autophagy, a degradative process that targets intracellular microbes. Although it is known that ActA prevents autophagic recognition of L. monocytogenes in epithelial cells by masking the bacterial surface with host factors, the relative roles of actin polymerisation and actin‐based motility in autophagy avoidance are unclear in macrophages. Using pharmacological inhibition of actin polymerisation and a collection of actA mutants, we found that actin polymerisation prevented the colocalisation of L. monocytogenes with polyubiquitin, the autophagy receptor p62, and the autophagy protein LC3 during macrophage infection. In addition, the ability of L. monocytogenes to stimulate actin polymerisation promoted autophagy avoidance and growth in macrophages in the absence of phospholipases C. Time‐lapse microscopy using green fluorescent protein‐LC3 macrophages and a probe for filamentous actin showed that bacteria undergoing actin‐based motility moved away from LC3‐positive membranes. Collectively, these results suggested that although actin polymerisation protects the bacterial surface from autophagic recognition, actin‐based motility allows escape of L. monocytogenes from autophagic membranes in the macrophage cytosol.     

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.     

The effect of diffusion,depolymerization and nucleation promoting factors on actin gel growth

In eukaryotic cells, localized actin polymerization is able to deform the plasma membrane and push the cell forward. Depolymerization of actin filaments and diffusion of actin monomers ensure the availability of monomers at sites of polymerization, and therefore these processes must play an active role in cellular actin dynamics. Here we reveal experimental evidence that actin gel growth can be limited by monomer diffusion, consistent with theoretical predictions. We study actin gels formed on beads coated with ActA (and ActA fragments), the bacterial factor responsible for actin-based movement of Listeria monocytogenes. We observe a saturation of gel thickness with increasing bead radius, the signature of diffusion control. Data analysis using an elastic model of actin gel growth gives an estimate of 2×10^–8 cm^–2 s^–1 for the diffusion coefficient of actin monomers through the gel, ten times less than in buffer, and in agreement with literature values in bulk cytoskeleton, providing corroboration of our model. The depolymerization rate of actin filaments and the elastic modulus of the gel are also evaluated. Furthermore, we qualitatively examine the different actin gels produced when ActA fragments interact with either VASP or the Arp2/3 complex.     

Listeria monocytogenes ActA is a key player in evading autophagic recognition

Autophagy is a pivotal bulk degradation system that eliminates undesirable molecules, damaged organelles, and misfolded protein aggregates in response to diverse stimuli, including infection. Autophagy acts to limit intracellular microbial growth but intracellular pathogens have evolved strategies to subvert host autophagic responses for their survival. We found that Listeria monocytogenes ActA, a surface protein required for actin polymerization and actin-based bacterial motility, plays a pivotal role in evading autophagy, but in a manner independent of bacterial motility. We show that L. monocytogenes exploits the biomimetic property of ActA to camouflage itself with host proteins comprised of Ena/VASP and the Arp2/3 complex, thereby escaping recognition by autophagy (Fig. 1).     

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.     

Quantitative insights into actin rearrangements and bacterial target site selection from Salmonella Typhimurium infection of micropatterned cells

Reorganization of the host cell actin cytoskeleton is crucial during pathogen invasion. We established micropatterned cells as a standardized infection model for cell invasion to quantitatively study actin rearrangements triggered by Salmonella Typhimurium (S. Tm). Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape avoiding strong cell‐to‐cell variations, a major limitation in classical cell culture conditions. S. Tm induced F‐actin‐rich ruffles and invaded micropatterned cells similar to unconstrained cells. Yet, standardized conditions allowed fast and unbiased comparison of cellular changes triggered by the SipA and SopE bacterial effector proteins. Intensity measurements in defined regions revealed that the content of pre‐existing F‐actin remained unchanged during infection, suggesting that newly polymerized F‐actin in bacteria‐triggered ruffles originates from the G‐actin pool. Analysing bacterial target sites, we found that bacteria did not show any preferences for the local actin cytoskeleton specificities. Rather, invasion was constrained to a specific ‘cell height’, due to flagella‐mediated near‐surface swimming. We found that invasion sites were similar to bacterial binding sites, indicating that S. Tm can induce a permissive invasion site wherever it binds. As micropatterned cells can be infected by many different pathogens they represent a valuable new tool for quantitative analysis of host–pathogen interactions.     

