scholarly journals Dot/Icm-Translocated Proteins Important for Biogenesis of theCoxiella burnetii-Containing Vacuole Identified by Screening of an Effector Mutant Sublibrary

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
Emerson Crabill ◽  
Whitman B. Schofield ◽  
Hayley J. Newton ◽  
Andrew L. Goodman ◽  
Craig R. Roy
Keyword(s):  
Loss Of Function ◽  
Intracellular Replication ◽  
Small Vacuole ◽  
Content Type ◽  
Type Iv ◽  
Cell Cytosol ◽  
Type Ivb Secretion ◽  
Translocated Proteins ◽  
Vacuole Biogenesis ◽  
Type Ivb Secretion System

ABSTRACTCoxiella burnetiiis an intracellular pathogen that replicates in a lysosome-derived vacuole. A determinant necessary forC. burnetiivirulence is the Dot/Icm type IVB secretion system (T4SS). The Dot/Icm system delivers more than 100 proteins, called type IV effectors (T4Es), across the vacuolar membrane into the host cell cytosol. Several T4Es have been shown to be important for vacuolar biogenesis. Here, transposon (Tn) insertion sequencing technology (INSeq) was used to identifyC. burnetiiNine Mile phase II mutants in an arrayed library, which facilitated the identification and clonal isolation of mutants deficient in 70 different T4E proteins. These effector mutants were screened in HeLa cells for deficiencies inCoxiella-containing vacuole (CCV) biogenesis. This screen identified and validated seven new T4Es that were important for vacuole biogenesis. Loss-of-function mutations incbu0414(coxH1),cbu0513,cbu0978(cem3),cbu1387(cem6),cbu1524(caeA),cbu1752, orcbu2028resulted in a small-vacuole phenotype. These seven mutant strains produced small CCVs in all cells tested, which included macrophage-like cells. Thecbu2028::Tn mutant, though unable to develop large CCVs, had intracellular replication rates similar to the rate of the parental strain ofC. burnetii, whereas the other six effector mutants defective in CCV biogenesis displayed significant reductions in intracellular replication. Vacuoles created by thecbu0513::Tn mutant did not accumulate lipidated microtubule-associated protein 1A/1B light chain 3 (LC3-II), suggesting a failure in fusion of the CCV with autophagosomes. These seven T4E proteins add to the growing repertoire ofC. burnetiifactors that contribute to CCV biogenesis.

scholarly journals Targeting of the Small GTPase Rab6A′ by the Legionella pneumophila Effector LidA

2013 ◽  
Vol 81 (6) ◽  
pp. 2226-2235 ◽  
Author(s):  
Yang Chen ◽  
Matthias P. Machner
Keyword(s):  
Legionella Pneumophila ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Host Cells ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Gtpase Activating Proteins ◽  
Active Pool ◽  
Cell Cytosol

ABSTRACTWhen the bacteriumLegionella pneumophila, the causative agent of Legionnaires' disease, is phagocytosed by alveolar macrophages, it delivers a large number of effector proteins through its Dot/Icm type IV secretion system into the host cell cytosol. Among those proteins is LidA, an effector that interacts with several host GTPases of the Rab family, including Rab6A′, a regulator of retrograde vesicle trafficking within eukaryotic cells. The effect of LidA on Rab6A′ function and the role of Rab6A′ forL. pneumophilagrowth within host cells has been unclear. Here, we show that LidA preferentially binds Rab6A′ in the active GTP-bound conformation. Rab6 binding occurred through the central region of LidA and followed a stoichiometry for LidA and Rab6A′ of 1:2. LidA maintained Rab6A′ in the active conformation by efficiently blocking the hydrolysis of GTP by Rab6A′, even in the presence of cellular GTPase-activating proteins, suggesting that the function of Rab6A′ must be important for efficient intracellular replication ofL. pneumophila. Accordingly, we found that production of constitutively inactive Rab6A′(T27N) but not constitutively active Rab6A′(Q72L) significantly reduced the ability ofL. pneumophilato initiate intracellular replication in human macrophages. Thus, the presence of an active pool of Rab6 within host cells early during infection is required to support efficient intracellular growth ofL. pneumophila.

scholarly journals Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Yoon-Suk Kang ◽  
James E. Kirby
Keyword(s):  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Life Cycles ◽  
Wild Type ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Reporter Systems ◽  
Brucella Neotomae

