scholarly journals mDia1 Assembles a Linear F-Actin Coat at Membrane Invaginations To Drive Listeria monocytogenes Cell-to-Cell Spreading

mBio ◽  
2021 ◽  
Author(s):  
Aaron S. Dhanda ◽  
A. Wayne Vogl ◽  
Fern Ness ◽  
Metello Innocenti ◽  
Julian A. Guttman
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Cell Movement ◽  
Cell Spreading ◽  
Membrane Protrusion ◽  
Propulsive Force ◽  
Comet Tail ◽  
Content Type ◽  
Host Plasma Membrane

Listeria monocytogenes spreads from one cell to another to colonize tissues. This cell-to-cell movement requires the propulsive force of an actin-rich comet tail behind the advancing bacterium, which ultimately distends the host plasma membrane into a slender bacterium-containing membrane protrusion.

scholarly journals The Host GTPase Arf1 and Its Effectors AP1 and PICK1 Stimulate Actin Polymerization and Exocytosis To Promote Entry of Listeria monocytogenes

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Susan Saila ◽  
Gaurav Chandra Gyanwali ◽  
Mazhar Hussain ◽  
Antonella Gianfelice ◽  
Keith Ireton
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Surface Protein ◽  
Dominant Negative ◽  
Host Cells ◽  
Filamin A ◽  
Content Type ◽  
Bacterial Surface ◽  
Dominant Negative Allele

ABSTRACT Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry through stimulation of localized actin polymerization and exocytosis. How actin cytoskeletal changes and exocytosis are controlled during entry is not well understood. Here, we demonstrate important roles for the host GTPase Arf1 and its effectors AP1 and PICK1 in actin polymerization and exocytosis during InlB-dependent uptake. Depletion of Arf1 by RNA interference (RNAi) or inhibition of Arf1 activity using a dominant-negative allele impaired InlB-dependent internalization, indicating an important role for Arf1 in this process. InlB stimulated an increase in the GTP-bound form of Arf1, demonstrating that this bacterial protein activates Arf1. RNAi and immunolocalization studies indicated that Arf1 controls exocytosis and actin polymerization during entry by recruiting the effectors AP1 and PICK1 to the plasma membrane. In turn, AP1 and PICK1 promoted plasma membrane translocation of both Filamin A (FlnA) and Exo70, two host proteins previously found to mediate exocytosis during InlB-dependent internalization (M. Bhalla, H. Van Ngo, G. C. Gyanwali, and K. Ireton, Infect Immun 87:e00689-18, 2018, https://doi.org/10.1128/IAI.00689-18). PICK1 mediated recruitment of Exo70 but not FlnA. Collectively, these results indicate that Arf1, AP1, and PICK1 stimulate exocytosis by redistributing FlnA and Exo70 to the plasma membrane. We propose that Arf1, AP1, and PICK1 are key coordinators of actin polymerization and exocytosis during infection of host cells by Listeria.

scholarly journals Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Aaron S. Dhanda ◽  
Connie Yu ◽  
Katarina T. Lulic ◽  
A. Wayne Vogl ◽  
Valentina Rausch ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Transfer Process ◽  
Bacterial Cell ◽  
Cell Spreading ◽  
Endocytic Pathway ◽  
Content Type ◽  
Caveolin 1 ◽  
Size Limit ◽  
Associated Proteins ◽  
Membrane Protrusions

ABSTRACT 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. IMPORTANCE Listeria 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.

scholarly journals Fluxes of Ca2+and K+Are Required for the Listeriolysin O-Dependent Internalization Pathway of Listeria monocytogenes

2013 ◽  
Vol 82 (3) ◽  
pp. 1084-1091 ◽  
Author(s):  
Stephen Vadia ◽  
Stephanie Seveau
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Early Stage ◽  
Severe Infection ◽  
Host Cells ◽  
Listeriolysin O ◽  
Content Type ◽  
Cell Plasma ◽  
Transmembrane Pores ◽  
Membrane Resealing