Role of host GTPases in infection by Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes induces internalization into mammalian cells and uses actin‐based motility to spread within tissues. Listeria accomplishes this intracellular life cycle by exploiting or antagonizing several host GTPases. Internalization into human cells is mediated by the bacterial surface proteins InlA or InlB. These two modes of uptake each require a host actin polymerization pathway comprised of the GTPase Rac1, nucleation promotion factors, and the Arp2/3 complex. In addition to Rac1, InlB‐mediated internalization involves inhibition of the GTPase Arf6 and participation of Dynamin and septin family GTPases. After uptake, Listeria is encased in host phagosomes. The bacterial protein GAPDH inactivates the human GTPase Rab5, thereby delaying phagosomal acquisition of antimicrobial properties. After bacterial‐induced destruction of the phagosome, cytosolic Listeria uses the surface protein ActA to stimulate actin‐based motility. The GTPase Dynamin 2 reduces the density of microtubules that would otherwise limit bacterial movement. Cell‐to‐cell spread results when motile Listeria remodel the host plasma membrane into protrusions that are engulfed by neighbouring cells. The human GTPase Cdc42, its activator Tuba, and its effector N‐WASP form a complex with the potential to restrict Listeria protrusions. Bacteria overcome this restriction through two microbial factors that inhibit Cdc42‐GTP or Tuba/N‐WASP interaction.     

Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells

Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram-positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C-terminal end. By immunofluorescence and immunogold labelling, we show that in wild-type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C-terminal region of internalin is necessary for cell-surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a‘domain-swapping’strategy - replacement of the cell wall anchor of InIA by the membrane anchor of ActA - we show that the reduced ability to adhere and enter cells of strains expressing InIA-ActA correlates with a lower amount of surface-exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.     

Listeria-derived ActA is an effective adjuvant for primary and metastatic tumor immunotherapy

Tumor immunotherapy is currently at the cusp of becoming an important aspect of comprehensive cancer treatment in the clinic. However, the need for improved adjuvants to augment immune responses against tumor antigens is always present. In this paper, we characterize the Listeria monocytogenes-derived actin-nucleating protein, ActA, as a novel adjuvant for use in tumor immunotherapy. ActA is a virulence factor that is expressed on the cell surface of L. monocytogenes and facilitates the production of actin tails that propel Listeria throughout the cytosol of an infected host cell. It is believed that this ActA-dependent cytosolic motility allows Listeria to evade adaptive host cell defenses and facilitates its invasion into a proximal uninfected host cell. However, there is evidence that ActA fused to a tumor antigen and delivered by L. monocytogenes can perform a beneficial function in tumor immunotherapy as an adjuvant. Our investigation of this adjuvant activity demonstrates that ActA, either fused to or administered as a mixture with a tumor antigen, can augment anti-tumor immune responses, break immune tolerance and facilitate tumor eradication, which suggests that ActA is not only an effective adjuvant in tumor immunotherapy but can also be applied in a number of therapeutic settings.     

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.     

The amino-terminal part of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-rich region acts as a stimulator

The intracellular bacterial pathogen Listeria monocytogenes moves inside the host-cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin-driven bacterial propulsion, we have now generated several L. monocytogenes strains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino-terminal part is critical for F-actin assembly and movement. The internal proline-rich repeats and the carboxy-terminal domains are not essential. However, in vitro motility assays have demonstrated that mutants lacking the proline-rich repeats domain of ActA moved two times slower (6±2 µm min⁻¹) than the wild type (13±3µm min−1}). In addition, phosphatase treatment of protein extracts of cells infected with the L. monocytogenes strains expressing the ActA variants suggested that phosphorylation may not be essential for ActA activity.     

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.     

Targeting of Listeria monocytogenes ActA protein to the plasma membrane as a tool to dissect both actin-based cell morphogenesis and ActA function.

Actin assembly on the surface of Listeria monocytogenes in the cytoplasm of infected cells provides a model to study actin-based motility and changes in cell shape. We have shown previously that the ActA protein, exposed on the bacterial surface, is required for polarized nucleation of actin filaments. To investigate whether plasma membrane-associated ActA can control the organization of microfilaments and cell shape, variants of ActA, in which the bacterial membrane signal had been replaced by a plasma membrane anchor sequence, were produced in mammalian cells. While both cytoplasmic and membrane-bound forms of ActA increased the F-actin content, only membrane-associated ActA caused the formation of plasma membrane extensions. This finding suggests that ActA acts as an actin filament nucleator and shows that permanent association with the inner face of the plasma membrane is required for changes in cell shape. Based on the observation that the amino-terminal segment of ActA and the remaining portion which includes the proline-rich repeats cause distinct phenotypic modifications in transfected cells, we propose a model in which two functional domains of ActA cooperate in the nucleation and dynamic turnover of actin filaments. The present approach is a new model system to dissect the mechanism of action of ActA and to further investigate interactions of the plasma membrane and the actin cytoskeleton during dynamic changes of cell shape.     

