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 Protein Kinase LegK2 Is a Type IV Secretion System Effector Involved in Endoplasmic Reticulum Recruitment and Intracellular Replication of Legionella pneumophila

2011 ◽  
Vol 79 (5) ◽  
pp. 1936-1950 ◽  
Author(s):  
Eva Hervet ◽  
Xavier Charpentier ◽  
Anne Vianney ◽  
Jean-Claude Lazzaroni ◽  
Christophe Gilbert ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv

ABSTRACTLegionella pneumophilais the etiological agent of Legionnaires' disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting theLegionellaphagosome. ManyLegionellaeffectors display distinctive eukaryotic domains, among which are protein kinase domains.In silicoanalysis andin vitrophosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemicL. pneumophilaLens strain. Except for LegK5, theLegionellaprotein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. ThelegK2mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant oflegK2exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed byL. pneumophilato establish a replicative niche and evade host cell defenses.

scholarly journals Genomic Insight into Symbiosis-Induced Insect Color Change by a Facultative Bacterial Endosymbiont, “CandidatusRickettsiella viridis”

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Naruo Nikoh ◽  
Tsutomu Tsuchida ◽  
Taro Maeda ◽  
Katsushi Yamaguchi ◽  
Shuji Shigenobu ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Effector Protein ◽  
Body Color ◽  
Content Type ◽  
Cellular Processes ◽  
Type Iv ◽  
Symbiont Genome

ABSTRACTMembers of the genusRickettsiellaare bacterial pathogens of insects and other arthropods. Recently, a novel facultative endosymbiont, “CandidatusRickettsiella viridis,” was described in the pea aphidAcyrthosiphon pisum, whose infection causes a striking host phenotype: red and green genetic color morphs exist in aphid populations, and upon infection with the symbiont, red aphids become green due to increased production of green polycyclic quinone pigments. Here we determined the complete genome sequence of the symbiont. The 1.6-Mb circular genome, harboring some 1,400 protein-coding genes, was similar to the genome of entomopathogenicRickettsiella grylli(1.6 Mb) but was smaller than the genomes of phylogenetically allied human pathogensCoxiella burnetii(2.0 Mb) andLegionella pneumophila(3.4 Mb). The symbiont’s metabolic pathways exhibited little complementarity to those of the coexisting primary symbiontBuchnera aphidicola, reflecting the facultative nature of the symbiont. The symbiont genome harbored neither polyketide synthase genes nor the evolutionarily allied fatty acid synthase genes that are suspected to catalyze the polycyclic quinone synthesis, indicating that the green pigments are produced not by the symbiont but by the host aphid. The symbiont genome retained many type IV secretion system genes and presumable effector protein genes, whose homologues inL. pneumophilawere reported to modulate a variety of the host's cellular processes for facilitating infection and virulence. These results suggest the possibility that the symbiont is involved in the green pigment production by affecting the host’s metabolism using the secretion machineries for delivering the effector molecules into the host cells.IMPORTANCEInsect body color is relevant to a variety of biological aspects such as species recognition, sexual selection, mimicry, aposematism, and crypsis. Hence, the bacterial endosymbiont “CandidatusRickettsiella viridis,” which alters aphid body color from red to green, is of ecological interest, given that different predators preferentially exploit either red- or green-colored aphids. Here we determined the complete 1.6-Mb genome of the symbiont and uncovered that, although the red-green color transition was ascribed to upregulated production of green polycyclic quinone pigments, the symbiont genome harbored few genes involved in the polycyclic quinone biosynthesis. Meanwhile, the symbiont genome contained type IV secretion system genes and presumable effector protein genes, whose homologues modulate eukaryotic cellular processes for facilitating infection and virulence in the pathogenLegionella pneumophila. We propose the hypothesis that the symbiont may upregulate the host’s production of polycyclic quinone pigments via cooption of secretion machineries and effector molecules for pathogenicity.

scholarly journals A New Method To Determine In Vivo Interactomes Reveals Binding of the Legionella pneumophila Effector PieE to Multiple Rab GTPases

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Aurélie Mousnier ◽  
Gunnar N. Schroeder ◽  
Charlotte A. Stoneham ◽  
Ernest C. So ◽  
James A. Garnett ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
New Method ◽  
Type Iv Secretion System ◽  
Rab Gtpases ◽  
Content Type ◽  
Type Iv

