Enteropathogenic Escherichia coli type III effectors alter cytoskeletal function and signalling in Saccharomyces cerevisiae

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2933-2945 ◽  
Author(s):  
Isabel Rodríguez-Escudero ◽  
Philip R. Hardwidge ◽  
César Nombela ◽  
Víctor J. Cid ◽  
B. Brett Finlay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Effector Proteins ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Genes Encoding ◽  
Cytoskeletal Function ◽  
Enterocyte Effacement ◽  
Functional Analyses ◽  
Map Kinase Pathways

Enteropathogenic Escherichia coli (EPEC) strains cause attaching/effacing lesions in enterocytes through the development of actin-supported pedestals at the site of bacterial adhesion. Pathogenesis requires a type III secretion system (TTSS), which injects into the host cell the intimin receptor, Tir, as well as other effectors called Esps (Escherichia secreted proteins). The genes encoding TTSS structural components and Esps are found within a pathogenicity island called the locus of enterocyte effacement (LEE). This paper describes the application of Saccharomyces cerevisiae as a model to probe the functions of LEE-encoded genes. In a systematic approach, the LEE-encoded translocator and effector proteins were endogenously expressed in yeast and their effects on cell growth, cytoskeletal function and signalling pathways were studied. EspD, EspG and Map inhibited growth by depolarizing the actin cortical cytoskeleton, whereas EspF expression altered the septin cytoskeleton. Specific yeast MAP kinase pathways were activated by EspF, EspG, EspH and Map. The yeast system was used to define functional domains in Map by expressing truncated versions; it was concluded that the C-terminal region of the protein is necessary for actin disruption and toxicity, but not for mitochondrial localization. The utility of the yeast model for functional analyses of EPEC pathogenesis is discussed.

scholarly journals Regulation of Expression and Secretion of NleH, a New Non-Locus of Enterocyte Effacement-Encoded Effector in Citrobacter rodentium

2008 ◽  
Vol 190 (7) ◽  
pp. 2388-2399 ◽  
Author(s):  
Víctor A. García-Angulo ◽  
Wanyin Deng ◽  
Nikhil A. Thomas ◽  
B. Brett Finlay ◽  
Jose L. Puente
Keyword(s):  
Escherichia Coli ◽  
Shigella Flexneri ◽  
Effector Proteins ◽  
Citrobacter Rodentium ◽  
Regulation Of Expression ◽  
Type Iii ◽  
Protein Levels ◽  
Amino Terminal ◽  
Genes Encoding ◽  
Enterocyte Effacement

ABSTRACT Together with enterohemorrhagic Escherichia coli and enteropathogenic Escherichia coli, Citrobacter rodentium is a member of the attaching-and-effacing (A/E) family of bacterial pathogens. A/E pathogens use a type III secretion system (T3SS) to translocate an assortment of effector proteins, encoded both within and outside the locus of enterocyte effacement (LEE), into the colonized host cell, leading to the formation of A/E lesions and disease. Here we report the identification and characterization of a new non-LEE encoded effector, NleH, in C. rodentium. NleH is conserved among A/E pathogens and shares identity with OspG, a type III secreted effector protein in Shigella flexneri. Downstream of nleH, genes encoding homologues of the non-LEE-encoded effectors EspJ and NleG/NleI are found. NleH secretion and translocation into Caco-2 cells requires a functional T3SS and signals located at its amino-terminal domain. Transcription of nleH is not significantly reduced in mutants lacking the LEE-encoded regulators Ler and GrlA; however, NleH protein levels are highly reduced in these strains, as well as in escN and cesT mutants. Inactivation of Lon, but not of ClpP, protease restores NleH levels even in the absence of CesT. Our results indicate that the efficient engagement of NleH in active secretion is needed for its stability, thus establishing a posttranslational regulatory mechanism that coregulates NleH levels with the expression of LEE-encoded proteins. A C. rodentium nleH mutant shows a moderate defect during the colonization of C57BL/6 mice at early stages of infection.

scholarly journals The Locus of Enterocyte Effacement-Encoded Effector Proteins All Promote Enterohemorrhagic Escherichia coli Pathogenicity in Infant Rabbits

