scholarly journals Gateway Entry Vector Library of Wolbachia pipientis Candidate Effectors from Strain wMel

2018 ◽  
Vol 7 (1) ◽  
Author(s):  
Irene L. G. Newton ◽  
Kathy B. Sheehan
Keyword(s):  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Wolbachia Pipientis ◽  
Entry Vector ◽  
Intracellular Symbiont ◽  
Host Cytoplasm ◽  
Type Iv ◽  
Gateway Entry Vector ◽  
Bacterial Effectors

Wolbachia pipientis is an intracellular symbiont that modifies host biology using a type IV secretion system to inject bacterial effectors into the host cytoplasm. We utilized a bioinformatics approach to predict Wolbachia effectors and cloned the candidates into an entry vector, which can be utilized for subsequent analyses.

scholarly journals Identification of ElpA, a Coxiella burnetii Pathotype-Specific Dot/Icm Type IV Secretion System Substrate

2015 ◽  
Vol 83 (3) ◽  
pp. 1190-1198 ◽  
Author(s):  
Joseph G. Graham ◽  
Caylin G. Winchell ◽  
Uma M. Sharma ◽  
Daniel E. Voth
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Secretion System ◽  
Terminal Region ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv

Coxiella burnetiicauses human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages,C. burnetiiuses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100C. burnetiiDot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in allC. burnetiiisolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizingproteinA). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function duringC. burnetiiinfection.

scholarly journals Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Julieta Aguilar ◽  
Todd A. Cameron ◽  
John Zupan ◽  
Patricia Zambryski
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cells ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Content Type ◽  
Protein Substrates ◽  
Type Iv

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain ofAgrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in theA. tumefaciensoctopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression followingvirinduction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles.vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiplevir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.IMPORTANCETransfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of theAgrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model ofA. tumefaciensattachment to a plant cell, whereA. tumefacienstakes advantage of the multiplevir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through thevir-T4SS. The T4SS ofA. tumefaciensis among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

scholarly journals Bacterial Energetic Requirements for Helicobacter pylori Cag Type IV Secretion System-Dependent Alterations in Gastric Epithelial Cells

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Aung Soe Lin ◽  
Samuel D. R. Dooyema ◽  
Arwen E. Frick-Cheng ◽  
M. Lorena Harvey ◽  
Giovanni Suarez ◽  
...  
Keyword(s):  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Gastric Epithelial Cells ◽  
Content Type ◽  
Type Iv ◽  
Mutant Strains ◽  
H Pylori ◽  
Tlr9 Activation ◽  
Bacterial Constituents

ABSTRACT Helicobacter pylori colonizes the stomach in about half of the world’s population. H. pylori strains containing the cag pathogenicity island (cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagβ, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori-induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagβ was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.

scholarly journals Novel Plasmid and Its Variant Harboring both ablaNDM-1Gene and Type IV Secretion System in Clinical Isolates of Acinetobacter lwoffii

2012 ◽  
Vol 56 (4) ◽  
pp. 1698-1702 ◽  
Author(s):  
Hongyan Hu ◽  
Yongfei Hu ◽  
Yuanlong Pan ◽  
Hui Liang ◽  
Haiyan Wang ◽  
...  
Keyword(s):  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv ◽  
Insertion Elements ◽  
Complete Sequences ◽  
Large Plasmids ◽  
P Type ◽  
T4ss Gene

ABSTRACTThe spread of theblaNDM-1gene is gaining worldwide attentions. This gene is usually carried by large plasmids and has been discovered in diverse bacteria since it was originally found inKlebsiella pneumoniae. Here we report the complete sequences of ablaNDM-1-bearing plasmid, pNDM-BJ01, and its variant, pNDM-BJ02, isolated from clinicalAcinetobacter lwoffiistrains. The plasmid pNDM-BJ01 is 47.3 kb in size and cannot be classified into any known plasmid incompatibility group, thus representing a novel plasmid with an unknown maintenance mechanism. This plasmid contains both ablaNDM-1gene and a type IV secretion system (T4SS) gene cluster. The T4SS is assigned to the P-type T4SS group, which usually encode a short, rigid pilus, and theblaNDM-1gene is located within a composite transposon flanked by two insertion elements of ISAba125. Plasmid pNDM-BJ02 is nearly identical to pNDM-BJ01 except that one copy of the ISAba125element is missing, and it is therefore regarded as a variant of pNDM-BJ01. Sequence alignment indicated that thisblaNDM-1-containing composite transposon, which can also be captured by other mobile elements, was probably a product of multiple recombination events and can move as a whole by transposition.

scholarly journals Brucella melitensis Invades Murine Erythrocytes during Infection

