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1.
Hepatology ; 65(1): 294-309, 2017 01.
Article in English | MEDLINE | ID: mdl-27770554

ABSTRACT

The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow-based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon-γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time-lapse recordings, we demonstrated "intracellular crawling" of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule-1 and stabilin-1 and was facilitated by the junctional complexes between HSECs. CONCLUSION: Lymphocyte migration is facilitated by the unique structure of HSECs. Intracellular crawling may contribute to optimal lymphocyte positioning in liver tissue during chronic hepatitis. (Hepatology 2017;65:294-309).


Subject(s)
Capillaries/cytology , Cell Movement , Endothelial Cells/physiology , Lymphocytes/physiology , Cytoplasm , Endothelium, Vascular/cytology , Humans , Liver/blood supply
2.
Biol Bull ; 231(1): 40-60, 2016 08.
Article in English | MEDLINE | ID: mdl-27638694

ABSTRACT

Particles present in diesel exhaust have been proposed as a significant contributor to the development of acute and chronic lung diseases, including respiratory infection and allergic asthma. Nanoceria (CeO2 nanoparticles) are used to increase fuel efficiency in internal combustion engines, are present in exhaust fumes, and could affect cells of the airway. Components from the environment such as biologically derived proteins, carbohydrates, and lipids can form a dynamic layer, commonly referred to as the "protein corona" which alters cellular nanoparticle interactions and internalization. Using confocal reflectance microscopy, we quantified nanoceria uptake by lung-derived cells in the presence and absence of a serum-derived protein corona. Employing mass spectrometry, we identified components of the protein corona, and demonstrated that the interaction between transferrin in the protein corona and the transferrin receptor is involved in mediating the cellular entry of nanoceria via clathrin-mediated endocytosis. Furthermore, under these conditions nanoceria does not affect cell growth, viability, or metabolism, even at high concentration. Alternatively, despite the antioxidant capacity of nanoceria, in serum-free conditions these nanoparticles induce plasma membrane disruption and cause changes in cellular metabolism. Thus, our results identify a specific receptor-mediated mechanism for nanoceria entry, and provide significant insight into the potential for nanoparticle-dependent toxicity.


Subject(s)
Cerium/toxicity , Clathrin/metabolism , Endocytosis , Metal Nanoparticles/toxicity , Protein Corona/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cerium/metabolism , Culture Media, Serum-Free , Humans , Protein Corona/chemistry , Receptors, Transferrin/metabolism , Transferrin/metabolism
3.
FEMS Microbiol Lett ; 362(11)2015 Jun.
Article in English | MEDLINE | ID: mdl-25953858

ABSTRACT

Salmonella can bind to the leaves of salad crops including lettuce and survive for commercially relevant periods. Previous studies have shown that younger leaves are more susceptible to colonization than older leaves and that colonization levels are dependent on both the bacterial serovar and the lettuce cultivar. In this study, we investigated the ability of two Lactuca sativa cultivars (Saladin and Iceberg) and an accession of wild lettuce (L. serriola) to support attachment of Salmonella enterica serovar Senftenberg, to the first and fifth to sixth true leaves and the associations between cultivar-dependent variation in plant leaf surface characteristics and bacterial attachment. Attachment levels were higher on older leaves than on the younger ones and these differences were associated with leaf vein and stomatal densities, leaf surface hydrophobicity and leaf surface soluble protein concentrations. Vein density and leaf surface hydrophobicity were also associated with cultivar-specific differences in Salmonella attachment, although the latter was only observed in the older leaves and was also associated with level of epicuticular wax.


Subject(s)
Bacterial Adhesion , Lactuca/microbiology , Plant Leaves/microbiology , Salmonella enterica/physiology , Colony Count, Microbial , Hydrophobic and Hydrophilic Interactions , Plant Leaves/anatomy & histology , Plant Leaves/chemistry , Salmonella enterica/growth & development , Salmonella enterica/isolation & purification , Salmonella enterica/ultrastructure , Waxes
4.
PLoS One ; 9(1): e80160, 2014.
Article in English | MEDLINE | ID: mdl-24454682