2005 ◽  
Vol 73 (3) ◽  
pp. 1466-1474 ◽  
Author(s):  
Jennifer M. Ritchie ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Effector Proteins ◽  
Enterohemorrhagic Escherichia Coli ◽  
Lesion Formation ◽  
Specific Effects ◽  
Genes Encoding ◽  
Organ Specific ◽  
Rabbit Intestine ◽  
Enterocyte Effacement

ABSTRACT The genes encoding the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of tir, which encodes one of these effector proteins, results in a profound reduction (∼10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in escN, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of escN reduced EHEC recovery from the rabbit intestine by ∼10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the espH and espF mutants was reduced throughout the intestine. In contrast, colonization by the map and espG mutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC espF mutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.

scholarly journals Distribution, Functional Expression, and Genetic Organization of Cif, a Phage-Encoded Type III-Secreted Effector from Enteropathogenic and Enterohemorrhagic Escherichia coli

2007 ◽  
Vol 190 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Estelle Loukiadis ◽  
Rika Nobe ◽  
Sylvia Herold ◽  
Clara Tramuta ◽  
Yoshitoshi Ogura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Expression ◽  
Effector Proteins ◽  
Enterohemorrhagic Escherichia Coli ◽  
Host Cells ◽  
E Coli ◽  
Type Iii ◽  
Related Phenotype ◽  
Enterocyte Effacement ◽  
Lambdoid Prophage

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.

scholarly journals CesD2 of Enteropathogenic Escherichia coli Is a Second Chaperone for the Type III Secretion Translocator Protein EspD

2003 ◽  
Vol 71 (4) ◽  
pp. 2130-2141 ◽  
Author(s):  
Bianca C. Neves ◽  
Rosanna Mundy ◽  
Liljana Petrovska ◽  
Gordon Dougan ◽  
Stuart Knutton ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Translocator Protein ◽  
Type Iii ◽  
Genes Encoding

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are extracellular pathogens that employ a type III secretion system to export translocator and effector proteins, proteins which facilitates colonization of the mucosal surface of the intestine via formation of attaching and effacing (A/E) lesions. The genes encoding the proteins for A/E lesion formation are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which contains eae encoding intimin as well as the type III secretion system and effector genes. Many type III secreted proteins are stabilized and maintained in a secretion-competent conformation in the bacterial cytosol by specific chaperone proteins. Three type III chaperones have been described thus far within the EPEC LEE region: CesD, for the translocator proteins EspB and EspD; CesT, for the effector proteins Tir and Map; and CesF, for EspF. In this study we report the characterization of CesD2 (previously Orf27), a second LEE-encoded chaperone for EspD. We show specific CesD2-EspD protein interaction which appears to be necessary for proper EspD secretion in vitro and pathogenesis in vivo as demonstrated in the A/E-lesion-forming mouse pathogen Citrobacter rodentium.

CesAB is an enteropathogenic Escherichia coli chaperone for the type-III translocator proteins EspA and EspB

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3639-3647 ◽  
Author(s):  
Elizabeth A. Creasey ◽  
Devorah Friedberg ◽  
Robert K. Shaw ◽  
Tatiana Umanski ◽  
Stuart Knutton ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Type Iii Secretion ◽  
Secretion System ◽  
Effector Proteins ◽  
Enteropathogenic Escherichia Coli ◽  
Type Iii ◽  
Mucosal Surfaces ◽  
Enterocyte Effacement ◽  
Bacterial Cytoplasm

Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that colonize mucosal surfaces of the intestine via formation of attaching and effacing (A/E) lesions. The genes responsible for induction of the A/E lesions are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes the adhesin intimin and the type III secretion system needle complex, translocator and effector proteins. One of the major EPEC translocator proteins, EspA, forms a filamentous conduit along which secreted proteins travel before they arrive at the translocation pore in the plasma membrane of the host cell, which is composed of EspB and EspD. Prior to secretion, many type III proteins, including translocators, are maintained in the bacterial cytoplasm by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins Tir, Map and EspF, and the translocator proteins EspD and EspB. In this study, CesAB (Orf3 of the LEE) was identified as a chaperone for EspA and EspB. Specific CesAB–EspA and CesAB–EspB protein interactions are demonstrated. CesAB was essential for stability of EspA within the bacterial cell prior to secretion. Furthermore, a cesAB mutant failed to secrete EspA, as well as EspB, to assemble EspA filaments, to induce A/E lesion following infection of HEp-2 cells and to adhere to, or cause haemolysis of, erythrocytes.

