scholarly journals Role of Listeriolysin O in Cell-to-Cell Spread ofListeria monocytogenes

2000 ◽  
Vol 68 (2) ◽  
pp. 999-1003 ◽  
Author(s):  
Margaret M. Gedde ◽  
Darren E. Higgins ◽  
Lewis G. Tilney ◽  
Daniel A. Portnoy
Keyword(s):  
Bacterial Pathogen ◽  
Listeriolysin O ◽  
Genetic Approach ◽  
Wild Type ◽  
Intracellular Growth ◽  
Double Membrane ◽  
Vacuolar Compartment ◽  
Oflisteria Monocytogenes ◽  
Cell Spread

ABSTRACT Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L. monocytogenes from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape ofL. monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread.

scholarly journals Mutagenesis of Active-Site Histidines ofListeria monocytogenes Phosphatidylinositol-Specific Phospholipase C: Effects on Enzyme Activity and Biological Function

1999 ◽  
Vol 67 (1) ◽  
pp. 182-186 ◽  
Author(s):  
Trudi Bannam ◽  
Howard Goldfine
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Intracellular Growth ◽  
Oflisteria Monocytogenes ◽  
Culture Supernatants ◽  
Derivatives Of ◽  
Cell Spread ◽  
Active Site Histidine

ABSTRACT Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C. PC-PLC, encoded by plcB, is a broad-range phospholipase, whereas PI-PLC, encoded by plcA, is specific for phosphatidylinositol. It was previously shown that PI-PLC plays a role in efficient escape of L. monocytogenes from the primary phagosome. To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis of plcA was performed. Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine. With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity. In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression. Derivatives of L. monocytogenes containing these specific plcA mutations were found to have phenotypes similar to that of the plcA deletion strain in an assay for escape from the primary vacuole, in intracellular growth in a murine macrophage cell line, and in a plaquing assay for cell-to-cell spread. Thus, catalytic activity of PI-PLC is required for all its intracellular functions.

scholarly journals Stress-Induced ClpP Serine Protease ofListeria monocytogenes Is Essential for Induction of Listeriolysin O-Dependent Protective Immunity

2001 ◽  
Vol 69 (8) ◽  
pp. 4938-4943 ◽  
Author(s):  
Olivier Gaillot ◽  
Søren Bregenholt ◽  
Francis Jaubert ◽  
James P. Di Santo ◽  
Patrick Berche
Keyword(s):  
Immune Response ◽  
Serine Protease ◽  
Protective Immunity ◽  
Listeriolysin O ◽  
Intracellular Growth ◽  
Lethal Challenge ◽  
Protective Antigens ◽  
Oflisteria Monocytogenes

ABSTRACT The stress-induced protease ClpP is required for virulence of the facultative intracellular pathogen Listeria monocytogenes. We previously found that in the absence of ClpP, the virulence of this pathogen was strongly reduced, mainly due to the decreased production of functional listeriolysin O (LLO), a major immunodominant virulence factor promoting intracellular growth. In this work, a clpP deletion mutant of L. monocytogenes was used to study the generation of anti-Listeria protective immunity. We found that ClpP is required for the intracellular growth of L. monocytogenes in resident macrophages in vivo. Mice infected with doses as high as 106 clpP mutant bacteria were not protected against a lethal challenge of wild-type bacteria and did not develop any detectable LLO-specific cytolytic T cells or antibodies, suggesting that the amount of LLO produced in infected mice under these conditions was too low to induce a specific immune response. However, in contrast to the results obtained with a mutant with a disrupted hly gene, this lack of protection was overcome by inoculation of very high infecting doses ofclpP mutant bacteria (5 × 108), thus producing sufficient amounts of LLO to stimulate anti-Listeria immunity. The role of ClpP was confirmed by showing that anti-Listeria immunity was restored in mice infected with a clpP-complemented mutant. These results indicate that the stress-induced serine protease ClpP is a potential target for modulating the presentation of protective antigens such as LLO and thereby the immune response against L. monocytogenes.

scholarly journals Motility-Independent Formation of Antibiotic-TolerantPseudomonas aeruginosaAggregates

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Sally Demirdjian ◽  
Hector Sanchez ◽  
Daniel Hopkins ◽  
Brent Berwin
Keyword(s):  
Biofilm Formation ◽  
Bacterial Pathogen ◽  
Aggregate Formation ◽  
Flagellar Motility ◽  
Wild Type ◽  
Chronic Infections ◽  
Content Type ◽  
Temporal Adaptation ◽  
Bacterial Aggregates