2014 ◽  
Vol 82 (9) ◽  
pp. 3927-3938 ◽  
Author(s):  
Marie-Alice Vitry ◽  
Delphine Hanot Mambres ◽  
Michaël Deghelt ◽  
Katrin Hack ◽  
Arnaud Machelart ◽  
...  
Keyword(s):  
Brucella Melitensis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Bacterial Cells ◽  
Content Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Time Point

ABSTRACTBrucellaspp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed thatBrucella melitensisis able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced followingBrucellavaccination. In contrast toBartonella, the type IV secretion system and flagellar expression are not critically required for the persistence ofBrucellain blood. ImageStream analysis of blood cells showed that following a brief extracellular phase,Brucellais associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated thatB. melitensisis able to invade erythrocytesin vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possibleBrucellatransmission by bloodsucking insects in nature.

scholarly journals Quorum-Sensing and BvrR/BvrS Regulation, the Type IV Secretion System, Cyclic Glucans, and BacA in the Virulence of Brucella ovis: Similarities to and Differences from Smooth Brucellae

2012 ◽  
Vol 80 (5) ◽  
pp. 1783-1793 ◽  
Author(s):  
Ana I. Martín-Martín ◽  
Pilar Sancho ◽  
María Jesús de Miguel ◽  
Luis Fernández-Lago ◽  
Nieves Vizcaíno
Keyword(s):  
Quorum Sensing ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Multiplication Rate ◽  
Content Type ◽  
Murine Macrophages ◽  
Brucella Ovis ◽  
Type Iv

ABSTRACTBrucella ovisis a rough bacterium—lacking O-polysaccharide chains in the lipopolysaccharide—that is virulent in its natural host and whose virulence mechanisms remain almost unexplored. In a search for additional traits that distinguishB. ovisfrom smoothBrucella, which require O-polysaccharide chains for virulence, we have analyzed the significance inB. ovisof the main virulence factors described for smoothBrucella. Attempts to obtain strains of virulentB. ovisstrain PA that are mutated in the BvrR/BvrS two-component regulatory system were unsuccessful, suggesting the requirement of that system forin vitrosurvival, while the inactivation ofbacA—in contrast to the results seen with smoothBrucella—did not affect splenic colonization in mice or behavior in J774.A1 murine macrophages. Defects in the synthesis of cyclic ß-1,2 glucans reduced the uptake ofB. ovisPA in macrophages and, although the intracellular multiplication rate was unaffected, led to attenuation in mice. Growth of strains with mutations in the type IV secretion system (encoded by thevirBoperon) and the quorum-sensing-related regulator VjbR was severely attenuated in the mouse model, and although the mutant strains internalized like the parental strain in J774.A1 murine macrophages, they were impaired for intracellular replication. As described forB. melitensis, VjbR regulates the transcription of thevirBoperon positively, and theN-dodecanoyl-dl-homoserine lactone (C12-HSL) autoinducer abrogates this effect. In contrast, no apparent VjbR-mediated regulation of thefliFflagellar gene was observed inB. ovis, probably due to the two deletions detected upstream offliF. These results, together with others reported in the text, point to similarities between rough virulentB. ovisand smoothBrucellaspecies as regards virulence but also reveal distinctive traits that could be related to the particular pathogenicity and host tropism characteristics ofB. ovis.

scholarly journals csrTRepresents a New Class ofcsrA-Like Regulatory Genes Associated with Integrative Conjugative Elements of Legionella pneumophila

2015 ◽  
Vol 198 (3) ◽  
pp. 553-564 ◽  
Author(s):  
Zachary D. Abbott ◽  
Kaitlin J. Flynn ◽  
Brenda G. Byrne ◽  
Sampriti Mukherjee ◽  
Daniel B. Kearns ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Legionella Pneumophila ◽  
Mobile Genetic Elements ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv ◽  
Horizontal Spread ◽  
Integrative Conjugative Elements