ABSTRACT

The type III protein secretion system is an important pathogenicity factor of enteropathogenic and enterohaemorrhagic Escherichia coli pathotypes. The genes encoding this apparatus are located on a pathogenicity island (the locus of enterocyte effacement) and are transcriptionally activated by the master regulator Ler. In each pathotype Ler is also known to regulate genes located elsewhere on the chromosome, but the full extent of the Ler regulon is unclear, especially for enteropathogenic E. coli. The Ler regulon was defined for two strains of E. coli: E2348/69 (enteropathogenic) and EDL933 (enterohaemorrhagic) in mid and late log phases of growth by DNA microarray analysis of the transcriptomes of wild-type and ler mutant versions of each strain. In both strains the Ler regulon is focused on the locus of enterocyte effacement - all major transcriptional units of which are activated by Ler, with the sole exception of the LEE1 operon during mid-log phase growth in E2348/69. However, the Ler regulon does extend more widely and also includes unlinked pathogenicity genes: in E2348/69 more than 50 genes outside of this locus were regulated, including a number of known or potential pathogenicity determinants; in EDL933 only 4 extra-LEE genes, again including known pathogenicity factors, were activated. In E2348/69, where the Ler regulon is clearly growth phase dependent, a number of genes including the plasmid-encoded regulator operon perABC, were found to be negatively regulated by Ler. Negative regulation by Ler of PerC, itself a positive regulator of the ler promoter, suggests a negative feedback loop involving these proteins.


Subject(s)
Enterohemorrhagic Escherichia coli/genetics , Enteropathogenic Escherichia coli/genetics , Escherichia coli Proteins/genetics , Oligonucleotide Array Sequence Analysis , Regulon/genetics , Trans-Activators/genetics , Adaptation, Physiological/genetics , Enterohemorrhagic Escherichia coli/physiology , Enteropathogenic Escherichia coli/physiology , Evolution, Molecular , Gene Transfer, Horizontal/genetics , Genome, Bacterial/genetics , Transcription, Genetic/genetics
5.
Biochem J ; 441(1): 247-53, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-21973189

ABSTRACT

The 5' end of the major RNA transcript of the LEE1 operon of enterohaemorrhagic Escherichia coli contains ~170 bases before the AUG translation start codon of the first recognized gene, ler. This unusually long leader sequence carries three potential alternative AUG start codons. Using a lac fusion expression vector, we confirmed that the ler gene AUG is functional for translation initiation, and we checked for translation initiation at the three alternative AUG codons. Whereas two of the alternative AUG codons appear incompetent for translation initiation, we detected strong initiation at the third AUG, which is followed by one AAA codon and a UAG stop codon. The location of this very short two-codon open reading frame with respect to the ler translation start appears to be critical. Hence mutations that destroy the UAG stop codon, or short deletions between the UAG stop codon and the ler translation initiation region, result in big effects on ler expression. In the context of the full-length LEE1 operon leader sequence, translation of this very short two-codon open reading frame is necessary for optimal expression of the ler gene and for the subsequent interactions of enterohaemorrhagic Escherichia coli with host target cells.


Subject(s)
Enterohemorrhagic Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Operon/physiology , Base Sequence , Codon, Initiator , Down-Regulation , Escherichia coli Proteins/genetics , Molecular Sequence Data , Operon/genetics , Promoter Regions, Genetic , Protein Biosynthesis
6.
Infect Immun ; 79(11): 4342-52, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21859856

ABSTRACT

Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella.


Subject(s)
Adhesins, Bacterial/metabolism , Bacterial Adhesion/physiology , Biofilms , Gene Expression Regulation, Bacterial/physiology , Membrane Proteins/metabolism , Salmonella typhimurium/metabolism , Adhesins, Bacterial/genetics , Alum Compounds , Animals , Bacterial Adhesion/genetics , Caco-2 Cells , Escherichia coli K12/metabolism , Humans , Immunoglobulin G , Membrane Proteins/genetics , Mice , Phylogeny , Salmonella typhimurium/genetics , Salmonella typhimurium/pathogenicity , Virulence
7.
Environ Microbiol ; 13(5): 1299-308, 2011 May.
Article in English | MEDLINE | ID: mdl-21349136