ABSTRACTPseudomonas aeruginosais a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate thatP. aeruginosaaggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections.IMPORTANCEIn this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated thatP. aeruginosaflagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation byP. aeruginosawas observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-typeP. aeruginosaand mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotileP. aeruginosabacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

scholarly journals Spatial, Temporal, and Functional Assessment of LC3-Dependent Autophagy inShigella flexneriDissemination

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Erin Weddle ◽  
Hervé Agaisse
Keyword(s):  
Colonic Mucosa ◽  
Defense Mechanism ◽  
Positive Impact ◽  
Shigella Flexneri ◽  
Wild Type ◽  
Content Type ◽  
Cell Defense ◽  
Membrane Protrusions ◽  
Cell Spread

ABSTRACTShigella flexneridisseminates within the colonic mucosa by displaying actin-based motility in the cytosol of epithelial cells. Motile bacteria form membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the bacteria escape, thereby achieving cell-to-cell spread. During dissemination,S. flexneriis targeted by LC3-dependent autophagy, a host cell defense mechanism against intracellular pathogens. TheS. flexneritype III secretion system effector protein IcsB was initially proposed to counteract the recruitment of the LC3-dependent autophagy machinery to cytosolic bacteria. However, a recent study proposed that LC3 was recruited to bacteria in DMVs formed during cell-to-cell spread. To resolve the controversy and clarify the role of autophagy inS. flexneriinfection, we tracked dissemination using live confocal microscopy and determined the spatial and temporal recruitment of LC3 to bacteria. This approach demonstrated that (i) LC3 was exclusively recruited to wild-type oricsBbacteria located in DMVs and (ii) theicsBmutant was defective in cell-to-cell spread due to failure to escape LC3-positive as well as LC3-negative DMVs. Failure ofS. flexnerito escape DMVs correlated with late LC3 recruitment, suggesting that LC3 recruitment is the consequence and not the cause of DMV escape failure. Inhibition of autophagy had no positive impact on the spreading of wild-type oricsBmutant bacteria. Our results unambiguously demonstrate that IcsB is required for DMV escape during cell-to-cell spread, regardless of LC3 recruitment, and do not support the previously proposed notion that autophagy countersS. flexneridissemination.

scholarly journals The Role of a Host-Induced Arginase of Xanthomonas oryzae pv. oryzae in Promoting Virulence on Rice

2019 ◽  
Vol 109 (11) ◽  
pp. 1869-1877
Author(s):  
Yuqiang Zhang ◽  
Guichun Wu ◽  
Ian Palmer ◽  
Bo Wang ◽  
Guoliang Qian ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Bacterial Blight ◽  
Wild Type Strain ◽  
Bacterial Pathogen ◽  
Extracellular Polysaccharide ◽  
Rice Cultivar ◽  
Xanthomonas Oryzae ◽  
Wild Type ◽  
Bacterial Blight Of Rice

The plant bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial blight of rice, which is one of the most destructive rice diseases prevalent in Asia and parts of Africa. Despite many years of research, how X. oryzae pv. oryzae causes bacterial blight of rice is still not completely understood. Here, we show that the loss of the rocF gene caused a significant decrease in the virulence of X. oryzae pv. oryzae in the susceptible rice cultivar IR24. Bioinformatics analysis demonstrated that rocF encodes arginase. Quantitative real-time PCR and Western blot assays revealed that rocF expression was significantly induced by rice and arginine. The rocF deletion mutant strain showed elevated sensitivity to hydrogen peroxide, reduced extracellular polysaccharide (EPS) production, and reduced biofilm formation, all of which are important determinants for the full virulence of X. oryzae pv. oryzae, compared with the wild-type strain. Taken together, the results of this study revealed a mechanism by which a bacterial arginase is required for the full virulence of X. oryzae pv. oryzae on rice because of its contribution to tolerance to reactive oxygen species, EPS production, and biofilm formation.

scholarly journals Role of Adhesin Release for Mucosal Colonization by a Bacterial Pathogen

2003 ◽  
Vol 197 (6) ◽  
pp. 735-742 ◽  
Author(s):  
Loïc Coutte ◽  
Sylvie Alonso ◽  
Nathalie Reveneau ◽  
Eve Willery ◽  
Brigitte Quatannens ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Wild Type ◽  
Early Step ◽  
Bacterial Surface ◽  
Extracellular Milieu ◽  
Wild Type Parent

