scholarly journals Actin Dynamics and Rho GTPases Regulate the Size and Formation of Parasitophorous Vacuoles Containing Coxiella burnetii

2009 ◽  
Vol 77 (10) ◽  
pp. 4609-4620 ◽  
Author(s):  
Milton Aguilera ◽  
Romina Salinas ◽  
Eliana Rosales ◽  
Sergio Carminati ◽  
Maria I. Colombo ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Rho Gtpases ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Latrunculin B ◽  
Constitutively Active Mutants ◽  
Infected Cells ◽  
Bacterial Protein Synthesis

ABSTRACT Q fever is a disease caused by Coxiella burnetii. In the host cell, this pathogen generates a large parasitophorous vacuole (PV) with lysosomal characteristics. Here we show that F-actin not only is recruited to but also is involved in the formation of the typical PV. Treatment of infected cells with F-actin-depolymerizing agents alters PV development. The small PVs formed in latrunculin B-treated cells were loaded with transferrin and Lysotracker and labeled with an antibody against cathepsin D, suggesting that latrunculin B did not affect vacuole cargo and its lysosomal characteristics. Nevertheless, the vacuoles were unable to fuse with latex bead phagosomes. It is known that actin dynamics are regulated by the Rho family GTPases. To assess the role of these GTPases in PV formation, infected cells were transfected with pEGFP expressing wild-type and mutant Rac1, Cdc42, and RhoA proteins. Rac1 did not show significant PV association. In contrast, PVs were decorated by both the wild types and constitutively active mutants of Cdc42 and RhoA. This association was inhibited by treatment of infected cells with chloramphenicol, suggesting a role for bacterial protein synthesis in the recruitment of these proteins. Interestingly, a decrease in vacuole size was observed in cells expressing dominant-negative RhoA; however, these small vacuoles accumulated transferrin, Lysotracker, and DQ-BSA. In summary, these results suggest that actin, likely modulated by the GTPases RhoA and Cdc42 and by bacterial proteins, is involved in the formation of the typical PV.

scholarly journals The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5

2020 ◽  
Vol 295 (21) ◽  
pp. 7391-7403 ◽  
Author(s):  
Eric Martinez ◽  
Sylvaine Huc-Brandt ◽  
Solène Brelle ◽  
Julie Allombert ◽  
Franck Cantet ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Coxiella Burnetii ◽  
Membrane Trafficking ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Host Protein ◽  
Host Cells ◽  
Intracellular Replication ◽  
Gtpase Activating Protein ◽  
Infected Cells

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system–mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host–pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein–protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

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.

scholarly journals Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase Signaling during Growth in Macrophages

2012 ◽  
Vol 80 (6) ◽  
pp. 1980-1986 ◽  
Author(s):  
Laura J. MacDonald ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Coxiella Burnetii ◽  
Alveolar Macrophages ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Bacterial Agent ◽  
Dependent Protein

ABSTRACTCoxiella burnetiiis the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in immunocompromised individuals. Following aerosol-mediated transmission,C. burnetiireplicates in alveolar macrophages in a unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The mechanisms ofC. burnetiiintracellular survival are poorly defined and a recent Q fever outbreak in the Netherlands emphasizes the need for better understanding this unique host-pathogen interaction. We recently demonstrated that inhibition of host cyclic AMP-dependent protein kinase (PKA) activity negatively impacts PV formation. In the current study, we confirmed PKA involvement in PV biogenesis and probed the role of PKA signaling duringC. burnetiiinfection of macrophages. Using PKA-specific inhibitors, we found the kinase was needed for biogenesis of prototypical PV andC. burnetiireplication. PKA and downstream targets were differentially phosphorylated throughout infection, suggesting prolonged regulation of the pathway. Importantly, the pathogen actively triggered PKA activation, which was also required for PV formation by virulentC. burnetiiisolates during infection of primary human alveolar macrophages. A subset of PKA-specific substrates were differentially phosphorylated duringC. burnetiiinfection, suggesting the pathogen uses PKA signaling to control distinct host cell responses. Collectively, the current results suggest a versatile role for PKA inC. burnetiiinfection and indicate virulent organisms usurp host kinase cascades for efficient intracellular growth.