ABSTRACTListeria monocytogenesis responsible for the life-threatening food-borne disease listeriosis. This disease mainly affects elderly and immunocompromised individuals, causing bacteremia and meningoencephalitis. In pregnant women,L. monocytogenesinfection leads to abortion and severe infection of the fetus or newborn. TheL. monocytogenesintracellular life cycle is critical for pathogenesis. Previous studies have established that the major virulence factor ofL. monocytogenes, the pore-forming toxin listeriolysin O (LLO), is sufficient to induceL. monocytogenesinternalization into human epithelial cell lines. This internalization pathway strictly requires the formation of LLO pores in the plasma membrane and can be stimulated by the heterologous pore-forming toxin pneumolysin, suggesting that LLO acts nonspecifically by forming transmembrane pores. The present work tested the hypothesis that Ca2+and K+fluxes subsequent to perforation by LLO controlL. monocytogenesinternalization. We report thatL. monocytogenesperforates the host cell plasma membrane in an LLO-dependent fashion at the early stage of invasion. In response to perforation, host cells undergo Ca2+-dependent but K+-independent resealing of their plasma membrane. In contrast to the plasma membrane resealing process, LLO-inducedL. monocytogenesinternalization requires both Ca2+and K+fluxes. Further linking ion fluxes to bacterial internalization, treating cells with a combination of Ca2+and K+ionophores but not with individual ionophores is sufficient to induce efficient internalization of large cargoes, such as 1-μm polystyrene beads and bacteria. We propose that LLO-inducedL. monocytogenesinternalization requires a Ca2+- and K+-dependent internalization pathway that is mechanistically distinct from the process of plasma membrane resealing.

HVEM Analysis of Microfilament Patterns in The Margins of Tissue Cells

1980 ◽  
Vol 38 ◽  
pp. 808-811
Author(s):  
Carol Allen
Keyword(s):  
Cell Movement ◽  
Cell Spreading ◽  
Living Cells ◽  
Tissue Cell ◽  
Large Sample Size ◽  
Cell Periphery ◽  
Whole Cell ◽  
Critical Point Drying ◽  
Lamellar Region ◽  
Tissue Cells

When provided with a suitable solid substrate, tissue cells undergo a rapid conversion from the spherical form expressed in suspension culture to a characteristic flattened morphology. As a result of this conversion, called cell spreading, the cell nucleus and organelles come to occupy a central region of “deep cytoplasm” which slopes steeply into a peripheral “lamellar” region less than 1 pm thick at its outer edge and generally free of cell organelles. Cell spreading is accomplished by a continuous outward repositioning of the lamellar margins. Cell translocation on the substrate results when the activity of the lamellae on one side of the cell become dominant. When this occurs, the cell is “polarized” and moves in the direction of the “leading lamellae”. Careful analysis of tissue cell locomotion by time-lapse microphotography (1) has shown that the deformational movements of the leading lamellae occur in a repeating cycle of advance and retreat in the direction of cell movement and that the rate of such deformations are positively correlated with the speed of cell movement. In the present study, the physical basis for these movements of the cell margin has been examined by comparative light microscopy of living cells with whole-mount electron microscopy of fixed cells. Ultrastructural observations were made on tissue cells grown on Formvar-coated grids, fixed with glutaraldehyde, further processed by critical-point drying, and then photographed in the High Voltage Electron Microscope. This processing and imaging system maintains the 3-dimensional organization of the whole cell, the relationship of the cell to the substrate, and affords a large sample size which facilitates quantitative analysis. Comparative analysis of film records of living cells with the whole-cell micrographs revealed that specific patterns of microfilament organization consistently accompany recognizable stages of lamellar formation and movement. The margins of spreading cells and the leading lamellae of locomoting cells showed a similar pattern of MF repositionings (Figs. 1-4). These results will be discussed in terms of a working model for the mechanics of lamellar motility which includes the following major features: (a) lamellar protrusion results when an intracellular force is exerted at a locally weak area of the cell periphery; (b) the association of cortical MFs with one another determines the local resistance to this force; (c) where MF-to-MF association is weak, the cell periphery expands and some cortical MFs are dragged passively forward; (d) contact of the expanded area with the substrate then triggers the lateral association and reorientation of these cortical MFs into MF bundles parallel to the direction of the expansion; and (e) an active interaction between these MF bundles associated with the cortex of the expanded lamellae and the cortical MFs which remained in the sub-lamellar region then pulls the latter MFs forward toward the expanded area. Thus, the advance of the cell periphery on the substrate occurs in two stages: a passive phase in which some cortical MFs are dragged outward by the force acting to expand the cell periphery, and an active phase in which additional cortical MFs are pulled forward by interaction with the first set. Subsequent interactions between peripheral microfilament bundles and filaments in the deeper cytoplasm could then transmit the advance gained by lamellar expansion to the bulk of the cytoplasm.

scholarly journals Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement

2013 ◽  
Vol 94 (3) ◽  
pp. 682-686 ◽  
Author(s):  
Kazuya Ishikawa ◽  
Kensaku Maejima ◽  
Ken Komatsu ◽  
Osamu Netsu ◽  
Takuya Keima ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Movement Protein ◽  
Rna Virus ◽  
Cell Movement ◽  
Plant Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Series Of Experiments ◽  
Fig Mosaic Virus

Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.

scholarly journals Physiological and Transcriptional Characterization of Persistent and Nonpersistent Listeria monocytogenes Isolates