ABSTRACT

The number of outbreaks and illness linked to the consumption of contaminated salad leaves have increased dramatically in the last decade. Escherichia coli and Salmonella enterica are the most common food-borne pathogens linked to consumption of fresh produce. Different serovars of S. enterica subspecies enterica have been shown to bind the surface of salad leaves, to exhibit tropism towards the stomata and to invade leaves and reach the underlying mesophyll. However the consequences of leaf invasion are not known. Here we show that following infiltration, serovars Typhimurium, Enteritidis, Heidelberg and Agona, as well as strains of S. enterica subspecies arizonae and diarizonae, survive in the mesophyll of Arabidopsis thaliana leaves but induce neither leaf chlorosis nor wilting. In contrast, S. Senftenberg induced strong leaf wilting 4 days post infiltration in A. thaliana accession Col-0 but not in accession Ws-0. Dead S. Senftenberg and bacterial lysates also induced leaf wilting. We found that mutations in the Arabidopsis pathogen associated molecular pattern (PAMP) recognition receptors (PRRs) FLS2, which recognizes flagellin, and EFR, which recognizes the bacterial elongation factor EF-Tu, had no effect on the wilting response of A. thaliana to S. Senftenberg. Infiltration of A. thaliana leaves with serovars Cannstatt, Krefeld and Liverpool, which like Senftenberg belong to Salmonella serogroup E(4) (O:1,3,19), also resulted in rapid leaf wilting, while all tested rough S. Senftenberg strains (lacking the O antigen) failed to elicit leaf wilting. These results suggest that the Salmonella O antigen 1,3,19 specifically triggers leaf chlorosis and wilting in A. thaliana.


Subject(s)
Arabidopsis/microbiology , Plant Leaves/microbiology , Salmonella enterica/classification , Salmonella enterica/pathogenicity , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Flagellin/metabolism , Genetic Variation , O Antigens/metabolism , Peptide Elongation Factor Tu/metabolism , Plant Diseases/microbiology , Protein Kinases/genetics , Receptors, Pattern Recognition/genetics , Salmonella enterica/genetics
8.
Environ Microbiol Rep ; 3(1): 112-7, 2011 Feb.
Article in English | MEDLINE | ID: mdl-23761239

ABSTRACT

Enterotoxigenic Escherichia coli (ETEC) causes child and travelers' diarrhea and is presumed to be water- and food-borne. Sporadic outbreaks were traced to consumption of contaminated fresh produce, particularly salad leaves as lettuce and parsley. Importantly, the mechanism by which ETEC binds salad leaves is not known. In this study we investigated the ability of clinical ETEC isolates to adhere to Eruca vesicaria (commonly known as rocket). Towards this end we inoculated pieces of cut E. vesicaria leaves with clinical ETEC isolates grown in Luria broth at 20°C, conditions that are not permissive for expression of the plasmid-encoded colonization factors and hence mimic the actual transmission pathways of ETEC through intake of contaminated food. We found that ETEC strains bind E. vesicaria at various efficiencies. Examination of representative strains by scanning electron microscopy revealed that they adhere to the E. vesicaria surface in a diffuse pattern by extended filaments resembling flagella. Using the prototype ETEC strain H10407 we found that it also binds to lettuce, basil and spinach leaves. Binding of H10407 was dependent on flagella as a fliC mutant attached to leaves at a much lower efficiency. Interestingly, under the employed environmental conditions EtpA, which forms a flagellar tip structure, and colonization factor I are dispensable for leaf attachment. The results show that ETEC can bind specifically to salad leaves, which might represent an important, yet less recognized, source of infection.

9.
Environ Microbiol Rep ; 3(5): 569-73, 2011 Oct.
Article in English | MEDLINE | ID: mdl-23761337

ABSTRACT

Fresh fruit and vegetables are important components of a healthy and balanced diet. However, they are increasingly being recognized as important vehicles for transmission of human pathogens that were traditionally classified as zoonotic. There is a significant gap in our knowledge and understanding of the mechanisms by which human pathogens colonize and survive on or in fruits and vegetables. In this study we investigated the binding of Salmonella enterica to tomato fruits (Solanum lycopersicum), which is becoming a major source of human infection. We report that Salmonella enterica serovars Typhimurium and Senftenberg bound to the surface of unripe tomatoes in an aggregative pattern, while serovar Thompson adhered diffusely. We found that while flagella did not have a role in binding, bcsC S. Typhimurim mutants, deficient in cellulose production, exhibited significantly reduced level of attachment to tomatoes. Trans complementation of the mutation restored adhesion to the wild-type level.

10.
Infection and Immunity ; 79(11): 4342-4352, 2011.
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP, SESSP-IBACERVO | ID: biblio-1063424

ABSTRACT

Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromisedindividuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance becausethey are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA,a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. Wedemonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice.Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinalCaco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG responsewhich provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced byadministering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadAhaving pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection againstSalmonella.