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors called adhesins. To exert these functions, adhesins are typically surface-exposed, although, surprisingly, some are also released into the extracellular milieu, the relevance of which has previously not been studied. To address the role of adhesin release in pathogenesis, we used Bordetella pertussis as a model, since its major adhesin, filamentous hemagglutinin (FHA), partitions between the bacterial surface and the extracellular milieu. FHA release depends on its maturation by the specific B. pertussis protease SphB1. We constructed SphB1-deficient mutants and found that they were strongly affected in their ability to colonize the mouse respiratory tract, although they adhered even better to host cells in vitro than their wild-type parent strain. The defect in colonization could be overcome by prior nasal instillation of purified FHA or by coinfection with FHA-releasing B. pertussis strains, but not with SphB1-producing FHA-deficient strains, ruling out a nonspecific effect of SphB1. These results indicate that the release of FHA is important for colonization, as it may facilitate the dispersal of bacteria from microcolonies and the binding to new sites in the respiratory tract.

scholarly journals Legionella pneumophila Entry GenertxA Is Involved in Virulence

2001 ◽  
Vol 69 (1) ◽  
pp. 508-517 ◽  
Author(s):  
Suat L. G. Cirillo ◽  
Luiz E. Bermudez ◽  
Sahar H. El-Etr ◽  
Gerald E. Duhamel ◽  
Jeffrey D. Cirillo
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Integrin Receptors ◽  
Legionnaires Disease ◽  
Cell Spread ◽  
Gain Access

ABSTRACT Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1,enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to β2 integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry genertxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.

Contribution of cysteine residue to the properties of Listeria monocytogenes listeriolysin O

2009 ◽  
Vol 55 (10) ◽  
pp. 1153-1159 ◽  
Author(s):  
Radosław Stachowiak ◽  
Jarosław Wiśniewski ◽  
Olga Osińska ◽  
Jacek Bielecki
Keyword(s):  
Listeria Monocytogenes ◽  
Hemolytic Activity ◽  
Cysteine Residue ◽  
Listeriolysin O ◽  
Wild Type ◽  
Gram Positive Bacteria ◽  
Culture Model ◽  
The Family ◽  
Kinetics Of

Listeriolysin (LLO) is the key virulence factor critical for Listeria monocytogenes pathogenesis. Listerial cytolysin belongs to the family of cholesterol-dependent cytolysins (CDCs), a group of pore-forming toxins produced by related gram-positive bacteria. Most CDCs contain a cysteine residue in the conserved undecapeptide — a sequence that is highly preserved among this group of proteins. Substitutions of cysteine do not always lead to loss of hemolytic activity, questioning the purpose of such strong conservation of this amino acid in the sequence of CDC. The properties of 3 L. monocytogenes strains, a wild type and 2 mutants expressing modified LLO within the cysteine residue, were analyzed in this work. The first of these mutants producing a toxin with cysteine to alanine substitution showed similar features to the wild type except that a thiol-reducing agent was not necessary for hemolytic activity. Another strain secreting LLO containing serine instead of cysteine exhibited strikingly different properties than the wild type. Modified toxin is independent of the reducing reagents, less stable, and shows accelerated kinetics of cytolysis in comparison with the unchanged protein. However, both mutant strains are less invasive in the cell culture model showing the important role of cysteine in L. monocytogenes virulence.

scholarly journals Recombinant Listeria monocytogenes Expressing a Cell Wall-Associated Listeriolysin O Is Weakly Virulent but Immunogenic

2009 ◽  
Vol 77 (10) ◽  
pp. 4371-4382 ◽  
Author(s):  
Javier A. Carrero ◽  
Boris Calderon ◽  
Hector Vivanco-Cid ◽  
Emil R. Unanue
Keyword(s):  
Listeria Monocytogenes ◽  
Listeriolysin O ◽  
Wild Type ◽  
Positive Bacterium ◽  
Cell Responses ◽  
A Cell ◽  
Weakly Virulent

ABSTRACT Listeriolysin O (LLO) is an essential virulence factor for the gram-positive bacterium Listeria monocytogenes. Our goal was to determine if altering the topology of LLO would alter the virulence and toxicity of L. monocytogenes in vivo. A recombinant strain was generated that expressed a surface-associated LLO (sLLO) variant secreted at 40-fold-lower levels than the wild type. In culture, the sLLO strain grew in macrophages, translocated to the cytosol, and induced cell death. However, the sLLO strain showed decreased infectivity, reduced lymphocyte apoptosis, and decreased virulence despite a normal in vitro phenotype. Thus, the topology of LLO in L. monocytogenes was a factor in the pathogenesis of the infection and points to a role of LLO secretion during in vivo infection. The sLLO strain was cleared by severe combined immunodeficient (SCID) mice. Despite the attenuation of virulence, the sLLO strain was immunogenic and capable of eliciting protective T-cell responses.

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