Actin-based motility is sufficient for bacterial membrane protrusion formation and host cell uptake

Shigella flexneri replicates in the cytoplasm of host cells, where it nucleates host cell actin filaments at one pole of the bacterial cell to form a 'comet tail' that propels the bacterium through the host's cytoplasm. To determine whether the ability to move by actin-based motility is sufficient for subsequent formation of membrane-bound protrusions and intercellular spread, we conferred the ability to nucleate actin on a heterologous bacterium, Escherichia coli . Previous work has shown that IcsA (VirG), the molecule that is necessary and sufficient for actin nucleation and actin-based motility, is distributed in a unipolar fashion on the surface of S. flexneri . Maintenance of the unipolar distribution of IcsA depends on both the S. flexneri outer membrane protease IcsP (SopA) and the structure of the lipopolysaccharide (LPS) in the outer membrane. We co-expressed IcsA and IcsP in two strains of E. coli that differed in their LPS structures. The E. coli were engineered to invade host cells by expression of invasin from Yersinia pseudotuberculosis and to escape the phagosome by incubation in purified listeriolysin O (LLO) from Listeria monocytogenes . All E. coli strains expressing IcsA replicated in host cell cytoplasm and moved by actin-based motility. Actin-based motility alone was sufficient for the formation of membrane protrusions and uptake by recipient host cells. The presence of IcsP and an elaborate LPS structure combined to enhance the ability of E. coli to form protrusions at the same frequency as S. flexneri , quantitatively reconstituting this step in pathogen intercellular spread in a heterologous organism. The frequency of membrane protrusion formation across all strains tested correlates with the efficiency of unidirectional actin-based movement, but not with bacterial speed.     

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.     

RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell

Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin-based motility, such as CSNK2B, Listeria monocytogenes requires specific host factors, such as CSNK1A1 in order to form productive membrane protrusions and spread from cell to cell.     

A role for ActA in epithelial cell invasion by Listeria monocytogenes

We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes . An in frame Δ actA mutant was constructed in a hyperinvasive strain of prfA * genotype, in which all genes of the PrfA-dependent virulence regulon, including actA , are highly expressed in vitro . Loss of ActA production in prfA * bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the Δ actA prfA * mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli. prfA * bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro ) or prfA *Δ actA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA * strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.     

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.     

The actin-based motility of the facultative intracellular pathogen Listeria monocytogenes

Summary
The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular parasite that invades and multiplies within diverse eukaryotic cell types. An essential pathogenicity determinant is its ability to move in the host cell cytoplasm and to spread within tissues by directly passing from one cell to another. The propulsive force for intracellular movement is thought to be generated by continuous actin assembly at the rear end of the bacterium. Moving bacteria that reach the plasma membrane induce the formation of long membranous protrusions that are internalized by neighbouring cells, thus mediating the spread of infection. The unrelated pathogens Shigella and Rickettsia use a similar process of actin-based motility to disseminate in infected tissues. This review focuses on the bacterial and cellular factors involved in the actin-based motility of L monocytogenes.     

The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity

Listeria monocytogenes has emerged as a remarkably tractable pathogen to dissect basic aspects of cell biology, intracellular pathogenesis, and innate and acquired immunity. In order to maintain its intracellular lifestyle, L. monocytogenes has evolved a number of mechanisms to exploit host processes to grow and spread cell to cell without damaging the host cell. The pore-forming protein listeriolysin O mediates escape from host vacuoles and utilizes multiple fail-safe mechanisms to avoid causing toxicity to infected cells. Once in the cytosol, the L. monocytogenes ActA protein recruits host cell Arp2/3 complexes and enabled/vasodilator-stimulated phosphoprotein family members to mediate efficient actin-based motility, thereby propelling the bacteria into neighboring cells. Alteration in any of these processes dramatically reduces the ability of the bacteria to establish a productive infection in vivo.     

Bacterial shape and ActA distribution affect initiation of Listeria monocytogenes actin-based motility

We have examined the process by which the intracellular bacterial pathogen Listeria monocytogenes initiates actin-based motility and determined the contribution of the variable surface distribution of the ActA protein to initiation and steady-state movement. To directly correlate ActA distributions to actin dynamics and motility of live bacteria, ActA was fused to a monomeric red fluorescent protein (mRFP1). Actin comet tail formation and steady-state bacterial movement rates both depended on ActA distribution, which in turn was tightly coupled to the bacterial cell cycle. Motility initiation was found to be a highly complex, multistep process for bacteria, in contrast to the simple symmetry breaking previously observed for ActA-coated spherical beads. F-actin initially accumulated along the sides of the bacterium and then slowly migrated to the bacterial pole expressing the highest density of ActA as a tail formed. Early movement was highly unstable with extreme changes in speed and frequent stops. Over time, saltatory motility and sensitivity to the immediate environment decreased as bacterial movement became robust at a constant steady-state speed.     

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.     

