On the generation of force required for actin-based motility.

The fundamental question of how forces are generated in a motile cell, a lamellipodium, and a comet tail is the subject of this note. It is now well established that cellular motility results from the polymerization of actin, the most abundant protein in eukaryotic cells, into an interconnected set of filaments. We portray this process in a continuum mechanics framework, claiming that polymerization promotes a mechanical swelling in a narrow zone around the nucleation loci, which ultimately results in cellular or bacterial motility. To this aim, a new paradigm in continuum multi-physics has been designed, departing from the well-known theory of Larché-Cahn chemo-transport-mechanics. In this note, we set up the theory of network growth and compare the outcomes of numerical simulations with experimental evidence.

The amount of Nck rather than N-WASP correlates with the rate of actin-based motility of Vaccinia virus.

IMPORTANCE: Vaccinia virus is a large double-stranded DNA virus and a close relative of Mpox and Variola virus, the causative agent of smallpox. During infection, Vaccinia hijacks its host's transport systems and promotes its spread into neighboring cells by recruiting a signaling network that stimulates actin polymerization. Over the years, Vaccinia has provided a powerful model to understand how signaling networks regulate actin polymerization. Nevertheless, we still lack important quantitative information about the system, including the precise number of viral and host molecules required to induce actin polymerization. Using quantitative fluorescence microscopy techniques, we have determined the number of viral and host signaling proteins accumulating on virions during their egress. Our analysis has uncovered two unexpected new aspects of this process: the number of viral proteins in the virion is not fixed and the velocity of virus movement depends on the level of a single adaptor within the signaling network.

In Vitro Roles of Burkholderia Intracellular Motility A (BimA) in Infection of Human Neuroblastoma Cell Line.

The bacterial pathogen Burkholderia pseudomallei causes human melioidosis, which can infect the brain, leading to encephalitis and brain abscesses. Infection of the nervous system is a rare condition but is associated with an increased risk of mortality. Burkholderia intracellular motility A (BimA) was reported to play an important role in the invasion and infection of the central nervous system in a mouse model. Thus, to gain insight of the cellular mechanisms underlying the pathogenesis of neurological melioidosis, we explored the human neuronal proteomics to identify the host factors that are up- and downregulated during Burkholderia infection. When infected the SH-SY5Y cells with B. pseudomallei K96243 wild-type (WT), 194 host proteins showed a fold change of >2 compared with uninfected cells. Moreover, 123 proteins showed a fold change of >2 when infected with a knockout bimA mutant (ΔbimA) mutant compared with WT. The differentially expressed proteins were mainly associated with metabolic pathways and pathways linked to human diseases. Importantly, we observed the downregulation of proteins in the apoptosis and cytotoxicity pathway, and in vitro investigation with the ΔbimA mutant revealed the association of BimA with the induction of these pathways. Additionally, we disclosed that BimA was not required for invasion into the neuron cell line but was necessary for effective intracellular replication and multinucleated giant cell (MNGC) formation. These findings show the extraordinary capacity of B. pseudomallei in subverting and interfering with host cellular systems to establish infection and extend our understanding of B. pseudomallei BimA involvement in the pathogenesis of neurological melioidosis. IMPORTANCE Neurological melioidosis, caused by Burkholderia pseudomallei, can result in severe neurological damage and enhance the mortality rate of melioidosis patients. We investigate the involvement of the virulent factor BimA, which mediates actin-based motility, in the intracellular infection of neuroblastoma SH-SY5Y cells. Using proteomics-based analysis, we provide a list of host factors exploited by B. pseudomallei. The expression level of selected downregulated proteins in neuron cells infected with the ΔbimA mutant was determined by quantitative reverse transcription-PCR and was consistent with our proteomic data. The role of BimA in the apoptosis and cytotoxicity of SH-SY5Y cells infected by B. pseudomallei was uncovered in this study. Additionally, our research demonstrates that BimA is required for successful intracellular survival and cell fusion upon infection of neuron cells. Our findings have significant implications for understanding the pathogenesis of B. pseudomallei infections and developing novel therapeutic strategies to combat this deadly disease.