Subject(s)
Mice , Adhesins, Bacterial/analysis , Adhesins, Bacterial/immunology , Adhesins, Bacterial/isolation & purification , Salmonella enterica/pathogenicity , Immunoglobulin G/immunology
11.
Environ Microbiol ; 12(9): 2385-97, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20636374

ABSTRACT

Much research into food-borne human pathogens has focused on transmission from foods of animal origin. However, recent investigations have identified fruits and vegetables are the source of many disease outbreaks. Now believed to be a much larger contributor to produce-associated outbreaks than previously reported, norovirus outbreaks are commonly caused by contamination of foods from hands of infected workers. Although infections with Shiga toxin-producing E. coli O157 have been linked to beef more often than to any other food product, severe outbreaks have been traced to consumption of contaminated radish sprouts and pre-packaged spinach. Similarly, while infections with Salmonella have mainly been linked to consumption of foods of animal origin, many outbreaks have been traced to contaminated fresh produce. E. coli O157 binds to lettuce leaves by alternative mechanisms involving the filamentous type III secretions system, flagella and the pilus curli. Association of Salmonella with fresh produce appears to be serovar-specific involving flagella, curli, cellulose, and O antigen capsule. A better understanding of plant, microbiological, environmental, processing and food handling factors that facilitate contamination will allow development of evidence-based policies, procedures and technologies aimed at reducing the risk of contamination of fresh produce.


Subject(s)
Food Microbiology , Foodborne Diseases/epidemiology , Fruit/microbiology , Vegetables/microbiology , Disease Outbreaks/prevention & control , Escherichia coli O157/isolation & purification , Foodborne Diseases/microbiology , Foodborne Diseases/prevention & control , Humans , Salmonella/isolation & purification
12.
ISME J ; 3(2): 261-5, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18830276

ABSTRACT

Contaminated salad leaves have emerged as important vehicles for the transmission of enteric pathogens to humans. A recent outbreak of Salmonella enterica serovar Senftenberg (S. Senftenberg) in the United Kingdom has been traced to the consumption of contaminated basil. Using the outbreak strain of S. Senftenberg, we found that it binds to basil, lettuce, rocket and spinach leaves showing a pattern of diffuse adhesion. Flagella were seen linking S. Senftenberg to the leaf epidermis, and the deletion of fliC (encoding phase-1 flagella) resulted in a significantly reduced level of adhesion. In contrast, although flagella linking S. enterica serovar Typhimurium to the basil leaf epidermis were widespread, deletion of fliC did not affect leaf attachment levels. These results implicate the role of flagella in Salmonella leaf attachment and suggest that different Salmonella serovars use strain-specific mechanisms to attach to salad leaves.


Subject(s)
Bacterial Adhesion , Barbarea/microbiology , Lactuca/microbiology , Ocimum basilicum/microbiology , Plant Leaves/microbiology , Salmonella enterica/physiology , Spinacia oleracea/microbiology , Disease Outbreaks , Flagella/genetics , Flagella/physiology , Humans , Salmonella Food Poisoning/epidemiology , Salmonella Food Poisoning/microbiology , Salmonella enterica/isolation & purification , United Kingdom/epidemiology
13.
Environ Microbiol Rep ; 1(4): 234-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-23765852

ABSTRACT

Enteroaggregative Escherichia coli (EAEC) are important human pathogens. However, their environmental reservoir is unknown. As fresh salad leaves are increasingly recognized as an important environmental vector for human pathogens, we investigated leaf attachment capability of EAEC strains. We found that binding of clinical EAEC isolates to leaves from Eruca vesicaria (commonly known as rocket or arugula) can be divided into high, moderate and low adherent phenotypes. Using the prototype EAEC strain 042 to investigate the underlining mechanisms involved in leaf attachment, we found small attached bacterial aggregates over the entire leaf surface and dense bacterial attachment to the guard cell of the stomata. An aaf 042 mutant lost the ability to bind the epidermis while retaining stomatal adherence. In contrast, a fliC 042 mutant retained the ability to bind the epidermis but lost stomatal tropism. These results show that multiple adherence factors are involved in the interaction of EAEC with leaves, that EAEC uses similar colonization factors to bind mucosal and leaf surfaces and that fresh produce might be an important reservoir of EAEC strains.

15.
J Mol Biol ; 378(1): 243-50, 2008 Apr 18.
Article in English | MEDLINE | ID: mdl-18353364

ABSTRACT

Enteropathogenic Escherichia coli utilise a filamentous type III secretion system to translocate effector proteins into host gut epithelial cells. The primary constituent of the extracellular component of the filamentous type III secretion system is EspA. This forms a long flexible helical conduit between the bacterium and host and has a structure almost identical to that of the flagella filament. We have inserted the D3 domain of FliCi (from Salmonella typhimurium) into the outer domain of EspA and have studied the structure and function of modified filaments when expressed in an enteropathogenic E. coli espA mutant. We found that the chimeric protein EspA-FliCi filaments were biologically active as they supported protein secretion and translocation [assessed by their ability to trigger actin polymerisation beneath adherent bacteria (fluorescent actin staining test)]. The expressed filaments were recognised by both EspA and FliCi antisera. Visualisation and analysis of the chimeric filaments by electron microscopy after negative staining showed that, remarkably, EspA filaments are able to tolerate a large protein insertion without a significant effect on their helical architecture.


