Bacteria-host-cell interactions at the plasma membrane: stories on actin cytoskeleton subversion

Exploitation of the host-cell actin cytoskeleton is pivotal for many microbial pathogens to enter cells, to disseminate within and between infected tissues, to prevent their uptake by phagocytic cells, or to promote intimate attachment to the cell surface. To accomplish this, these pathogens have evolved common as well as unique strategies to modulate actin dynamics at the plasma membrane, which will be discussed here, exemplified by a number of well-studied bacterial pathogens.     

Pathogenicity of bacteria and the actin cytoskeleton

Actin system of eukaryotic cells creates the driving force for alteration of the phagocytic cytoplasmatic membrane shape, which is needed for cell movement in the space and for microorganism capturing. Manipulation by actin cytoskeleton mediated through specialized bacterial products can promote proliferation of bacteria in the host. Published reports indicate that bacterial regulation of the actin system activity can be carried out by two modes: 1) by bacterial interactions with surface receptors regulating the cytoskeleton status and 2) by introduction of bacterial products targeted to the cytoskeleton components into the cells. Intracellular pathogens (Legionella) possess ligands which interact with eukaryotic receptors and type IV secretion system fit for translocation of heretofore unknown effector molecules into the cytoplasm. This can result in stimulation of actin polymerization activity and accelerated phagocytosis of the bacteria with rapid multiplication in tissues. By contrast, representatives of extracellular pathogens (Clostridium) produce substances penetrating inside the eukaryotic cells and destroying the actin network, thus making capturing and intracellular digestion of these microorganisms impossible.     

Actin-based motility of intracellular bacteria, and polarized surface distribution of the bacterial effector molecules

Several intracellular bacterial pathogens, including species of Listeria, Rickettsia, Shigella, Mycobacteria, and Burkholderia, have evolved mechanisms to exploit the actin polymerization machinery of their hosts to induce actin-based motility, enabling these pathogens to spread between host cells without exposing themselves to the extracellular milieu. Efficient cell-to-cell spread requires directional motility, which the bacteria may achieve by concentrating the effector molecules at one pole of their cell body, thereby restricting polymerization of monomeric actin into actin tails to this pole. The study of the molecular processes involved in the initiation of actin tail formation at the bacterial surface, and subsequent actin-based motility, has provided much insight into the pathogenesis of infections caused by these bacteria and into the cell biology of actin dynamics. Concomitantly, this field of research has provided an opportunity to understand the mechanisms whereby bacteria can achieve a polarized distribution of surface proteins. This review will describe the process of actin-based motility of intracellular bacteria, and the mechanisms by which bacteria can obtain a polarized distribution of their surface proteins.     

From filaments to function: The role of the plant actin cytoskeleton in pathogen perception,signaling and immunity

The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. In recent years,research in both plants and animal systems have described a function for actin as the ideal surveillance platform, linking the function and activity of primary physiological processes to the immune system. In this review, we will highlight recent advances that have defined the regulation and breadth of function of the actin cytoskeleton as a network required for defense signaling following pathogen infection. Coupled with an overview of recent work demonstrating specific targeting of the plant actin cytoskeleton by a diversity of pathogens,including bacteria, fungi and viruses, we will highlight the importance of actin as a key signaling hub in plants, one that mediates surveillance of cellular homeostasis and the activation of specific signaling responses following pathogen perception. B4 ased on the studies highlighted herein, we propose a working model that posits changes in actin filament organization is in and of itself a highly specific signal, which induces, regulates and physically directs stimulus-specific signaling processes, most importantly, those associated with response to pathogens.     

Integration of linear and dendritic actin nucleation in Nck-induced actin comets

The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails—dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens.     

Viral transport and the cytoskeleton

In the past decade, studies into the way in which intracellular bacterial pathogens hijack and subvert their hosts have provided many important insights into regulation of the actin cytoskeleton and cell motility, in addition to increasing our understanding of the infection process. Viral pathogens, however, may ultimately unlock more cellular secrets as they are even more dependent on their hosts during their life cycle.     

Surfing pathogens and the lessons learned for actin polymerization

A number of unrelated bacterial species as well as vaccinia virus (ab)use the process of actin polymerization to facilitate and enhance their infection cycle. Studies into the mechanism by which these pathogens hijack and control the actin cytoskeleton have provided many interesting insights into the regulation of actin polymerization in migrating cells. This review focuses on what we have learnt from the actin-based motilities of Listeria, Shigella and vaccinia and discusses what we would still like to learn from our nasty friends, including enteropathogenic Escherichia coli and Rickettsia     

Entrapment of intracytosolic bacteria by septin cage-like structures

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.     

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.     

Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility

Diverse intracellular pathogens subvert the host actin-polymerization machinery to drive movement within and between cells during infection. Rickettsia in the spotted fever group (SFG) are Gram-negative, obligate intracellular bacterial pathogens that undergo actin-based motility and assemble distinctive 'comet tails' that consist of long, unbranched actin filaments. Despite this distinct organization, it was proposed that actin in Rickettsia comet tails is nucleated by the host Arp2/3 complex and the bacterial protein RickA, which assemble branched actin networks. However, a second bacterial gene, sca2, was recently implicated in actin-tail formation by R. rickettsii. Here, we demonstrate that Sca2 is a bacterial actin-assembly factor that functionally mimics eukaryotic formin proteins. Sca2 nucleates unbranched actin filaments, processively associates with growing barbed ends, requires profilin for efficient elongation, and inhibits the activity of capping protein, all properties shared with formins. Sca2 localizes to the Rickettsia surface and is sufficient to promote the assembly of actin filaments in cytoplasmic extract. These results suggest that Sca2 mimics formins to determine the unique organization of actin filaments in Rickettsia tails and drive bacterial motility, independently of host nucleators.     