Confined keratocytes mimic in vivo migration and reveal volume-speed relationship.

Fish basal epidermal cells, known as keratocytes, are well-suited for cell migration studies. In vitro, isolated keratocytes adopt a stereotyped shape with a large fan-shaped lamellipodium and a nearly spherical cell body. However, in their native in vivo environment, these cells adopt a significantly different shape during their rapid migration toward wounds. Within the epidermis, keratocytes experience two-dimensional (2D) confinement between the outer epidermal cell layer and the basement membrane; these two deformable surfaces constrain keratocyte cell bodies to be flatter in vivo than in isolation. In vivo keratocytes also exhibit a relative elongation of the front-to-back axis and substantially more lamellipodial ruffling, as compared to isolated cells. We have explored the effects of 2D confinement, separated from other in vivo environmental cues, by overlaying isolated cells with an agarose hydrogel with occasional spacers, or with a ceiling made of polydimethylsiloxane (PDMS) elastomer. Under these conditions, isolated keratocytes more closely resemble the in vivo migratory shape phenotype, displaying a flatter apical-basal axis and a longer front-to-back axis than unconfined keratocytes. We propose that 2D confinement contributes to multiple dimensions of in vivo keratocyte shape determination. Further analysis demonstrates that confinement causes a synchronous 20% decrease in both cell speed and volume. Interestingly, we were able to replicate the 20% decrease in speed using a sorbitol hypertonic shock to shrink the cell volume, which did not affect other aspects of cell shape. Collectively, our results suggest that environmentally imposed changes in cell volume may influence cell migration speed, potentially by perturbing physical properties of the cytoplasm.

Interferon receptor-deficient mice are susceptible to eschar-associated rickettsiosis.

Arthropod-borne rickettsial pathogens cause mild and severe human disease worldwide. The tick-borne pathogen Rickettsia parkeri elicits skin lesions (eschars) and disseminated disease in humans; however, inbred mice are generally resistant to infection. We report that intradermal infection of mice lacking both interferon receptors (Ifnar1-/-;Ifngr1-/-) with as few as 10 R. parkeri elicits eschar formation and disseminated, lethal disease. Similar to human infection, eschars exhibited necrosis and inflammation, with bacteria primarily found in leukocytes. Using this model, we find that the actin-based motility factor Sca2 is required for dissemination from the skin to internal organs, and the outer membrane protein OmpB contributes to eschar formation. Immunizing Ifnar1-/-;Ifngr1-/- mice with sca2 and ompB mutant R. parkeri protects against rechallenge, revealing live-attenuated vaccine candidates. Thus, Ifnar1-/-;Ifngr1-/- mice are a tractable model to investigate rickettsiosis, virulence factors, and immunity. Our results further suggest that discrepancies between mouse and human susceptibility may be due to differences in interferon signaling.

Tick bites allow disease-causing microbes, including multiple species of Rickettsia bacteria, to pass from arthropods to humans. Being exposed to Rickettsia parkeri, for example, can cause a scab at the bite site, fever, headache and fatigue. To date, no vaccine is available against any of the severe diseases caused by Rickettsia species. Modelling human infections in animals could help to understand and combat these illnesses. R. parkeri is a good candidate for such studies, as it can give insight into more severe Rickettsia infections while being comparatively safer to handle. However, laboratory mice are resistant to this species of bacteria, limiting their use as models. To explore why this is the case, Burke et al. probed whether an immune mechanism known as interferon signalling protects laboratory rodents against R. parkeri. During infection, the immune system releases molecules called interferons that stick to 'receptors' at the surface of cells, triggering defense mechanisms that help to fight off an invader. Burke et al. injected R. parkeri into the skin of mice that had or lacked certain interferon receptors, showing that animals without two specific receptors developed scabs and saw the disease spread through their body. Further investigation showed that two R. parkeri proteins, known as OmpB or Sca2, were essential for the bacteria to cause skin lesions and damage internal organs. Burke et al. then used R. parkeri that lacked OmpB or Sca2 to test whether these modified, inoffensive microbes could act as 'vaccines'. And indeed, vulnerable laboratory mice which were first exposed to the mutant bacteria were then able to survive the 'normal' version of the microbe. Together, this work reveals that interferon signalling protects laboratory mice against R. parkeri infections. It also creates an animal model that can be used to study disease and vaccination.

Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions.

In the outer membrane of gram-negative bacteria, O-antigen segments of lipopolysaccharide (LPS) form a chemomechanical barrier, whereas lipid A moieties anchor LPS molecules. Upon infection, human guanylate binding protein-1 (hGBP1) colocalizes with intracellular gram-negative bacterial pathogens, facilitates bacterial killing, promotes activation of the lipid A sensor caspase-4, and blocks actin-driven dissemination of the enteric pathogen Shigella. The underlying molecular mechanism for hGBP1's diverse antimicrobial functions is unknown. Here, we demonstrate that hGBP1 binds directly to LPS and induces "detergent-like" LPS clustering through protein polymerization. Binding of polymerizing hGBP1 to the bacterial surface disrupts the O-antigen barrier, thereby unmasking lipid A, eliciting caspase-4 recruitment, enhancing antibacterial activity of polymyxin B, and blocking the function of the Shigella outer membrane actin motility factor IcsA. These findings characterize hGBP1 as an LPS-binding surfactant that destabilizes the rigidity of the outer membrane to exert pleiotropic effects on the functionality of gram-negative bacterial cell envelopes.

Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading.

Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens.IMPORTANCEListeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1-based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1-based internalization event can exceed the theoretical size limit for this endocytic pathway.

Towards a structural understanding of the remodeling of the actin cytoskeleton.

Actin filaments (F-actin) are a key component of eukaryotic cells. Whether serving as a scaffold for myosin or using their polymerization to push onto cellular components, their function is always related to force generation. To control and fine-tune force production, cells have a large array of actin-binding proteins (ABPs) dedicated to control every aspect of actin polymerization, filament localization, and their overall mechanical properties. Although great advances have been made in our biochemical understanding of the remodeling of the actin cytoskeleton, the structural basis of this process is still being deciphered. In this review, we summarize our current understanding of this process. We outline how ABPs control the nucleation and disassembly, and how these processes are affected by the nucleotide state of the filaments. In addition, we highlight recent advances in the understanding of actomyosin force generation, and describe recent advances brought forward by the developments of electron cryomicroscopy.

Optimized fixation of actin filaments for improved indirect immunofluorescence staining of rickettsiae.

OBJECTIVE: The objective was to investigate fixative solutions: 3.7% formaldehyde, 4% paraformaldehyde, 4% paraformaldehyde in the cytoskeletal buffer and 4% paraformaldehyde in PHEM buffer (containing PIPES, HEPES, EGTA and MgCl2), applicable for immunofluorescence assay. RESULTS: Herein we optimized this serological technique, testing four fixative solutions, for the sensitive detection of rickettsial antigens, and preservation of intracellular structures of the host cells, particularly filamentous actin. Rickettsial antigens were presented equally well both with formaldehyde and all paraformaldehyde-based fixations, but only protocol with 4% paraformaldehyde in PHEM buffer allowed accurate imaging of actin filaments, and simultaneously allows monitoring of rickettsiae using actin-based motility during infection inside the host cells.

WIPF2 promotes Shigella flexneri actin-based motility and cell-to-cell spread.

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin-based motility and formation of membrane protrusions at cell-cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell-to-cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation-promoting factor N-WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N-WASP exists as a complex with WIP. The involvement of WIP in N-WASP-dependent actin-based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT-29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non-motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N-WASP to the bacterial pole, and among N-WASP-positive bacteria, a decrease in actin tail-positive bacteria, suggesting that WIPF2 is required for N-WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.

Evolutionary Perspectives on the Moonlighting Functions of Bacterial Factors That Support Actin-Based Motility.