Evidence implicating the 5' untranslated region of Listeria monocytogenes actA in the regulation of bacterial actin-based motility

The ActA protein of Listeria monocytogenes is a major virulence factor, essential for the recruitment and polymerization of host actin filaments that lead to intracellular motility and cell-to-cell spread of bacteria within the infected host. The expression of actA is tightly regulated and is strongly induced only when L. monocytogenes is within the host cytosol. Intracellular induction of actA expression is mediated through a single promoter element that directs the expression of a messenger RNA with a long (150 bp) 5' untranslated region (UTR). Deletion of the actA+3 to +130 upstream region was found to result in bacterial mutants that were no longer capable of intracellular actin recruitment or cell-to-cell spread, thus indicating that this region is important for actA expression. L. monocytogenes strains that contained smaller deletions (21-23 bp) within the actA upstream region demonstrated a range of actA expression levels that coincided with the amount of bacterial cell-to-cell spread observed within infected monolayers. A correlation appeared to exist between levels of actA expression and the ability of L. monocytogenes to transition from uniform actin accumulation surrounding individual bacteria (actin clouds) to directional assembly and the formation of actin tails. Bacterial mutants containing deletions that most significantly altered the predicted secondary structure of the actA mRNA 5' UTR had the largest reductions in actA expression. These results suggest that the actA 5' UTR is required for maximal ActA synthesis and that a threshold level of ActA synthesis must be achieved to promote the transition from bacteria-associated actin clouds to directional actin assembly and movement.     

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 .     

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.     

Molecular mechanisms of cell–cell spread of intracellular bacterial pathogens

Several bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and Rickettsia spp., have evolved mechanisms to actively spread within human tissues. Spreading is initiated by the pathogen-induced recruitment of host filamentous (F)-actin. F-actin forms a tail behind the microbe, propelling it through the cytoplasm. The motile pathogen then encounters the host plasma membrane, forming a bacterium-containing protrusion that is engulfed by an adjacent cell. Over the past two decades, much progress has been made in elucidating mechanisms of F-actin tail formation. Listeria and Shigella produce tails of branched actin filaments by subverting the host Arp2/3 complex. By contrast, Rickettsia forms tails with linear actin filaments through a bacterial mimic of eukaryotic formins. Compared with F-actin tail formation, mechanisms controlling bacterial protrusions are less well understood. However, recent findings have highlighted the importance of pathogen manipulation of host cell–cell junctions in spread. Listeria produces a soluble protein that enhances bacterial protrusions by perturbing tight junctions. Shigella protrusions are engulfed through a clathrin-mediated pathway at ‘tricellular junctions’—specialized membrane regions at the intersection of three epithelial cells. This review summarizes key past findings in pathogen spread, and focuses on recent developments in actin-based motility and the formation and internalization of bacterial protrusions.     

Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility

The Listeria monocytogenes surface protein ActA mediates actin-based motility by interacting with a number of host cytoskeletal components, including Ena/VASP family proteins, which in turn interact with actin and the actin-binding protein profilin. We employed a bidirectional genetic approach to study Ena/VASP's contribution to L. monocytogenes movement and pathogenesis. We generated an ActA allelic series within the defined Ena/VASP-binding sites and introduced the resulting mutant L. monocytogenes into cell lines expressing different Ena/VASP derivatives. Our findings indicate that Ena/VASP proteins contribute to the persistence of both speed and directionality of L. monocytogenes movement. In the absence of the Ena/VASP proline-rich central domain, speed consistency decreased by sixfold. In addition, the Ena/VASP F-actin-binding region increased directionality of bacterial movement by fourfold. We further show that both regions of Ena/VASP enhanced L. monocytogenes cell-to-cell spread to a similar degree, although the Ena/VASP F-actin-binding region did so in an ActA-independent manner. Surprisingly, our ActA allelic series enabled us to uncouple L. monocytogenes speed from directionality although both were controlled by Ena/VASP proteins. Lastly, we showed the pathogenic relevance of these findings by the observation that L. monocytogenes lacking ActA Ena/VASP-binding sites were up to 400-fold less virulent during an adaptive immune response.     

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.     

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.     

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.     

Interactions of Listeria monocytogenes with mammalian cells during entry and actin-based movement: bacterial factors, cellular ligands and signaling.

Although <50 kb of its 3.3 megabase genome is known, Listeria monocytogenes has received much attention and an impressive amount of data has contributed in raising this bacterium among the best understood intracellular pathogens. The mechanisms that Listeria uses to enter cells, escape from the phagocytic vacuole and spread from one cell to another using an actin-based motility process have been analysed in detail. Several bacterial proteins contributing to these events have been identified, including the invasion proteins internalin A (InlA) and B (InlB), the secreted pore-forming toxin listeriolysin O (LLO) which promotes the escape from the phagocytic vacuole, and the surface protein ActA which is required for actin polymerization and bacterial movement. While LLO and ActA are critical for the infectious process and are not redundant with other listerial proteins, the precise role of InlA and InlB in vivo remains unclear. How InlA, InlB, LLO or ActA interact with the mammalian cells is beginning to be deciphered. The picture that emerges is that this bacterium uses general strategies also used by other invasive bacteria but has evolved a panel of specific tools and tricks to exploit mammalian cell functions. Their study may lead to a better understanding of important questions in cell biology such as ligand receptor signalling and dynamics of actin polymerization in mammalian cells.     

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.