scholarly journals Enteropathogenic Escherichia coli Type III Effectors EspG and EspG2 Disrupt the Microtubule Network of Intestinal Epithelial Cells

2005 ◽  
Vol 73 (7) ◽  
pp. 4385-4390 ◽  
Author(s):  
Robert K. Shaw ◽  
Katherine Smollett ◽  
Jennifer Cleary ◽  
Junkal Garmendia ◽  
Ania Straatman-Iwanowska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Effector Proteins ◽  
Intestinal Epithelial ◽  
Enteropathogenic Escherichia Coli ◽  
Microtubule Cytoskeleton ◽  
Microtubule Network ◽  
Type Iii Effectors ◽  
Type Iii

ABSTRACT Enteropathogenic Escherichia coli infection of intestinal epithelial cells leads to localized depletion of the microtubule cytoskeleton, an effect that is dependent on delivery of type III translocated effector proteins EspG and Orf3 (designated EspG2) to the site of depletion. Microtubule depletion involved disruption rather than displacement of microtubules.

scholarly journals Bioinformatics-Enabled Identification of the HrpL Regulon and Type III Secretion System Effector Proteins of Pseudomonas syringae pv. phaseolicola 1448A

2006 ◽  
Vol 19 (11) ◽  
pp. 1193-1206 ◽  
Author(s):  
Monica Vencato ◽  
Fang Tian ◽  
James R. Alfano ◽  
C. Robin Buell ◽  
Samuel Cartinhour ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Candidate Gene Approach ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Genes Encoding

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.

scholarly journals Differential Modulation by Ca2+ of Type III Secretion of Diffusely Adhering Enteropathogenic Escherichia coli

2003 ◽  
Vol 71 (4) ◽  
pp. 1725-1732 ◽  
Author(s):  
Tina Ide ◽  
Silke Michgehl ◽  
Sabine Knappstein ◽  
Gerhard Heusipp ◽  
M. Alexander Schmidt
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Host Cells ◽  
Chromosomal Locus ◽  
Type Iii ◽  
Calcium Chelation ◽  
Host Plasma Membrane ◽  
Enterocyte Effacement

ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains are a common cause of persistent diarrhea among infants, primarily in developing countries. The pathogenicity of EPEC is associated with the expression and secretion of bacterial proteins encoded by the chromosomal locus of enterocyte effacement (LEE). The LEE-encoded type III-secreted proteins EspA, EspB, and EspD are part of a molecular syringe, which is used by EPEC to translocate effector proteins directly into the cytoplasm of host cells. The type III-secreted translocated intimin receptor (Tir) protein is thought to be delivered by an Esp-dependent mechanism into the host cell, and this is followed by insertion into the host plasma membrane, where the protein serves as the receptor for intimin, an afimbrial bacterial adhesin. Type III secretion is subject to environmental regulation, and secretion can be induced in vitro by growing bacteria in cell culture medium. In this study we found that Ca2+ is involved in the regulation of type III secretion both in classical locally adherent EPEC and in atypical diffusely adherent EPEC. Interestingly, we observed contrasting secretion of Esp proteins and Tir in response to Ca2+. While the secretion of Tir is clearly enhanced and the protein is integrated into HeLa membranes under calcium chelation conditions, Esp secretion is strongly reduced under these conditions. These data suggest that under Ca2+-depleted conditions Tir might be secreted into the medium and integrated into host membranes by an Esp-independent mechanism, without the need for a functional type III translocation machinery.

Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

1991 ◽  
Vol 227 (3) ◽  
pp. 438-445 ◽  
Author(s):  
Mitsuhiro Itaya ◽  
Dorothy McKelvin ◽  
Sunil K. Chatterjie ◽  
Robert J. Crouch
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Salmonella Typhimurium ◽  
Rnase H ◽  
Genes Encoding

scholarly journals Genome context influences evolutionary flexibility of nearly identical type III effectors in two phytopathogenic Pseudomonads

2021 ◽  
Author(s):  
David A Baltrus ◽  
Qian Feng ◽  
Brian H Kvitko
Keyword(s):  
Pseudomonas Syringae ◽  
Evolutionary Dynamics ◽  
Effector Proteins ◽  
In Planta ◽  
Genomic Context ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Genome Context ◽  
The Impact ◽  
Multiple Type

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and highlights how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

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