Various bacterial pathogens display an intracellular lifestyle and spread from cell to cell through actin-based motility (ABM). ABM requires actin polymerization at the bacterial pole and is mediated by the expression of bacterial factors that hijack the host cell actin nucleation machinery or exhibit intrinsic actin nucleation properties. It is increasingly recognized that bacterial ABM factors, in addition to having a crucial task during the intracellular phase of infection, display "moonlighting" adhesin functions, such as bacterial aggregation, biofilm formation, and host cell adhesion/invasion. Here, we review our current knowledge of ABM factors and their additional functions, and we propose that intracellular ABM functions have evolved from ancestral, extracellular adhesin functions.

The hitchhiker's guide to parasite dissemination.

Toxoplasma gondii (T. gondii) is a parasitic protist that can infect nearly all nucleated cell types and tissues of warm-blooded vertebrate hosts. T. gondii utilises a unique form of gliding motility to cross cellular barriers, enter tissues, and penetrate host cells, thus enhancing spread within an infected host. However, T. gondii also disseminates by hijacking the migratory abilities of infected leukocytes. Traditionally, this process has been viewed as a route to cross biological barriers such as the blood-brain barrier. Here, we review recent findings that challenge this view by showing that infection of monocytes downregulates the program of transendothelial migration. Instead, infection by T. gondii enhances Rho-dependent interstitial migration of monocytes and macrophages, which enhances dissemination within tissues. Collectively, the available evidence indicates that T. gondii parasites use multiple means to disseminate within the host, including enhanced motility in tissues and translocation across biological barriers.

Listeria monocytogenes hijacks CD147 to ensure proper membrane protrusion formation and efficient bacterial dissemination.

Efficient cell-to-cell transfer of Listeria monocytogenes (L. monocytogenes) requires the proper formation of actin-rich membrane protrusions. To date, only the host proteins ezrin, the binding partner of ezrin, CD44, as well as cyclophilin A (CypA) have been identified as crucial components for L. monocytogenes membrane protrusion stabilization and, thus, efficient cell-to-cell movement of the microbes. Here, we examine the classical binding partner of CypA, CD147, and find that this membrane protein is also hijacked by the bacteria for their cellular dissemination. CD147 is enriched at the plasma membrane surrounding the membrane protrusions as well as the resulting invaginations generated in neighboring cells. In cells depleted of CD147, these actin-rich structures appear similar to those generated in CypA depleted cells as they are significantly shorter and more contorted as compared to their straighter counterparts formed in wild-type control cells. The presence of malformed membrane protrusions hampers the ability of L. monocytogenes to efficiently disseminate from CD147-depleted cells. Our findings uncover another important host protein needed for L. monocytogenes membrane protrusion formation and efficient microbial dissemination.

Burkholderia pseudomallei BimC Is Required for Actin-Based Motility, Intracellular Survival, and Virulence.

The intracellular pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans and various animals, is capable of survival and movement within the cytoplasm of host cells by a process known as actin-based motility. The bacterial factor BimA is required for actin-based motility through its direct interaction with actin, and by mediating actin polymerization at a single pole of the bacterium to promote movement both within and between cells. However, little is known about the other bacterial proteins required for this process. Here, we have investigated the role of the bimC gene (bpss1491) which lies immediately upstream of the bimA gene (bpss1492) on the B. pseudomallei chromosome 2. Conserved amongst all B. pseudomallei, B. mallei and B. thailandensis strains sequenced to date, this gene encodes an iron-binding protein with homology to a group of proteins known as the bacterial autotransporter heptosyltransferase (BAHT) family. We have constructed a B. pseudomallei bimC deletion mutant and demonstrate that it is defective in intracellular survival in HeLa cells, but not in J774.1 macrophage-like cells. The bimC mutant is defective in cell to cell spread as demonstrated by ablation of plaque formation in HeLa cells, and by the inability to form multi-nucleated giant cells in J774.1 cells. These phenotypes in intracellular survival and cell to cell spread are not due to the loss of expression and polar localization of the BimA protein on the surface of intracellular bacteria, however they do correlate with an inability of the bacteria to recruit and polymerize actin. Furthermore, we also establish a role for bimC in virulence of B. pseudomallei using a Galleria mellonella larvae model of infection. Taken together, our findings indicate that B. pseudomallei BimC plays an important role in intracellular behavior and virulence of this emerging pathogen.