Subject(s)
Enteropathogenic Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Flagellin/chemistry , Flagellin/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Amino Acid Sequence , Escherichia coli Proteins/genetics , Flagellin/genetics , HeLa Cells , Humans , Microscopy, Electron , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Recombinant Fusion Proteins/genetics , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism
16.
Appl Environ Microbiol ; 74(9): 2908-14, 2008 May.
Article in English | MEDLINE | ID: mdl-18310437

ABSTRACT

Enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens that use a filamentous type III secretion system (fT3SS) for colonization of the gut epithelium. In this study we have shown that EHEC O157 and O26 strains use the fT3SS apparatus for attachment to leaves. Leaf attachment was independent of effector protein translocation.


Subject(s)
Bacterial Adhesion/physiology , Enterohemorrhagic Escherichia coli/physiology , Escherichia coli Proteins/metabolism , Lactuca/microbiology , Plant Leaves/microbiology , Colony Count, Microbial , Escherichia coli Proteins/genetics , Gene Deletion
17.
Cell Microbiol ; 10(7): 1429-41, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18331467

ABSTRACT

Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium. We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway.


Subject(s)
Actins/metabolism , Bacteria/pathogenicity , Bacterial Adhesion/physiology , Bacterial Proteins/metabolism , Stress Fibers/metabolism , 3T3 Cells , Actin Depolymerizing Factors/metabolism , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Bacteria/metabolism , Bacterial Proteins/genetics , Enzyme Activation , GTP-Binding Proteins , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Phylogeny , Sequence Alignment , Signal Transduction/physiology , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases/metabolism
18.
J Bacteriol ; 190(6): 2221-6, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18178741

ABSTRACT

Enteropathogenic Escherichia coli employs a filamentous type III secretion system, made by homopolymerization of the translocator protein EspA. In this study, we have shown that the N-terminal region of EspA has a role in EspA's protein stability, interaction with the CesAB chaperone, and filament biogenesis and function.


Subject(s)
Amino Acids/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Amino Acids/genetics , Amino Acids/physiology , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Fluorescent Antibody Technique , HeLa Cells , Humans , Models, Biological , Mutation , Protein Structure, Tertiary
19.
Microbes Infect ; 8(8): 2220-7, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16781180

ABSTRACT

The enteropathogenic Escherichia coli (EPEC) effector protein EspG, like the Shigella effector VirA, functions through disruption of the host cell microtubule network. Reports have differed as to whether the EspG homologue, EspG2, is also responsible for microtubule disruption. In this study we show that following translocation, EspG2 and VirA are localised under adherent bacteria and able to restore the microtubule disruption phenotype to an espG/espG2 double EPEC mutant. The espG/espG2 double mutant produced A/E lesions similar to wild-type EPEC on human intestinal in vitro organ cultures. Determining the distribution of espG and espG2 among clinical EPEC isolates revealed two different types of espG (espG alpha and espG beta) and espG2 (intact and pseudo genes), which were associated with specific EPEC serotypes and closely followed the EPEC lineage. This investigation has established a role for EspG2 in the disruption of the microtubule network and associated different espG and espG2 types with different groups of EPEC.


Subject(s)
Escherichia coli Proteins/analysis , Escherichia coli Proteins/physiology , Escherichia coli/chemistry , Escherichia coli/pathogenicity , Virulence Factors/genetics , Caco-2 Cells , Cytoplasm/chemistry , DNA, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Deletion , Genetic Complementation Test , Humans , Intestinal Mucosa/microbiology , Intestinal Mucosa/ultrastructure , Microscopy, Confocal , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Microtubules/metabolism , Organ Culture Techniques
20.
J Bacteriol ; 188(8): 3110-5, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16585770

ABSTRACT

EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.


Subject(s)
Carrier Proteins/metabolism , Escherichia coli Proteins/metabolism , Amino Acid Motifs , Carrier Proteins/chemistry , Epithelial Cells/chemistry , Epithelial Cells/microbiology , Escherichia coli Proteins/chemistry , HeLa Cells , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins , Microscopy, Confocal , Protein Binding , Sorting Nexins , Vesicular Transport Proteins , src Homology Domains
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