A role for host cell exocytosis in InlB‐mediated internalisation of Listeria monocytogenes

The bacterial surface protein InlB mediates internalisation of Listeria monocytogenes into human cells through interaction with the host receptor tyrosine kinase, Met. InlB‐mediated entry requires localised polymerisation of the host actin cytoskeleton. Apart from actin polymerisation, roles for other host processes in Listeria entry are unknown. Here, we demonstrate that exocytosis in the human cell promotes InlB‐dependent internalisation. Using a probe consisting of VAMP3 with an exofacial green fluorescent protein tag, focal exocytosis was detected during InlB‐mediated entry. Exocytosis was dependent on Met tyrosine kinase activity and the GTPase RalA. Depletion of SNARE proteins by small interfering RNA demonstrated an important role for exocytosis in Listeria internalisation. Depletion of SNARE proteins failed to affect actin filaments during internalisation, suggesting that actin polymerisation and exocytosis are separable host responses. SNARE proteins were required for delivery of the human GTPase Dynamin 2, which promotes InlB‐mediated entry. Our results identify exocytosis as a novel host process exploited by Listeria for infection.     

Mechanism of polarization of Listeria monocytogenes surface protein ActA

The polar distribution of the ActA protein on the surface of the Gram-positive intracellular bacterial pathogen, Listeria monocytogenes, is required for bacterial actin-based motility and successful infection. ActA spans both the bacterial membrane and the peptidoglycan cell wall. We have directly examined the de novo ActA polarization process in vitro by using an ActA-RFP (red fluorescent protein) fusion. After induction of expression, ActA initially appeared at distinct sites along the sides of bacteria and was then redistributed over the entire cylindrical cell body through helical cell wall growth. The accumulation of ActA at the bacterial poles displayed slower kinetics, occurring over several bacterial generations. ActA accumulated more efficiently at younger, less inert poles, and proper polarization required an optimal balance between protein secretion and bacterial growth rates. Within infected host cells, younger generations of L. monocytogenes initiated motility more quickly than older ones, consistent with our in vitro observations of de novo ActA polarization. We propose a model in which the polarization of ActA, and possibly other Gram-positive cell wall-associated proteins, may be a direct consequence of the differential cell wall growth rates along the bacterium and dependent on the relative rates of protein secretion, protein degradation and bacterial growth.     

Listeria monocytogenes Exploits Normal Host Cell Processes to Spread from Cell to Cell✪

The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1-2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.     

Erratum to: Florez-McClure ML,Hohsfield LA,Fonte G,Bealor MT,Link CD. Decreased insulin-receptor signaling promotes the autophagic degradation of β-amyloid peptide in C. elegans. Autophagy 2007; 3:569-80

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.     

CNF and DNT

The actin cytoskeleton of mammalian cells is involved in many processes that affect the growth and colonization of bacteria, such as migration of immune cells, phagocytosis by macrophages, secretion of cytokines, maintenance of epithelial barrier functions and others. With respect to these functions, it is not surprising that many bacterial protein toxins, which are important virulence factors and causative agents of human and/or animal diseases, target the actin cytoskeleton of the host. Some toxins target actin directly, such as the C2 toxin produced by Clostridium botulinum. Moreover, bacterial toxins target the cytoskeleton indirectly by modifying actin regulators such as the low-molecular-mass guanosine triphosphate (GTP)-binding proteins of the Rho family. Remarkably, toxins affect these GTPases in a bidirectional manner. Some toxins inhibit and some activate the GTPases. Here we review the Rho-activating toxins CNF1 and CNF2 (cytotoxic necrotizing factors) from Escherichia coli, the Yersinia CNF_Y and the dermonecrotic toxin (DNT) from Bordetella species. We describe and compare their uptake into mammalian cells, mode of action, structure–function relationship, substrate specificity and role in diseases.