Listeria monocytogenes cell-to-cell spread in epithelia is heterogeneous and dominated by rare pioneer bacteria.

Listeria monocytogenes hijacks host actin to promote its intracellular motility and intercellular spread. While L. monocytogenes virulence hinges on cell-to-cell spread, little is known about the dynamics of bacterial spread in epithelia at a population level. Here, we use live microscopy and statistical modeling to demonstrate that L. monocytogenes cell-to-cell spread proceeds anisotropically in an epithelial monolayer in culture. We show that boundaries of infection foci are irregular and dominated by rare pioneer bacteria that spread farther than the rest. We extend our quantitative model for bacterial spread to show that heterogeneous spreading behavior can improve the chances of creating a persistent L. monocytogenes infection in an actively extruding epithelium. Thus, our results indicate that L. monocytogenes cell-to-cell spread is heterogeneous, and that rare pioneer bacteria determine the frontier of infection foci and may promote bacterial infection persistence in dynamic epithelia. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Building a dendritic actin filament network branch by branch: models of filament orientation pattern and force generation in lamellipodia.

We review mathematical and computational models of the structure, dynamics, and force generation properties of dendritic actin networks. These models have been motivated by the dendritic nucleation model, which provided a mechanistic picture of how the actin cytoskeleton system powers cell motility. We describe how they aimed to explain the self-organization of the branched network into a bimodal distribution of filament orientations peaked at 35° and - 35° with respect to the direction of membrane protrusion, as well as other patterns. Concave and convex force-velocity relationships were derived, depending on network organization, filament, and membrane elasticity and accounting for actin polymerization at the barbed end as a Brownian ratchet. This review also describes models that considered the kinetics and transport of actin and diffuse regulators and mechanical coupling to a substrate, together with explicit modeling of dendritic networks.

Baculovirus Actin-Based Motility Drives Nuclear Envelope Disruption and Nuclear Egress.

Viruses that replicate in the host cell nucleus face challenges in usurping cellular pathways to enable passage through the nuclear envelope [1]. Baculoviruses are enveloped, double-stranded DNA viruses that infect lepidopteran insects and are tools for protein expression, cell transduction, and pest management [2-4]. The type species Autographa californica M nucleopolyhedrovirus (AcMNPV) shares with other pathogens an ability to assemble host actin monomers (G-actin) into actin filaments (F-actin) to drive motility [5]. During early infection, actin-based motility in the cytoplasm speeds AcMNPV transit to the nucleus and passage through nuclear pores, enabling nuclear ingress [6, 7]. During late infection, AcMNPV assembles F-actin within the nucleus [8], which is essential for virus production [9, 10]. However, the function of nuclear F-actin is poorly understood [11], and its mechanistic role in AcMNPV infection was unknown. We show that AcMNPV mobilizes actin within the nucleus to promote egress. AcMNPV nucleocapsids exhibit intranuclear actin-based motility, mediated by the viral protein P78/83 and the host Arp2/3 complex. Viral motility drives transit to the nuclear periphery and is required for viruses to enter protrusions of the nuclear envelope. Moreover, actin polymerization is necessary for viral disruption of nuclear envelope integrity during egress. In the cytoplasm, viruses use actin-based motility to reach the plasma membrane to enable budding. Our results demonstrate that pathogens can harness actin polymerization to disrupt the nuclear envelope. Employing actin for nuclear envelope disruption may reflect viral appropriation of normal functions of nuclear actin in nuclear envelope integrity, stability, and remodeling.

RECON-Dependent Inflammation in Hepatocytes Enhances Listeria monocytogenes Cell-to-Cell Spread.