2011 ◽  
Vol 77 (18) ◽  
pp. 6559-6569 ◽  
Author(s):  
Edward M. Fox ◽  
Nola Leonard ◽  
Kieran Jordan
Keyword(s):  
Listeria Monocytogenes ◽  
Antimicrobial Agents ◽  
Cetylpyridinium Chloride ◽  
Phenotype Microarray ◽  
Content Type ◽  
Benzethonium Chloride ◽  
Transcriptomic Sequencing ◽  
Ammonium Compounds ◽  
Compare Gene Expression

ABSTRACTThis study aimed to characterize physiological differences between persistent and presumed nonpersistentListeria monocytogenesstrains isolated at processing facilities and to investigate the molecular basis for this by transcriptomic sequencing. Full metabolic profiles of two strains, one persistent and one nonpersistent, were initially screened using Biolog's Phenotype MicroArray (PM) technology. Based on these results, in which major differences from selected antimicrobial agents were detected, another persistent strain and two nonpersistent strains were characterized using two antimicrobial PMs. Resistance to quaternary ammonium compounds (QACs) was shown to be higher among persistent strains. Growth of persistent and nonpersistent strains in various concentrations of the QACs benzethonium chloride (BZT) and cetylpyridinium chloride (CPC) was determined. Transcriptomic sequencing of a persistent and a presumed nonpersistent strain was performed to compare gene expression among these strains in the presence and absence of BZT. Two strains, designated “frequent persisters” because they were the most frequently isolated at the processing facility, showed overall higher resistance to QACs. Transcriptome analysis showed that BZT induced a complex peptidoglycan (PG) biosynthesis response, which may play a key role in BZT resistance. Comparison of persistent and nonpersistent strains indicated that transcription of many genes was upregulated among persistent strains. This included three gene operons:pdu,cob-cbi, andeut. These genes may play a role in the persistence ofL. monocytogenesoutside the human host.

scholarly journals Genome Sequencing Identifies Two Nearly Unchanged Strains of Persistent Listeria monocytogenes Isolated at Two Different Fish Processing Plants Sampled 6 Years Apart

2013 ◽  
Vol 79 (9) ◽  
pp. 2944-2951 ◽  
Author(s):  
Anne Holch ◽  
Kristen Webb ◽  
Oksana Lukjancenko ◽  
David Ussery ◽  
Benjamin M. Rosenthal ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Random Amplified Polymorphic Dna ◽  
The United States ◽  
Genome Comparison ◽  
Physiological Factors ◽  
Content Type ◽  
Human Pathogenic Bacterium ◽  
A Genome ◽  
Genome Analyses

ABSTRACTListeria monocytogenesis a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for twoL. monocytogenesstrains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate usingin silicoanalyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistentL. monocytogenesstrain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene forinlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774,lmo2775, and the 3′-terminal part oflmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype.

scholarly journals Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

2015 ◽  
Vol 83 (8) ◽  
pp. 3233-3242 ◽  
Author(s):  
Lena Meyer ◽  
Jeanette E. Bröms ◽  
Xijia Liu ◽  
Martin E. Rottenberg ◽  
Anders Sjöstedt
Keyword(s):  
Listeria Monocytogenes ◽  
Francisella Tularensis ◽  
Hela Cells ◽  
Cell Types ◽  
Metabolic Adaptation ◽  
Content Type ◽  
Bacterial Uptake ◽  
J774 Cells ◽  
Cell Cytosol ◽  
Successful Replication

Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. ForFrancisella tularensis, many genes of theFrancisellapathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) ofF. tularensis; five selected FPI mutants thereof, i.e., ΔiglA, ΔiglÇ ΔiglG, ΔiglI, and ΔpdpEstrains; andListeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC−/−BMDM, MyD88−/−BMDM, J774 cells, or HeLa cells, LVS, ΔpdpEand ΔiglGmutants, andL. monocytogenesreplicated efficiently in all five cell types, whereas the ΔiglAand ΔiglCmutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC−/−BMDM, and HeLa cells. In contrast to the rapid replication in other cell types,L. monocytogenesshowed no replication in MyD88−/−BMDM and LVS showed no replication in either BMDM or MyD88−/−BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartmentper seare important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.

scholarly journals Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Mario Codemo ◽  
Sandra Muschiol ◽  
Federico Iovino ◽  
Priyanka Nannapaneni ◽  
Laura Plant ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Plasma Membrane ◽  
Streptococcus Pneumoniae ◽  
Extracellular Vesicles ◽  
Factor H ◽  
Host Cells ◽  
Immunomodulatory Effects ◽  
Content Type ◽  
Associated Proteins ◽  
Tract Infections

ABSTRACTGram-positive bacteria, including the major respiratory pathogenStreptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.IMPORTANCEStreptococcus pneumoniaeis a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections.

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