ABSTRACT We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

scholarly journals Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

2014 ◽  
Vol 83 (2) ◽  
pp. 661-670 ◽  
Author(s):  
Charles L. Larson ◽  
Paul A. Beare ◽  
Daniel E. Voth ◽  
Dale Howe ◽  
Diane C. Cockrell ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Parasitophorous Vacuole ◽  
Coiled Coil ◽  
Bacterial Pathogen ◽  
Effector Proteins ◽  
Intracellular Replication ◽  
Intracellular Growth ◽  
Content Type ◽  
Cell Cytosol ◽  
Pv Generation

The intracellular bacterial pathogenCoxiella burnetiidirects biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supportsC. burnetiireplication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promoteC. burnetiiintracellular growth and PV expansion. We predict additionalC. burnetiieffectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predictedC. burnetiiT4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion byC. burnetiiduring infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termedCoxiellavacuolarprotein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins.C. burnetiiΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpEmutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpDand ΔcvpEmutants rescued intracellular growth and PV generation, whereas the growth ofC. burnetiiΔcvpBand ΔcvpCwas rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicateC. burnetiiencodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.

scholarly journals Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Justin A. McDonough ◽  
Hayley J. Newton ◽  
Scott Klum ◽  
Rachel Swiss ◽  
Hervé Agaisse ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Factors ◽  
Content Type ◽  
Host Determinants ◽  
Genome Wide ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System ◽  
Bacterial Effector Proteins

ABSTRACTCoxiella burnetiiis an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used byC. burnetiito translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required forC. burnetiiintracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication ofC. burnetiiwas measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogatedC. burnetiireplication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles withinC. burnetii-infected cells. Silencing of theSTX17gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containingC. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed forC. burnetiireplication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate thatC. burnetiivacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur duringC. burnetiiinfection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication.IMPORTANCEQ fever in humans is caused by the bacteriumCoxiella burnetii. Infection withC. burnetiiis marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets ofC. burnetiiinfection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of theC. burnetiivacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.

scholarly journals Polar targeting and assembly of theLegionellaDot/Icm type IV secretion system (T4SS) by T6SS-related components

2018 ◽  
Author(s):  
KwangCheol C. Jeong ◽  
Jacob Gyore ◽  
Lin Teng ◽  
Debnath Ghosal ◽  
Grant J. Jensen ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Legionnaires Disease ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

SummaryLegionella pneumophila, the causative agent of Legionnaires’ disease, survives and replicates inside amoebae and macrophages by injecting a large number of protein effectors into the host cells’ cytoplasm via the Dot/Icm type IVB secretion system (T4BSS). Previously, we showed that the Dot/Icm T4BSS is localized to both poles of the bacterium and that polar secretion is necessary for the proper targeting of theLegionellacontaining vacuole (LCV). Here we show that polar targeting of the Dot/Icm core-transmembrane subcomplex (DotC, DotD, DotF, DotG and DotH) is mediated by two Dot/Icm proteins, DotU and IcmF, which are able to localize to the poles ofL. pneumophilaby themselves. Interestingly, DotU and IcmF are homologs of the T6SS components TssL and TssM, which are part of the T6SS membrane complex (MC). We propose thatLegionellaco-opted these T6SS components to a novel function that mediates subcellular localization and assembly of this T4SS. Finally, in depth examination of the biogenesis pathway revealed that polar targeting and assembly of theLegionellaT4BSS apparatus is mediated by an innovative “outside-inside” mechanism.

scholarly journals Induction of Rapid Cell Death by an Environmental Isolate of Legionella pneumophila in Mouse Macrophages

2013 ◽  
Vol 81 (9) ◽  
pp. 3077-3088 ◽  
Author(s):  
Lili Tao ◽  
Wenhan Zhu ◽  
Bi-Jie Hu ◽  
Jie-Ming Qu ◽  
Zhao-Qing Luo
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Immune Surveillance ◽  
Human Macrophage ◽  
Aqueous Environment ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Extensive Cell Death ◽  
Extensive Cell

ABSTRACTLegionella pneumophila, the etiological agent for Legionnaires' disease, is ubiquitous in the aqueous environment, where it replicates as an intracellular parasite of free-living protozoa. Our understanding ofL. pneumophilapathogenicity is obtained mostly from study of derivatives of several clinical isolates, which employ almost identical virulent determinants to exploit host functions. To determine whether environmentalL. pneumophilaisolates interact similarly with the model host systems, we analyzed intracellular replication of several recently isolated such strains and found that these strains cannot productively grow in bone marrow-derived macrophages of A/J mice, which are permissive for all examined laboratory strains. By focusing on one strain called LPE509, we found that its deficiency in intracellular replication in primary A/J macrophages is not caused by the lack of important pathogenic determinants because this strain replicates proficiently in two protozoan hosts and the human macrophage U937 cell. We also found that in the early phase of infection, the trafficking of this strain in A/J macrophages is similar to that of JR32, a derivative of strain Philadelphia 1. Furthermore, infection of these cells by LPE509 caused extensive cell death in a process that requires the Dot/Icm type IV secretion system. Finally, we showed that the cell death is caused neither by the activation of the NAIP5/NLRC4 inflammasome nor by the recently described caspase 11-dependent pathway. Our results revealed that some environmentalL. pneumophilastrains are unable to overcome the defense conferred by primary macrophages from mice known to be permissive for laboratoryL. pneumophilastrains. These results also suggest the existence of a host immune surveillance mechanism differing from those currently known in responding toL. pneumophilainfection.