ABSTRACTBacterial evolution is accelerated by mobile genetic elements. To spread horizontally and to benefit the recipient bacteria, genes encoded on these elements must be properly regulated. Among the legionellae are multiple integrative conjugative elements (ICEs) that each encode a paralog of the broadly conserved regulatorcsrA. Using bioinformatic analyses, we deduced that specificcsrAparalogs are coinherited with particular lineages of the type IV secretion system that mediates horizontal spread of its ICE, suggesting a conserved regulatory interaction. As a first step to investigate the contribution ofcsrAregulators to this class of mobile genetic elements, we analyzed here the activity of thecsrAparalog encoded onLegionella pneumophilaICE-βox. Deletion of this gene, which we namecsrT, had no observed effect under laboratory conditions. However, ectopic expression ofcsrTabrogated the protection to hydrogen peroxide and macrophage degradation that ICE-βox confers toL. pneumophila. When ectopically expressed,csrTalso repressedL. pneumophilaflagellin production and motility, a function similar to the core genome's canonicalcsrA. Moreover,csrTrestored the repression of motility tocsrAmutants ofBacillus subtilis, a finding consistent with the predicted function of CsrT as an mRNA binding protein. Since all known ICEs of legionellae encode coinheritedcsrA-type IV secretion system pairs, we postulate that CsrA superfamily proteins regulate ICE activity to increase their horizontal spread, thereby expandingL. pneumophilaversatility.IMPORTANCEICEs are mobile DNA elements whose type IV secretion machineries mediate spread among bacterial populations. All surveyed ICEs within theLegionellagenus also carry paralogs of the essential life cycle regulatorcsrA. It is striking that thecsrAloci could be classified into distinct families based on either their sequence or the subtype of the adjacent type IV secretion system locus. To investigate whether ICE-encodedcsrAparalogs are bona fide regulators, we analyzed ICE-βox as a model system. When expressed ectopically, itscsrAparalog inhibited multiple ICE-βox phenotypes, as well as the motility of not onlyLegionellabut alsoBacillus subtilis. Accordingly, we predict that CsrA regulators equip legionellae ICEs to promote their spread via dedicated type IV secretion systems.

scholarly journals New Complementation Constructs for Inducible and Constitutive Gene Expression in Neisseria gonorrhoeae and Neisseria meningitidis

2012 ◽  
Vol 78 (9) ◽  
pp. 3068-3078 ◽  
Author(s):  
Meghan E. Ramsey ◽  
Kathleen T. Hackett ◽  
Chaitra Kotha ◽  
Joseph P. Dillard
Keyword(s):  
Gene Expression ◽  
Neisseria Gonorrhoeae ◽  
Neisseria Meningitidis ◽  
Inducible Promoter ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv ◽  
Low Concentrations ◽  
Constitutive Gene Expression

ABSTRACTWe have created new complementation constructs for use inNeisseria gonorrhoeaeandNeisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genesigaandtrpB. In order to increase the available options for gene expression inNeisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-β-d-thiogalactopyranoside-induciblelacpromoter, which has been widely used inNeisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcalopaBgene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter inNeisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenicNeisseriaat very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy inN. gonorrhoeaeas well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.

scholarly journals Effectors of the Stenotrophomonas maltophilia Type IV Secretion System Mediate Killing of Clinical Isolates of Pseudomonas aeruginosa

mBio ◽  
2021 ◽  
Author(s):  
Megan Y. Nas ◽  
Jeffrey Gabell ◽  
Nicholas P. Cianciotto
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Stenotrophomonas Maltophilia ◽  
Opportunistic Pathogen ◽  
Lung Infection ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv

S. maltophilia is an increasingly important opportunistic pathogen. Inherently resistant to many antibiotics, S. maltophilia is often associated with lung infection, being, among other things, a complicating factor in cystic fibrosis patients.

scholarly journals Maintenance of Type IV Secretion Function During Helicobacter pylori Infection in Mice

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e03147-20
Author(s):  
Emma C. Skoog ◽  
Miriam E. Martin ◽  
Roberto M. Barrozo ◽  
Lori M. Hansen ◽  
Lucy P. Cai ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Murine Model ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Biologically Relevant ◽  
Type Iv ◽  
H Pylori

ABSTRACTThe Helicobacter pylori type IV secretion system (T4SS) encoded on the cag pathogenicity island (cagPAI) secretes the CagA oncoprotein and other effectors into the gastric epithelium. During murine infection, T4SS function is lost in an immune-dependent manner, typically as a result of in-frame recombination in the middle repeat region of cagY, though single nucleotide polymorphisms (SNPs) in cagY or in other essential genes may also occur. Loss of T4SS function also occurs in gerbils, nonhuman primates, and humans, suggesting that it is biologically relevant and not simply an artifact of the murine model. Here, we sought to identify physiologically relevant conditions under which T4SS function is maintained in the murine model. We found that loss of H. pylori T4SS function in mice was blunted by systemic Salmonella coinfection and completely eliminated by dietary iron restriction. Both have epidemiologic parallels in humans, since H. pylori strains from individuals in developing countries, where iron deficiency and systemic infections are common, are also more often cagPAI+ than strains from developed countries. These results have implications for our fundamental understanding of the cagPAI and also provide experimental tools that permit the study of T4SS function in the murine model.IMPORTANCE The type IV secretion system (T4SS) is the major Helicobacter pylori virulence factor, though its function is lost during murine infection. Loss of function also occurs in gerbils and in humans, suggesting that it is biologically relevant, but the conditions under which T4SS regulation occurs are unknown. Here, we found that systemic coinfection with Salmonella and iron deprivation each promote retention of T4SS function. These results improve our understanding of the cag pathogenicity island (cagPAI) and provide experimental tools that permit the study of T4SS function in the murine model.

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