scholarly journals The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.

1996 ◽  
Vol 135 (3) ◽  
pp. 647-660 ◽  
Author(s):  
G A Smith ◽  
J A Theriot ◽  
D A Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Consensus Sequence ◽  
Wild Type ◽  
Movement Rate ◽  
Bacterial Surface ◽  
Rate Dependent ◽  
Repeat Domain ◽  
Low Levels ◽  
Rate Of Movement

The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.

scholarly journals Listeria monocytogenes σB Regulates Stress Response and Virulence Functions

2003 ◽  
Vol 185 (19) ◽  
pp. 5722-5734 ◽  
Author(s):  
Mark J. Kazmierczak ◽  
Sharon C. Mithoe ◽  
Kathryn J. Boor ◽  
Martin Wiedmann
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Stress Response ◽  
Consensus Sequence ◽  
Virulence Gene ◽  
Wild Type ◽  
Promoter Regions ◽  
Virulence Gene Expression ◽  
Stress Response Genes ◽  
In The Wild

ABSTRACT While the stress-responsive alternative sigma factor σB has been identified in different species of Bacillus, Listeria, and Staphylococcus, theσ B regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify σB-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidateσ B-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted σB-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ΔsigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significantσ B-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting σB-dependent expression, 54 were preceded by a sequence resembling the σB promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the σB-dependent nature of a subset of eight selected promoter regions. Notably, theσ B-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, σB also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest thatσ B contributes to L. monocytogenes gene expression during infection.

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 Invasion of Epithelial Cells Requires the MEK-1/ERK-2 Mitogen-Activated Protein Kinase Pathway

1998 ◽  
Vol 66 (3) ◽  
pp. 1106-1112 ◽  
Author(s):  
Patrick Tang ◽  
Claire L. Sutherland ◽  
Michael R. Gold ◽  
B. Brett Finlay
Keyword(s):  
Listeria Monocytogenes ◽  
Epithelial Cells ◽  
Map Kinase ◽  
Actin Polymerization ◽  
Map Kinases ◽  
Specific Inhibitor ◽  
Bacterial Invasion ◽  
Wild Type ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

ABSTRACT PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blocked Listeria monocytogenesinvasion into HeLa epithelial cells. The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug. Previously, we reported that L. monocytogenes could activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P. Tang, I. Rosenshine, P. Cossart, and B. B. Finlay, Infect. Immun. 64:2359–2361, 1996). We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-type L. monocytogenes. Mutants lacking functional LLO (hly mutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times. Two inhibitors of L. monocytogenesinvasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-type L. monocytogenes and hly mutants. However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation. These results suggest that MEK-1 and ERK-2 activities are essential for L. monocytogenes invasion into host epithelial cells. This is the first report to show that a MAP kinase pathway is required for bacterial invasion.

Actin Dynamics and Force Generation in the Motility of Listeria Monocytogenes

1997 ◽  
Vol 3 (S2) ◽  
pp. 209-210
Author(s):  
David C. Fung ◽  
Julie A. Theriot
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Time Lapse ◽  
Actin Dynamics ◽  
Cell Protein ◽  
Animal Cells ◽  
Free System ◽  
Bacterial Surface ◽  
Egg Extracts ◽  
Serious Disease

The gram-positive bacterium Listeria monocytogenes is one of several intracellular pathogens which can move about within its host's cytoplasm using a form of actin-based motility. This motility plays an important role in the virulence of the microbe, which can cause serious disease in humans. Actin is a host-cell protein whose polymerization is required for the locomotion of animal cells. L. monocytogenes exploits this normal cellular machinery for its own movement by creating a “comet” tail of cross-linked actin filaments behind it. Actin polymerization occurs at the bacterial surface and is required to drive the bacterium forward.We are interested in how L. monocytogenes induces the polymerization of actin and how this polymerization can lead to the generation of force. To study this, we have used time-lapse videomicroscopy and a cell-free system derived from Xenopus egg extracts. We have observed rare instances of a saltatory movement of L. monocytogenes in these extracts.

scholarly journals HET-E and HET-D Belong to a New Subfamily of WD40 Proteins Involved in Vegetative Incompatibility Specificity in the Fungus Podospora anserina

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 71-81
Author(s):  
Eric Espagne ◽  
Pascale Balhadère ◽  
Marie-Louise Penin ◽  
Christian Barreau ◽  
Béatrice Turcq
Keyword(s):  
Genetic Difference ◽  
Podospora Anserina ◽  
Wild Type ◽  
Incompatible Interaction ◽  
Vegetative Incompatibility ◽  
Repeat Domain ◽  
Upper Face ◽  
E1a Gene ◽  
Type Strains ◽  
Wd40 Repeats

Abstract Vegetative incompatibility, which is very common in filamentous fungi, prevents a viable heterokaryotic cell from being formed by the fusion of filaments from two different wild-type strains. Such incompatibility is always the consequence of at least one genetic difference in specific genes (het genes). In Podospora anserina, alleles of the het-e and het-d loci control heterokaryon viability through genetic interactions with alleles of the unlinked het-c locus. The het-d2Y gene was isolated and shown to have strong similarity with the previously described het-e1A gene. Like the HET-E protein, the HET-D putative protein displayed a GTP-binding domain and seemed to require a minimal number of 11 WD40 repeats to be active in incompatibility. Apart from incompatibility specificity, no other function could be identified by disrupting the het-d gene. Sequence comparison of different het-e alleles suggested that het-e specificity is determined by the sequence of the WD40 repeat domain. In particular, the amino acids present on the upper face of the predicted β-propeller structure defined by this domain may confer the incompatible interaction specificity.