Interaction of vaccinia virus with the actin cytoskeleton

Vaccinia virus infection results in large rearrangements of the host actin cytoskeleton including the formation of actin tails that are strikingly similar to those seen inListeria, Shigella andRickettsia infections. Using actin polymerization as the driving force the intracellular enveloped form of the vaccinia virus (IEV) is propelled on the tip of actin tails at a speed of 2.8 μm/min, both intra- and intercellularly. The similarities between the actin-based motility of the vaccinia virus,Listeria, Shigella andRickettsia suggest that intracellular pathogens have developed a common strategy to exploit the actin cytoskeleton of the host to facilitate their intercellular spread. This review focuses on our current understanding of the interactions between the vaccinia virus,and the actin cytoskeleton. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

Direct modulation of the host cell cytoskeleton by Salmonella actin-binding proteins

Invasive Salmonella trigger their own uptake into non-phagocytic eukaryotic cells by delivering virulence proteins that stimulate signaling pathways and remodel the actin cytoskeleton. It has recently emerged that Salmonella encodes two actin-binding proteins, SipC and SipA, which together efficiently nucleate actin polymerization and stabilize the resulting supramolecular filament architecture. Therefore, Salmonella might directly initiate actin polymerization independently of the cellular Arp2/3 complex early in the cell entry process. This is an unprecedented example of a direct intervention strategy to facilitate entry of a pathogen into a target cell. Here, we discuss the Salmonella actin-binding proteins and how they might function in combination with entry effectors that stimulate Rho GTPases. We propose that membrane-targeted bacterial effector proteins might trigger actin polymerization through diverse mechanisms during cell entry by bacterial pathogens.     

Actin regulation in the malaria parasite

Many intracellular pathogens hijack host cell actin or its regulators for cell-to-cell spreading. In marked contrast, apicomplexan parasites, obligate intracellular, single cell eukaryotes that are phylogenetically older than the last common ancestor of animals and plants, employ their own actin cytoskeleton for active motility through tissues and invasion of host cells. A hallmark of actin-based motility of the malaria parasite is a minimal set of proteins that potentially regulate microfilament dynamics. An intriguing feature of the Plasmodium motor machinery is the virtual absence of elongated filamentous actin in vivo. Despite this unusual actin regulation sporozoites, the transmission stages that are injected into the mammalian host by Anopheles mosquitoes, display fast (1-3 μm/s) extracellular motility. Experimental genetics and analysis of recombinant proteins have recently contributed to clarify some of the cellular roles of apicomplexan actin monomer- and filament-binding proteins in parasite life cycle progression. These studies established that the malaria parasite employs multiple proteins that bind actin to form pools of readily polymerizable monomers, a prerequisite for fast formation of actin polymers. The motile extracellular stages of Plasmodium parasites are an excellent in vivo model system for functional characterization of actin regulation in single cell eukaryotes.     

Actin filament nucleation by endosomes, lysosomes and secretory vesicles

Intracellular pathogens such as Listeria monocytogenes and vaccinia virus propel themselves through the cytoplasm of mammalian cells by nucleating actin filaments. Recently, actin assembly has also been shown to power the movement of intracellular vesicles, and this may be a mechanism underlying endomembrane movement in a variety of physiological contexts. Surprisingly, class I myosins have been found to play important roles in both actin nucleation and endomembrane trafficking.     

Regulating cellular actin assembly

Cellular actin assembly is tightly regulated. The study of pathogen motility has led to the identification of several cellular factors that are critical for controlling this process. Pathogens such as Listeria require Ena/VASP and Arp2/3 proteins to translate actin polymerization into movement. Recent work has extended these observations and uncovered some similarities and surprising differences in the way cells and pathogens utilize the actin cytoskeleton.     

A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.     

Small GTP-binding proteins of the Rho- and Ras-subfamilies are not involved in the actin rearrangements induced by attaching and effacing Escherichia coli

Attaching and effacing Escherichia coli (AEEC) are extracellular pathogens that induce the formation of actin-rich structures at their sites of attachment to eukaryotic host cells. We analysed whether small GTP-binding proteins of the Rho- and Ras-subfamilies, which control the cellular actin system, are essential for these bacterial-induced microfilament reorganizations. For this purpose we specifically inactivated them using the Clostridium difficile toxins TcdB-10463 and TcdB-1470. Such treatment led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the bacterial-induced actin rearrangements. Our data therefore indicate that the microfilament reorganizations induced by AEEC are independent of those small GTP-binding proteins that under normal conditions control the dynamics and maintenance of the actin cytoskeleton.     

Polymer motors: pushing out the front and pulling up the back

Mechanical work in cells is performed by specialized motor proteins that operate in a continuous mechanochemical cycle. Less complex, but still efficient, 'one-shot' motors evolved based on the assembly and disassembly of polymers. We review the mechanisms of pushing and pulling by actin and microtubule filaments and the organizational principles of actin networks. We show how these polymer force generators are used for the propulsion of intracellular pathogens, protrusion of lamellipodia and mitotic movements. We discuss several examples of cellular forces generated by the assembly and disassembly of polymer gels.     

Exploitation of the ubiquitin system by invading bacteria

A variety of bacterial intracellular pathogens target the host cell ubiquitin system during invasion, a process that involves transient but fundamental changes in the actin cytoskeleton and plasma membrane. These changes are induced by bacterial proteins, which can be surface associated, secreted or injected directly into the host cell. Here, the invasion strategies of two extensively studied intracellular bacteria, Salmonella enterica serovar Typhimurium and Listeria monocytogenes, are used to illustrate some of the diverse ways by which bacterial pathogens intersect the host cell ubiquitin pathway.     

Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells

The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.