ABSTRACTLegionella pneumophila, the causative agent of Legionnaires’ disease, uses the Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effectors into host cells, where they subvert host cell signaling. The function and host cell targets of most effectors remain unknown. PieE is a 69-kDa Dot/Icm effector containing three coiled-coil (CC) regions and 2 transmembrane (TM) helices followed by a fourth CC region. Here, we report that PieE dimerized by an interaction between CC3 and CC4. We found that ectopically expressed PieE localized to the endoplasmic reticulum (ER) and induced the formation of organized smooth ER, while following infection PieE localized to theLegionella-containing vacuole (LCV). To identify the physiological targets of PieE during infection, we established a new purification method for which we created an A549 cell line stably expressing theEscherichia colibiotin ligase BirA and infected the cells withL. pneumophilaexpressing PieE fused to a BirA-specific biotinylation site and a hexahistidine tag. Following tandem Ni2+nitrilotriacetic acid (NTA) and streptavidin affinity chromatography, the effector-target complexes were analyzed by mass spectrometry. This revealed interactions of PieE with multiple host cell proteins, including the Rab GTPases 1a, 1b, 2a, 5c, 6a, 7, and 10. Binding of the Rab GTPases, which was validated by yeast two-hybrid binding assays, was mediated by the PieE CC1 and CC2. In summary, using a novel, highly specific strategy to purify effector complexes from infected cells, which is widely applicable to other pathogens, we identified PieE as a multidomain LCV protein with promiscuous Rab GTPase-binding capacity.IMPORTANCEThe respiratory pathogenLegionella pneumophilauses the Dot/Icm type IV secretion system to translocate more than 300 effector proteins into host cells. The function of most effectors in infection remains unknown. One of the bottlenecks for their characterization is the identification of target proteins. Frequently usedin vitroapproaches are not applicable to all effectors and suffer from high rates of false positives or missed interactions, as they are not performed in the context of an infection. Here, we determine key functional domains of the effector PieE and describe a new method to identify host cell targets under physiological infection conditions. Our approach, which is applicable to other pathogens, uncovered the interaction of PieE with several proteins involved in membrane trafficking, in particular Rab GTPases, revealing new details of theLegionellainfection strategy and demonstrating the potential of this method to greatly advance our understanding of the molecular basis of infection.

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 IcmF and DotU Are Required for Optimal Effector Translocation and Trafficking of the Legionella pneumophila Vacuole

2004 ◽  
Vol 72 (10) ◽  
pp. 5972-5982 ◽  
Author(s):  
Susan M. VanRheenen ◽  
Guillaume Duménil ◽  
Ralph R. Isberg
Keyword(s):  
Legionella Pneumophila ◽  
Severe Form ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Intracellular Growth ◽  
Macrophage Infection ◽  
Type Iv ◽  
Bacterial Mutants ◽  
Bacterial Replication

ABSTRACT The gram-negative bacterium Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease, characterized by bacterial replication within alveolar macrophages. Prior to intracellular replication, the vacuole harboring the bacterium must first escape trafficking to the host lysosome, a process that is dependent on the Dot/Icm type IV secretion system. To identify genes required for intracellular growth, bacterial mutants were isolated that were delayed in escape from the macrophage but which retain a minimally functional Dot/Icm machinery. The mutations were found in eight distinct genes, including three genes known to be required for optimal intracellular growth. Two of these genes, icmF and dotU, are located at one end of a cluster of genes that encode the type IV secretion system, yet both icmF and dotU lack orthologs in other type IV translocons. DotU protein is degraded in the early postexponential phase in wild-type L. pneumophila and at all growth phases in an icmF mutant. IcmF contains an extracytoplasmic domain(s) based on accessibility to a membrane-impermeant amine-reactive reagent. In the absence of either gene, L. pneumophila targets inappropriately to LAMP-1-positive compartments during macrophage infection, is defective in the formation of replicative vacuoles, and is impaired in the translocation of the effector protein SidC. Therefore, although IcmF and DotU do not appear to be part of the core type IV secretion system, these proteins are necessary for an efficiently functioning secretion apparatus.

scholarly journals The crystal structure of LidA, a translocated substrate of the Legionella pneumophila type IV secretion system

2013 ◽  
Vol 4 (12) ◽  
pp. 897-900 ◽  
Author(s):  
Geng Meng ◽  
Xiaojing An ◽  
Sheng Ye ◽  
Yong Liu ◽  
Wenzhuang Zhu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv
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