scholarly journals Optimal Flow—A Pilot Study Balancing Sheep Movement and Welfare in Abattoirs

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 344
Author(s):  
Melissa J. Starling ◽  
Elyssa Payne ◽  
Paul McGreevy
Keyword(s):  
Pilot Study ◽  
Additional Source ◽  
Movement Rate ◽  
Optimal Outcomes ◽  
Optimal Flow ◽  
Livestock Movement ◽  
Trade Offs ◽  
Behavioural Interactions ◽  
Handling Practices ◽  
Rate Of Movement

Abattoirs are faced with the challenge of moving livestock efficiently through the plant, while also engaging in handling practices that assure good animal welfare. Achieving optimal outcomes for both of these goals can bring them into conflict. An additional source of conflict can arise from the design of the abattoir. These problems are compounded by the dearth of research available to inform how livestock should be handled to achieve all of these goals. We applied the concept of ‘Optimal Flow’ to describe conditions under which rate of movement is maximised while overt signs of distress in sheep are minimised. Effectively, this represents the point at which trade-offs between speed and welfare converge. The current pilot study examined the behavioural interactions between humans (n = 5), livestock herding dogs (n = 7), and sheep (n = 3235) in a large Australian abattoir to describe the factors associated with an increase or decrease in rate of sheep movement per minute. It revealed that distress behaviours in sheep were associated with dog presence and with a decrease in livestock movement rate. However, we found that as sheep density increased, there was increased livestock movement rate as well as an elevated incidence of distress behaviours. Optimal Flow at this abattoir was achieved by maintaining sheep at lower densities. Our report discusses the possible confounds in this interpretation.

scholarly journals α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 771
Author(s):  
Julen Rodríguez-Castejón ◽  
Ana Alarcia-Lacalle ◽  
Itziar Gómez-Aguado ◽  
Mónica Vicente-Pascual ◽  
María Ángeles Solinís Aspiazu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fabry Disease ◽  
Viral Vector ◽  
Viral Gene ◽  
Lysosomal Storage ◽  
Wild Type ◽  
Dose Administration ◽  
Viral Gene Therapy ◽  
Liver Transaminases ◽  
Low Levels

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.

scholarly journals Renal Dnase1 expression is regulated by FGF23 but loss of Dnase1 does not alter renal phosphate handling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniela Egli-Spichtig ◽  
Martin Y. H. Zhang ◽  
Alfred Li ◽  
Eva Maria Pastor Arroyo ◽  
Nati Hernando ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Actin Polymerization ◽  
Fibroblast Growth Factor 23 ◽  
Wild Type ◽  
Vitamin D Metabolism ◽  
Endocrine Hormone ◽  
Dnase 1 ◽  
Sodium Dependent ◽  
Renal Tubular ◽  
High Phosphate

AbstractFibroblast growth factor 23 (FGF23) is a bone-derived endocrine hormone that regulates phosphate and vitamin D metabolism. In models of FGF23 excess, renal deoxyribonuclease 1 (Dnase1) mRNA expression is downregulated. Dnase-1 is an endonuclease which binds monomeric actin. We investigated whether FGF23 suppresses renal Dnase-1 expression to facilitate endocytic retrieval of renal sodium dependent phosphate co-transporters (NaPi-IIa/c) from the brush border membrane by promoting actin polymerization. We showed that wild type mice on low phosphate diet and Fgf23−/− mice with hyperphosphatemia have increased renal Dnase1 mRNA expression while in Hyp mice with FGF23 excess and hypophosphatemia, Dnase1 mRNA expression is decreased. Administration of FGF23 in wild type and Fgf23−/− mice lowered Dnase1 expression. Taken together, our data shows that Dnase1 is regulated by FGF23. In 6-week-old Dnase1−/− mice, plasma phosphate and renal NaPi-IIa protein were significantly lower compared to wild-type mice. However, these changes were transient, normalized by 12 weeks of age and had no impact on bone morphology. Adaptation to low and high phosphate diet were similar in Dnase1−/− and Dnase1+/+ mice, and loss of Dnase1 gene expression did not rescue hyperphosphatemia in Fgf23−/− mice. We conclude that Dnase-1 does not mediate FGF23-induced inhibition of renal tubular phosphate reabsorption.

scholarly journals Platelet and Erythrocyte Extravasation across Inflamed Corneal Venules Depend on CD18, Neutrophils and Mast Cell Degranulation

2021 ◽  
Vol 22 (14) ◽  
pp. 7360
Author(s):  
Angie De La Cruz ◽  
Aubrey Hargrave ◽  
Sri Magadi ◽  
Justin A. Courson ◽  
Paul T. Landry ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Types ◽  
Mast Cell Degranulation ◽  
Wild Type ◽  
Delayed Wound Healing ◽  
Corneal Epithelial ◽  
Corneal Abrasion ◽  
Low Levels ◽  
Endothelial Pores

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the β2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.

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