The oxidoreductase RECON is a high-affinity cytosolic sensor of bacterium-derived cyclic dinucleotides (CDNs). CDN binding inhibits RECON's enzymatic activity and subsequently promotes inflammation. In this study, we sought to characterize the effects of RECON on the infection cycle of the intracellular bacterium Listeria monocytogenes, which secretes cyclic di-AMP (c-di-AMP) into the cytosol of infected host cells. Here, we report that during infection of RECON-deficient hepatocytes, which exhibit hyperinflammatory responses, L. monocytogenes exhibits significantly enhanced cell-to-cell spread. Enhanced bacterial spread could not be attributed to alterations in PrfA or ActA, two virulence factors critical for intracellular motility and intercellular spread. Detailed microscopic analyses revealed that in the absence of RECON, L. monocytogenes actin tail lengths were significantly longer and there was a larger number of faster-moving bacteria. Complementation experiments demonstrated that the effects of RECON on L. monocytogenes spread and actin tail lengths were linked to its enzymatic activity. RECON enzyme activity suppresses NF-κB activation and is inhibited by c-di-AMP. Consistent with these previous findings, we found that augmented NF-κB activation in the absence of RECON caused enhanced L. monocytogenes cell-to-cell spread and that L. monocytogenes spread correlated with c-di-AMP secretion. Finally, we discovered that, remarkably, increased NF-κB-dependent inducible nitric oxide synthase expression and nitric oxide production were responsible for promoting L. monocytogenes cell-to-cell spread. The work presented here supports a model whereby L. monocytogenes secretion of c-di-AMP inhibits RECON's enzymatic activity, drives augmented NF-κB activation and nitric oxide production, and ultimately enhances intercellular spread.IMPORTANCE To date, bacterial CDNs in eukaryotes are solely appreciated for their capacity to activate cytosolic sensing pathways in innate immunity. However, it remains unclear whether pathogens that actively secrete CDNs benefit from this process. Here, we provide evidence that secretion of CDNs leads to enhancement of L. monocytogenes cell-to-cell spread. This is a heretofore-unknown role of these molecules and suggests L. monocytogenes may benefit from their secretion in certain contexts. Molecular characterization revealed that, surprisingly, nitric oxide was responsible for the enhanced spread. Pathogens act to prevent nitric oxide production or, like L. monocytogenes, they have evolved to resist its direct antimicrobial effects. This study provides evidence that intracellular bacterial pathogens not only tolerate nitric oxide, which is inevitably encountered during infection, but can also capitalize on the changes this pleiotropic molecule enacts on the host cell.

Role of Sca2 and RickA in the Dissemination of Rickettsia parkeri in Amblyomma maculatum.

The Gram-negative obligate intracellular bacterium Rickettsia parkeri is an emerging tick-borne human pathogen. Recently, R. parkeri Sca2 and RickA have been implicated in adherence and actin-based motility in vertebrate host cell infection models; however, the rickettsia-derived factors essential to tick infection are unknown. Using R. parkeri mutants lacking functional Sca2 or RickA to compare actin polymerization, replication, and cell-to-cell spread in vitro, similar phenotypes in tick and mammalian cells were observed. Specifically, actin polymerization in cultured tick cells is controlled by the two separate proteins in a time-dependent manner. To assess the role of Sca2 and RickA in dissemination in the tick host, Rickettsia-free Amblyomma maculatum, the natural vector of R. parkeri, was exposed to wild-type, R. parkeri rickA::tn, or R. parkeri sca2::tn bacteria, and individual tick tissues, including salivary glands, midguts, ovaries, and hemolymph, were analyzed at 12 h and after continued bloodmeal acquisition for 3 or 7 days postexposure. Initially, ticks exposed to wild-type R. parkeri had the highest rickettsial load across all organs; however, rickettsial loads decreased and wild-type rickettsiae were cleared from the ovaries at 7 days postexposure. In contrast, ticks exposed to R. parkeririckA::tn or R. parkerisca2::tn had comparatively lower rickettsial loads, but bacteria persisted in all organs for 7 days. These data suggest that while RickA and Sca2 function in actin polymerization in tick cells, the absence of these proteins did not change dissemination patterns within the tick vector.

Loss of Actin-Based Motility Impairs Ectromelia Virus Release In Vitro but Is Not Critical to Spread In Vivo.

Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted.