scholarly journals Metabolic Control of Virulence Genes in Brucella abortus: HutC Coordinates virB Expression and the Histidine Utilization Pathway by Direct Binding to Both Promoters

2009 ◽  
Vol 192 (1) ◽  
pp. 217-224 ◽  
Author(s):  
Rodrigo Sieira ◽  
Gastón M. Arocena ◽  
Lucas Bukata ◽  
Diego J. Comerci ◽  
Rodolfo A. Ugalde
Keyword(s):  
Brucella Abortus ◽  
Cell Envelope ◽  
Host Cells ◽  
Adaptive Responses ◽  
Secretion Systems ◽  
Mobility Shift ◽  
Type Iv ◽  
Dnase I Footprinting ◽  
Direct Binding ◽  
Utilization Pathway

ABSTRACT Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. The virB operon of Brucella abortus codes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that the virB operon of B. abortus is tightly regulated within the host cells. In order to identify factors implicated in the control of virB expression, we searched for proteins of Brucella that directly bind to the virB promoter (P virB ). Using different procedures, we isolated a 27-kDa protein that binds specifically to P virB . This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses of virB and hut promoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of the virB operon, whereas it represses the hut genes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in both hut and virB promoters. Taken together, these results indicate that Brucella coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses.

scholarly journals Postreplication Roles of theBrucellaVirB Type IV Secretion System Uncovered via Conditional Expression of the VirB11 ATPase

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Erin P. Smith ◽  
Cheryl N. Miller ◽  
Robert Child ◽  
Jennifer A. Cundiff ◽  
Jean Celli
Keyword(s):  
Brucella Abortus ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Type Iv ◽  
Conditional Expression ◽  
Bacterial Replication

ABSTRACTBrucella abortus, the bacterial agent of the worldwide zoonosis brucellosis, primarily infects host phagocytes, where it undergoes an intracellular cycle within a dedicated membrane-bound vacuole, theBrucella-containing vacuole (BCV). Initially of endosomal origin (eBCV), BCVs are remodeled into replication-permissive organelles (rBCV) derived from the host endoplasmic reticulum, a process that requires modulation of host secretory functions via delivery of effector proteins by theBrucellaVirB type IV secretion system (T4SS). Following replication, rBCVs are converted into autophagic vacuoles (aBCVs) that facilitate bacterial egress and subsequent infections, arguing that the bacterium sequentially manipulates multiple cellular pathways to complete its cycle. The VirB T4SS is essential for rBCV biogenesis, as VirB-deficient mutants are stalled in eBCVs and cannot mediate rBCV biogenesis. This has precluded analysis of whether the VirB apparatus also drives subsequent stages of theBrucellaintracellular cycle. To address this issue, we have generated aB. abortusstrain in which VirB T4SS function is conditionally controlled via anhydrotetracycline (ATc)-dependent complementation of a deletion of thevirB11gene encoding the VirB11 ATPase. We show in murine bone marrow-derived macrophages (BMMs) that early VirB production is essential for optimal rBCV biogenesis and bacterial replication. Transient expression ofvirB11prior to infection was sufficient to mediate normal rBCV biogenesis and bacterial replication but led to T4SS inactivation and decreased aBCV formation and bacterial release, indicating that these postreplication stages are also T4SS dependent. Hence, our findings support the hypothesis of additional, postreplication roles of type IV secretion in theBrucellaintracellular cycle.IMPORTANCEMany intracellular bacterial pathogens encode specialized secretion systems that deliver effector proteins into host cells to mediate the multiple stages of their intracellular cycles. Because these intracellular events occur sequentially, classical genetic approaches cannot address the late roles that these apparatuses play, as secretion-deficient mutants cannot proceed past their initial defect. Here we have designed a functionally controllable VirB type IV secretion system (T4SS) in the bacterial pathogenBrucella abortusto decipher its temporal requirements during the bacterium’s intracellular cycle in macrophages. By controlling production of the VirB11 ATPase, which energizes the T4SS, we show not only that this apparatus is required early to generate theBrucellareplicative organelle but also that it contributes to completion of the bacterium’s cycle and bacterial egress. Our findings expand upon the pathogenic functions of theBrucellaVirB T4SS and illustrate targeting of secretion ATPases as a useful strategy to manipulate the activity of bacterial secretion systems.

scholarly journals Epistatic Interplay between Type IV Secretion Effectors Engages the Small GTPase Rab2 in the Brucella Intracellular Cycle

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Erin P. Smith ◽  
Alexis Cotto-Rosario ◽  
Elizabeth Borghesan ◽  
Kiara Held ◽  
Cheryl N. Miller ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Bacterial Pathogens ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iv ◽  
Infectious Cycle

ABSTRACT Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. While the function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium Brucella abortus, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in Brucella replication. Defects in rBCV biogenesis and Brucella replication caused by deletion of bspB were dependent on the host GTPase Rab2a and suppressed by the deletion of ricA, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and Brucella intracellular replication were abolished upon deletion of both bspB and ricA, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the Brucella intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA’s functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs Brucella replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors. IMPORTANCE Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium Brucella abortus delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.

Positive and negative transcriptional elements of the human type IV collagenase gene

1990 ◽  
Vol 10 (12) ◽  
pp. 6524-6532
Author(s):  
S M Frisch ◽  
J H Morisaki
Keyword(s):  
Core Promoter ◽  
Type Iv Collagenase ◽  
Mobility Shift ◽  
Specific Expression ◽  
Enhancer Element ◽  
Type Iv ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift ◽  
Transcriptional Elements ◽  
Mcf 7

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.

scholarly journals Molecular architecture of theLegionellaDot/Icm type IV secretion system

2018 ◽  
Author(s):  
Debnath Ghosal ◽  
Yi-Wei Chang ◽  
Kwang Cheol Jeong ◽  
Joseph P. Vogel ◽  
Grant J. Jensen
Keyword(s):  
Legionella Pneumophila ◽  
Cell Envelope ◽  
Secretion System ◽  
Host Cells ◽  
Molecular Architecture ◽  
Intact Cells ◽  
Type Iv ◽  
Type Ivb Secretion ◽  
Electron Cryotomography ◽  
First Time

AbstractLegionella pneumophilasurvives and replicates inside host cells by secreting ~300 effectors through the Dot/Icm type IVB secretion system (T4BSS). Understanding this machine’s structure is challenging because of its large number of components (27) and integration into all layers of the cell envelope. Previously we overcame this obstacle by imaging the Dot/Icm T4BSS in its native state within intact cells through electron cryotomography. Here we extend our observations by imaging a stabilized mutant that yielded a higher resolution map. We describe for the first time the presence of a well-ordered central channel that opens up into a windowed large (~32 nm wide) secretion chamber with an unusual 13-fold symmetry. We then dissect the complex by matching proteins to densities for many components, including all those with periplasmic domains. The placement of known and predicted structures of individual proteins into the map reveals the architecture of the T4BSS and provides a roadmap for further investigation of this amazing specialized secretion system.

scholarly journals Export von Gefahrgut: Helicobacter pylori und sein CagA-Protein

BIOspektrum ◽  
2020 ◽  
Vol 26 (6) ◽  
pp. 597-599
Author(s):  
Clara Lettl ◽  
Wolfgang Fischer
Keyword(s):  
Helicobacter Pylori ◽  
Pathogenic Bacteria ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Bacterial Protein ◽  
Secretion Systems ◽  
Unusual Structure ◽  
Type Iv ◽  
Single Protein ◽  
Protein Transporters

Abstract Pathogenic bacteria often utilize type IV secretion systems to interact with host cells and to modify their microenvironment in a favourable way. The human pathogen Helicobacter pylori produces such a system to inject only a single protein, CagA, into gastric cells, but this injection represents a major risk factor for gastric cancer development. Here, we discuss the unusual structure of the Cag secretion nanomachine and other features that make it unique among bacterial protein transporters.

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 Differential Requirements for VirB1 and VirB2 during Brucella abortus Infection

2004 ◽  
Vol 72 (9) ◽  
pp. 5143-5149 ◽  
Author(s):  
Andreas B. den Hartigh ◽  
Yao-Hui Sun ◽  
David Sondervan ◽  
Niki Heuvelmans ◽  
Marjolein O. Reinders ◽  
...  
Keyword(s):  
Mouse Model ◽  
Brucella Abortus ◽  
Persistent Infection ◽  
Host Cells ◽  
Intracellular Replication ◽  
Bacterial Surface ◽  
Type Iv

ABSTRACT The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.

scholarly journals Using an Optimal Set of Features with a Machine Learning-Based Approach to Predict Effector Proteins forLegionella pneumophila

2018 ◽  
Author(s):  
Zhila Esna Ashari ◽  
Kelly A. Brayton ◽  
Shira L. Broschat
Keyword(s):  
Machine Learning ◽  
Legionella Pneumophila ◽  
De Novo ◽  
Effector Proteins ◽  
Machine Learning Algorithms ◽  
Host Cells ◽  
Support Vector ◽  
Secretion Systems ◽  
Type Iv ◽  
Optimal Set

AbstractType IV secretion systems exist in a number of bacterial pathogens and are used to secrete effector proteins directly into host cells in order to change their environment making the environment hospitable for the bacteria. In recent years, several machine learning algorithms have been developed to predict effector proteins, potentially facilitating experimental verification. However, inconsistencies exist between their results. Previously we analysed the disparate sets of predictive features used in these algorithms to determine an optimal set of 370 features for effector prediction. This work focuses on the best way to use these optimal features by designing three machine learning classifiers, comparing our results with those of others, and obtaining de novo results. We chose the pathogenLegionella pneumophilastrain Philadelphia-1, a cause of Legionnaires’ disease, because it has many validated effector proteins and others have developed machine learning prediction tools for it. While all of our models give good results indicating that our optimal features are quite robust, Model 1, which uses all 370 features with a support vector machine, has slightly better accuracy. Moreover, Model 1 predicted 760 effector proteins, more than any other study, 315 of which have been validated. Although the results of our three models agree well with those of other researchers, their models only predicted 126 and 311 candidate effectors.

scholarly journals Pseudomonas aeruginosa ExsA Regulates a Metalloprotease, ImpA, That Inhibits Phagocytosis of Macrophages

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Zhenyang Tian ◽  
Sen Cheng ◽  
Bin Xia ◽  
Yongxin Jin ◽  
Fang Bai ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Critical Role ◽  
Surface Protein ◽  
Host Cells ◽  
Bacterial Cells ◽  
Mobility Shift ◽  
Content Type ◽  
Direct Binding ◽  
Mobility Shift Assay ◽  
Opportunistic Pathogenic

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogenic bacterium whose type III secretion system (T3SS) plays a critical role in acute infections. Translocation of the T3SS effectors into host cells induces cytotoxicity. In addition, the T3SS promotes the intracellular growth of P. aeruginosa during host infections. The T3SS regulon genes are regulated by an AraC-type regulator, ExsA. In this study, we found that an extracellular metalloprotease encoded by impA (PA0572) is under the regulation of ExsA. An ExsA consensus binding sequence was identified upstream of the impA gene, and direct binding of the site by ExsA was demonstrated via an electrophoretic mobility shift assay. We further demonstrate that secreted ImpA cleaves the macrophage surface protein CD44, which inhibits the phagocytosis of the bacterial cells by macrophages. Combined, our results reveal a novel ExsA-regulated virulence factor that cooperatively inhibits the functions of macrophages with the T3SS.

scholarly journals A MarR-Type Regulator Directly Activates Transcription from the Brucella abortus virB Promoter by Sharing a Redundant Role with HutC

2012 ◽  
Vol 194 (23) ◽  
pp. 6431-6440 ◽  
Author(s):  
Rodrigo Sieira ◽  
Gastón M. Arocena ◽  
Angeles Zorreguieta ◽  
Diego J. Comerci ◽  
Rodolfo A. Ugalde
Keyword(s):  
Transcription Factors ◽  
Dna Sequences ◽  
Cell Envelope ◽  
Regulatory Element ◽  
Dependent Manner ◽  
Secretion Systems ◽  
Mobility Shift ◽  
Content Type ◽  
Environmental Signals

ABSTRACTType IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genusBrucellaessentially depends on the integrity of the T4SS-encodingvirBoperon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of thevirBgenes that were isolated from total protein extracts ofBrucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of thevirBpromoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNAin vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activatedvirBexpression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of thevirBpromoter and is probably involved in coordinating gene expression in response to specific environmental signals.

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