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 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 AtlA Functions as a Peptidoglycan Lytic Transglycosylase in the Neisseria gonorrhoeae Type IV Secretion System

2007 ◽  
Vol 189 (15) ◽  
pp. 5421-5428 ◽  
Author(s):  
Petra L. Kohler ◽  
Holly L. Hamilton ◽  
Karen Cloud-Hansen ◽  
Joseph P. Dillard
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Lytic Transglycosylases ◽  
Type Iv ◽  
Lytic Transglycosylase ◽  
Enzymatic Function ◽  
Type Iv Secretion Systems

ABSTRACT Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.

scholarly journals Transcriptional and translational responsiveness of the Neisseria gonorrhoeae type IV secretion system to conditions of host infections

2021 ◽  
Author(s):  
Melanie M. Callaghan ◽  
Amy K. Klimowicz ◽  
Abigail C. Shockey ◽  
John Kane ◽  
Caitlin S. Pepperell ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Protein Levels ◽  
Type Iv ◽  
Coupling Protein ◽  
Host Infection ◽  
System Coupling ◽  
Translational Mechanisms

AbstractThe type IV secretion system of Neisseria gonorrhoeae translocates single-stranded DNA into the extracellular space, facilitating horizontal gene transfer and initiating biofilm formation. Expression of this system has been observed to be low under laboratory conditions, and multiple levels of regulation have been identified. We used a translational fusion of lacZ to traD, the gene for the type IV secretion system coupling protein, to screen for increased type IV secretion system expression. We identified several physiologically relevant conditions, including surface adherence, decreased manganese or iron, and increased zinc or copper, which increase gonococcal type IV secretion system protein levels through transcriptional and/or translational mechanisms. These metal treatments are reminiscent of the conditions in the macrophage phagosome. The ferric uptake regulator, Fur, was found to repress traD transcript levels, but to also have a second role, acting to allow TraD protein levels to increase only in the absence of iron. To better understand type IV secretion system regulation during infection, we examined transcriptomic data from active urethral infection samples from five men. These data demonstrated differential expression of 20 of 21 type IV secretion system genes during infection, indicating upregulation of genes necessary for DNA secretion during host infection.

scholarly journals Increased Expression of the Type IV Secretion System in Piliated Neisseria gonorrhoeae Variants

2010 ◽  
Vol 192 (7) ◽  
pp. 1912-1920 ◽  
Author(s):  
Wilmara Salgado-Pabón ◽  
Ying Du ◽  
Kathleen T. Hackett ◽  
Katelynn M. Lyons ◽  
Cindy Grove Arvidson ◽  
...  
Keyword(s):  
Growth Rate ◽  
Neisseria Gonorrhoeae ◽  
Antibiotic Resistance Genes ◽  
Gene Cassette ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Chromosomal Dna ◽  
Translational Machinery ◽  
Type Iv

ABSTRACT Neisseria gonorrhoeae produces a type IV secretion system that secretes chromosomal DNA. The secreted DNA is active in the transformation of other gonococci in the population and may act to transfer antibiotic resistance genes and variant alleles for surface antigens, as well as other genes. We observed that gonococcal variants that produced type IV pili secreted more DNA than variants that were nonpiliated, suggesting that the process may be regulated. Using microarray analysis, we found that a piliated strain showed increased expression of the gene for the putative type IV secretion coupling protein TraD, whereas a nonpiliated variant showed increased expression of genes for transcriptional and translational machinery, consistent with its higher growth rate compared to that of the piliated strain. These results suggested that type IV secretion might be controlled by either traD expression or growth rate. A mutant with a deletion in traD was found to be deficient in DNA secretion. Further mutation and complementation analysis indicated that traD is transcriptionally and translationally coupled to traI, which encodes the type IV secretion relaxase. We were able to increase DNA secretion in a nonpiliated strain by inserting a gene cassette with a strong promoter to drive the expression of the putative operon containing traI and traD. Together, these data suggest a model in which the type IV secretion system apparatus is made constitutively, while its activity is controlled through regulation of traD and traI.

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.

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