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.     

Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK(-) bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles.     

Structure of Internalin InlK from the Human Pathogen Listeria monocytogenes

Listeria monocytogenes is a human pathogen that employs a wide variety of virulence factors in order to adhere to, invade, and replicate within target cells. Internalins play key roles in processes ranging from adhesion to receptor recognition and are thus essential for infection. Recently, InlK, a surface-associated internalin, was shown to be involved in Listeria's ability to escape from autophagy by recruitment of the major vault protein (MVP) to the bacterial surface. Here, we report the structure of InlK, which harbors four domains arranged in the shape of a “bent arm”. The structure supports a role for the “elbow” of InlK in partner recognition, as well as of two Ig-like pedestals intercalated by hinge regions in the projection of InlK away from the surface of the bacterium. The unusual fold and flexibility of InlK could be essential for MVP binding and concealment from recognition by molecules involved in the autophagic process.     

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.     

Listeria monocytogenes evades killing by autophagy during colonization of host cells

Listeria monocytogenes is an intracellular pathogen that is able to colonize the cytosol of macrophages. Here we examined the interaction of this pathogen with autophagy, a host cytosolic degradative pathway that constitutes an important component of innate immunity towards microbial invaders. L. monocytogenes infection induced activation of the autophagy system in macrophages. At 1 h post infection (p.i.), a population of intracellular bacteria ( approximately 37%) colocalized with the autophagy marker LC3. These bacteria were within vacuoles and were targeted by autophagy in an LLO-dependent manner. At later stages in infection (by 4 h p.i.), the majority of L. monocytogenes escaped into the cytosol and rapidly replicated. At these times, less than 10% of intracellular bacteria colocalized with LC3. We found that ActA expression was sufficient to prevent autophagy of bacteria in the cytosol of macrophages. Surprisingly, ActA expression was not strictly necessary, indicating that other virulence factors were involved. Accordingly, we also found a role for the bacterial phospholipases, PI-PLC and PC-PLC, in autophagy evasion, as bacteria lacking phospholipase expression were targeted by autophagy at later times in infection. Together, our results demonstrate that L. monocytogenes utilizes multiple mechanisms to avoid destruction by the autophagy system during colonization of macrophages.     

p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways

Autophagy is an important mechanism of innate immune defense. We have recently shown that autophagy components are recruited with septins, a new and increasingly characterized cytoskeleton component, to intracytosolic Shigella that have started to polymerize actin. On the other hand, intracytosolic Listeria avoids autophagy recognition by expressing ActA, a bacterial effector required for actin polymerization. Here, we exploit Shigella and Listeria as intracytosolic tools to characterize different pathways of selective autophagy. We show that the ubiquitin-binding adaptor proteins p62 and NDP52 target Shigella to an autophagy pathway dependent upon septin and actin. In contrast, p62 or NDP52 targets the Listeria ActA mutant to an autophagy pathway independent of septin or actin. TNF-α, a host cytokine produced upon bacterial infection, stimulates p62-mediated autophagic activity and restricts the survival of Shigella and the Listeria ActA mutant. These data provide a new molecular framework to understand the emerging complexity of autophagy and its ability to achieve specific clearance of intracytosolic bacteria.     

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.     

Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection

Autophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a DeltaactA mutant strain, impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria colocalize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a DeltaplcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy.     

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.     

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.     

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.     

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).     

Actin-based bacterial motility: towards a definition of the minimal requirements

At the border line between microbiology and cell biology, the spectacular capacity o f some intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and several Rickettsias, to use actin polymerization as a driving force for intracellular movement, cell-to-cell spreading and dissemination within the infected tissue is being increasingly studied. Now that it is possible to manipulate the bacterial surface proteins involved in this process - ActA o f L. monocytogenes and IcsA of S. flexneri - these bacterial systems are providing experimental models in which to investigate the role o f actin filament dynamics in cell motility.     

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.     

Avoiding death by autophagy: interactions of Listeria monocytogenes with the macrophage autophagy system

Autophagy restricts the growth of a variety of intracellular pathogens. However, cytosol-adapted pathogens have evolved ways to evade restriction by this innate immune mechanism. Listeria monocytogenes is a Gram-positive bacterial pathogen that utilizes a cholesterol-dependent pore-forming toxin, listeriolysin O (LLO), to escape from the phagosome. Autophagy targets L. monocytogenes in LLO-damaged phagosomes and also in the cytosol under some experimental conditions. However, this bacterium has evolved multiple mechanisms to evade restriction by autophagy, including actin-based motility in the cytosol and an as yet undefined mechanism mediated by bacterial phospholipases C (PLCs). A population of L. monocytogenes with inefficient LLO activity forms Spacious Listeria-containing Phagosomes (SLAPs), which are autophagosome-like compartments that do not mature, allowing slow bacterial growth within enlarged vesicles. SLAPs may represent a stalemate between bacterial LLO action and the host autophagy system, resulting in persistent infection.     

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.     

Listeria monocytogenes Evades Killing by Autophagy During Colonization of Host Cells

Listeria monocytogenes is an intracellular pathogen that is able to colonize the cytosol of macrophages. Here we examined the interaction of this pathogen with autophagy, a host cytosolicdegradative pathway that constitutes an important component of innate immunity towards microbial invaders. L. monocytogenes infection induced activation of the autophagy system in macrophages. At 1 h post infection (p.i.), a population of intracellular bacteria (~37%) colocalized with the autophagy marker LC3. These bacteria were within vacuoles and were targeted by autophagy in an LLO-dependent manner. At later stages in infection (by 4 h p.i.), the majority of L. monocytogenes escaped into the cytosol and rapidly replicated. At these times, less than 10% of intracellular bacteria colocalized with LC3. We found that ActA expression was sufficient to prevent autophagy of bacteria in the cytosol of macrophages. Surprisingly, ActA expression was not strictly necessary, indicating that other virulence factors were involved. Accordingly, we also found a role for the bacterial phospholipases, PI-PLC and PC-PLC, in autophagy evasion, as bacteria lacking phospholipase expression were targeted by autophagy at later times in infection. Together, our results demonstratethat L. monocytogenes utilizes multiple mechanisms to avoid destruction by the autophagy system during colonization of macrophages.     

The actin-polymerization protein from Listeria ivanovii is a large repeat protein which shows only limited amino acid sequence homology to actA from Listeria monocytogenes

Abstract Within infected eukaryotic cells the two pathogenic Listeria species, L. monocytogenes and L. ivanovii , induce polymerization of cellular actin and the formation of a propulsive actin tail at one bacterial pole. For L. monocytogenes it has been shown that the product of the listerial actA gene is required for this process which is regarded as a model for actin-based motility. We have now cloned and sequenced a functionally analogous gene from L. ivanovii ; its product, as deduced from the DNA sequence, is considerably larger (108 kDa) than L. monocytogenes ActA (67 kDa) and shares only a limited amino acid sequence homology (46% similarity on average) with the latter protein. This is the first example of a virulence gene product from L. ivanovii which is significantly different from its L. monocytogenes counterpart. Comparison of the two ActA proteins gives new insight into the structure of this class of actin-polymerization proteins, in particular with respect to their proline-rich repeat region.     

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 .