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 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.

scholarly journals Contributions of lipopolysaccharide and the type IVB secretion system to Coxiella burnetii vaccine efficacy and reactogenicity

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Carrie M. Long ◽  
Paul A. Beare ◽  
Diane C. Cockrell ◽  
Jonathan Fintzi ◽  
Mahelat Tesfamariam ◽  
...  
Keyword(s):  
Phase I ◽  
Coxiella Burnetii ◽  
Vaccine Efficacy ◽  
Q Fever ◽  
Secretion System ◽  
Cell Vaccine ◽  
Post Vaccination ◽  
Whole Cell Vaccine ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

AbstractCoxiella burnetii is the bacterial causative agent of the zoonosis Q fever. The current human Q fever vaccine, Q-VAX®, is a fixed, whole cell vaccine (WCV) licensed solely for use in Australia. C. burnetii WCV administration is associated with a dermal hypersensitivity reaction in people with pre-existing immunity to C. burnetii, limiting wider use. Consequently, a less reactogenic vaccine is needed. Here, we investigated contributions of the C. burnetii Dot/Icm type IVB secretion system (T4BSS) and lipopolysaccharide (LPS) in protection and reactogenicity of fixed WCVs. A 32.5 kb region containing 23 dot/icm genes was deleted in the virulent Nine Mile phase I (NMI) strain and the resulting mutant was evaluated in guinea pig models of C. burnetii infection, vaccination-challenge, and post-vaccination hypersensitivity. The NMI ∆dot/icm strain was avirulent, protective as a WCV against a robust C. burnetii challenge, and displayed potentially altered reactogenicity compared to NMI. Nine Mile phase II (NMII) strains of C. burnetii that produce rough LPS, were similarly tested. NMI was significantly more protective than NMII as a WCV; however, both vaccines exhibited similar reactogenicity. Collectively, our results indicate that, like phase I LPS, the T4BSS is required for full virulence by C. burnetii. Conversely, unlike phase I LPS, the T4BSS is not required for vaccine-induced protection. LPS length does not appear to contribute to reactogenicity while the T4BSS may contribute to this response. NMI ∆dot/icm represents an avirulent phase I strain with full vaccine efficacy, illustrating the potential of genetically modified C. burnetii as improved WCVs.

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 Noncanonical Inhibition of mTORC1 byCoxiella burnetiiPromotes Replication within a Phagolysosome-Like Vacuole

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Charles L. Larson ◽  
Kelsi M. Sandoz ◽  
Diane C. Cockrell ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Secretion System ◽  
Effector Proteins ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Content Type ◽  
Mechanistic Target Of Rapamycin ◽  
Infected Cells ◽  
Bacterial Replication

ABSTRACTThe Q fever agentCoxiella burnetiiis a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify theCoxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report thatC. burnetiiinhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However,C. burnetiidid not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 byC. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulatesC. burnetiiintracellular growth.IMPORTANCECoxiella burnetiiis an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of theCoxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report thatC. burnetiigrowth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefitC. burnetiigrowth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate thatC. burnetiiinhibition of mTORC1 without accelerated autophagy promotes bacterial growth.

scholarly journals Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing Constitutive Fusion of Autophagosomes with the Coxiella -Containing Vacuole

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Lara J. Kohler ◽  
Shawna R. Reed ◽  
Shireen A. Sarraf ◽  
David D. Arteaga ◽  
Hayley J. Newton ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Host Response ◽  
Galleria Mellonella ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Effector Protein ◽  
Autophagy Pathway ◽  
Host Tolerance ◽  
Bacterial Effector Proteins

ABSTRACT Coxiella burnetii replicates in an acidified lysosome-derived vacuole. Biogenesis of the Coxiella -containing vacuole (CCV) requires bacterial effector proteins delivered into host cells by the Dot/Icm secretion system. Genetic and cell biological analysis revealed that an effector protein called Cig2 promotes constitutive fusion of autophagosomes with the CCV to maintain this compartment in an autolysosomal stage of maturation. This distinguishes the CCV from other pathogen-containing vacuoles that are targeted by the host autophagy pathway, which typically confers host resistance to infection by delivering the pathogen to a toxic lysosomal environment. By maintaining the CCV in an autolysosomal stage of maturation, Cig2 enabled CCV homotypic fusion and enhanced bacterial virulence in the Galleria mellonella (wax moth) model of infection by a mechanism that decreases host tolerance. Thus, C. burnetii residence in an autolysosomal organelle alters host tolerance of infection, which indicates that Cig2-dependent manipulation of a lysosome-derived vacuole influences the host response to infection. IMPORTANCE Coxiella burnetii is an obligate, intracellular bacterial pathogen that replicates inside a unique, lysosome-like compartment called the Coxiella -containing vacuole (CCV). Over 130 bacterial effector proteins are delivered into the host cell cytosol by the C. burnetii Dot/Icm type IV secretion system. Although the Dot/Icm system is essential for pathogenesis, the functions of most effectors remain unknown. Here we show that the effector protein Cig2 is essential for converting the CCV to an organelle that is similar to the autolysosome. Cig2 function promotes constitutive fusion between the CCV and autophagosomes generated by selective autophagy. Cig2-directed biogenesis of an autolysosomal vacuole is essential for the unique fusogenic properties of the CCV and for virulence in an animal model of disease. This work highlights how bacterial subversion of the host autophagy pathway can influence the cell biological properties of the CCV and influence the host response to infection.

scholarly journals Coxiella burnetii type IVB secretion system region I genes are expressed early during the infection of host cells

2010 ◽  
Vol 311 (1) ◽  
pp. 61-69 ◽  
Author(s):  
John K. Morgan ◽  
Brandon E. Luedtke ◽  
Herbert A. Thompson ◽  
Edward I. Shaw
Keyword(s):  
Coxiella Burnetii ◽  
Secretion System ◽  
Host Cells ◽  
Region I ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

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 The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection

2016 ◽  
Vol 84 (9) ◽  
pp. 2524-2533 ◽  
Author(s):  
Mary M. Weber ◽  
Robert Faris ◽  
Erin J. van Schaik ◽  
Juanita Thrasher McLachlan ◽  
William U. Wright ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Rho Gtpase ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Secretion System ◽  
Effector Proteins ◽  
Gtpase Activity ◽  
Content Type

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial forC. burnetiiintracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment ofCoxiella burnetiiinfection and virulence in mammalian cell culture and mouse models of infection.

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