Structure of the Legionella Dot/Icm type IV secretion system in situ by electron cryotomography

2016 ◽  
Author(s):  
Debnath Ghosal ◽  
Yi-Wei Chang ◽  
Kwangcheol C. Jeong ◽  
Joseph P. Vogel ◽  
Grant J. Jensen
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Secretion Systems ◽  
Type Iv ◽  
Type Ivb Secretion ◽  
Structural Preservation ◽  
Electron Cryotomography ◽  
Type Ivb Secretion System

AbstractType IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence homology with type IVA secretion systems (T4ASSs), its overall structure shows remarkable similarities to two previously imaged T4ASSs, suggesting shared aspects of mechanism. However, compared to one of these, the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, it is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.

scholarly journals A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Laura F. Fielden ◽  
Jennifer H. Moffatt ◽  
Yilin Kang ◽  
Michael J. Baker ◽  
Chen Ai Khoo ◽  
...  
Keyword(s):  
Host Cell ◽  
Outer Membrane ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Effector Proteins ◽  
Mitochondrial Outer Membrane ◽  
Intracellular Replication ◽  
Content Type ◽  
Multimeric Complex ◽  
Type Ivb Secretion

ABSTRACT Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella. Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial C oxiella effector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3×FLAG or MceA tagged with 2×hemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection.

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 Dot/Icm Type IVB Secretion System Requirements for Coxiella burnetii Growth in Human Macrophages

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Paul A. Beare ◽  
Stacey D. Gilk ◽  
Charles L. Larson ◽  
Joshua Hill ◽  
Christopher M. Stead ◽  
...  
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Secretion System ◽  
Mononuclear Phagocytes ◽  
Productive Infection ◽  
Content Type ◽  
Human Macrophages ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

ABSTRACTCentral to Q fever pathogenesis is replication of the causative agent,Coxiella burnetii, within a phagolysosome-like parasitophorous vacuole (PV) in mononuclear phagocytes.C. burnetiimodulates PV biogenesis and other host cell functions, such as apoptotic signaling, presumably via the activity of proteins delivered to the host cytosol by a Dot/Icm type IVB secretion system (T4BSS). In this study, we utilized aC. burnetiistrain carrying IcmD inactivated by theHimar1transposon to investigate the requirements for Dot/Icm function inC. burnetiiparasitism of human THP-1 macrophage-like cells. TheicmD::Tn mutant failed to secrete characterized T4BSS substrates, a defect that correlated with deficient replication, PV development, and apoptosis protection. Restoration of type IVB secretion and intracellular growth of theicmD::Tn mutant required complementation withicmD,-J, and-B, indicating a polar effect of the transposon insertion on downstreamdot/icmgenes. Induction oficmDJBexpression at 1 day postinfection resulted inC. burnetiireplication and PV generation. Collectively, these data prove that T4BSS function is required for productive infection of human macrophages byC. burnetii. However, illustrating the metabolic flexibility ofC.burnetti, theicmD::Tn mutant could replicate intracellularly when sequestered in a PV generated by wild-type bacteria, where Dot/Icm function is provided intrans, and within a phenotypically similar PV generated by the protozoan parasiteLeishmania amazonensis, where host cells are devoid of Dot/Icm T4BSS effector proteins.IMPORTANCECoxiella burnetii, the cause of human Q fever, is the only bacterial pathogen known to replicate in a vacuole resembling a phagolysosome. The organism manipulates host macrophages to promote the biogenesis of a vacuolar compartment permissive for growth. By analogy to the well-established cellular microbiology ofLegionella pneumophila, the Dot/Icm type IVB secretion system ofC. burnetiiis implicated as a critical virulence factor in host cell modification that delivers proteins with effector functions directly into the host cell cytosol. Using new genetic tools, we verify that Dot/Icm function is essential for productive infection of human macrophages byC. burnetii. Interestingly, despite the production of homologous secretion systems,L. pneumophilaandC. burnetiihave strikingly different temporal requirements for Dot/Icm function during their respective infectious cycles.

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