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 Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase

2010 ◽  
Vol 192 (23) ◽  
pp. 6154-6159 ◽  
Author(s):  
Stacey D. Gilk ◽  
Paul A. Beare ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Fluorescent Protein ◽  
De Novo ◽  
Cholesterol Metabolism ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Cholesterol Biosynthesis ◽  
Brefeldin A ◽  
Ergosterol Biosynthesis

ABSTRACT Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.

scholarly journals Development of anEx VivoTissue Platform To Study the Human Lung Response to Coxiella burnetii

2016 ◽  
Vol 84 (5) ◽  
pp. 1438-1445 ◽  
Author(s):  
Joseph G. Graham ◽  
Caylin G. Winchell ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Coxiella Burnetii ◽  
Lung Tissue ◽  
Human Lung ◽  
Q Fever ◽  
Ex Vivo ◽  
Parasitophorous Vacuole ◽  
Inflammasome Activation ◽  
Human Lung Tissue ◽  
Content Type

Coxiella burnetiiis an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells,C. burnetiigenerates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes.C. burnetiirequires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand howC. burnetiievades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infectionin vitroand found that avirulentC. burnetiitriggers sustained interleukin-1β (IL-1β) production. Here, we evaluated infection ofex vivohuman lung tissue, defining a valuable approach for characterizingC. burnetiiinteractions with a human host. Within whole lung tissue,C. burnetiipreferentially replicated in hAMs. Additionally, IL-1β production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur followingC. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response toC. burnetii.

scholarly journals The Coxiella burnetii Ankyrin Repeat Domain-Containing Protein Family Is Heterogeneous, with C-Terminal Truncations That Influence Dot/Icm-Mediated Secretion

2009 ◽  
Vol 191 (13) ◽  
pp. 4232-4242 ◽  
Author(s):  
Daniel E. Voth ◽  
Dale Howe ◽  
Paul A. Beare ◽  
Joseph P. Vogel ◽  
Nathan Unsworth ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Coxiella Burnetii ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Protein Family ◽  
Ankyrin Repeat ◽  
Reference Isolate ◽  
Type Iv

ABSTRACT Coxiella burnetii is an obligate intracellular bacterium that directs biogenesis of a parasitophorous vacuole (PV) for replication. Effectors of PV maturation are likely translocated into the host cytosol by a type IV secretion system (T4SS) with homology to the Dot/Icm apparatus of Legionella pneumophila. Since secreted bacterial virulence factors often functionally mimic the activities of host proteins, prokaryotic proteins with eukaryotic features are considered candidate T4SS substrates. Genes encoding proteins with eukaryotic-type ankyrin repeat domains (Anks) were identified upon genome sequencing of the C. burnetii Nine Mile reference isolate, which is associated with a case of human acute Q fever. Interestingly, recent genome sequencing of the G and K isolates, derived from human chronic endocarditis patients, and of the Dugway rodent isolate revealed remarkable heterogeneity in the Ank gene family, with the Dugway isolate harboring the largest number of full-length Ank genes. Using L. pneumophila as a surrogate host, we identified 10 Dugway Anks and 1 Ank specific to the G and K endocarditis isolates translocated into the host cytosol in a Dot/Icm-dependent fashion. A 10-amino-acid C-terminal region appeared to be necessary for translocation, with some Anks also requiring the chaperone IcmS for secretion. Ectopically expressed Anks localized to a variety of subcellular regions in mammalian cells, including microtubules, mitochondria, and the PV membrane. Collectively, these data suggest that C. burnetii isolates translocate distinct subsets of the Ank protein family into the host cytosol, where they modulate diverse functions, some of which may be unique to C. burnetii pathotypes.

scholarly journals Sustained Axenic Metabolic Activity by the Obligate Intracellular Bacterium Coxiella burnetii

2008 ◽  
Vol 190 (9) ◽  
pp. 3203-3212 ◽  
Author(s):  
Anders Omsland ◽  
Diane C. Cockrell ◽  
Elizabeth R. Fischer ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Metabolic Activity ◽  
De Novo ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Chloride Concentration ◽  
Citrate Buffer ◽  
Cell Variant

ABSTRACT Growth of Coxiella burnetii, the agent of Q fever, is strictly limited to colonization of a viable eukaryotic host cell. Following infection, the pathogen replicates exclusively in an acidified (pH 4.5 to 5) phagolysosome-like parasitophorous vacuole. Axenic (host cell free) buffers have been described that activate C. burnetii metabolism in vitro, but metabolism is short-lived, with bacterial protein synthesis halting after a few hours. Here, we describe a complex axenic medium that supports sustained (>24 h) C. burnetii metabolic activity. As an initial step in medium development, several biological buffers (pH 4.5) were screened for C. burnetii metabolic permissiveness. Based on [35S]Cys-Met incorporation, C. burnetii displayed optimal metabolic activity in citrate buffer. To compensate for C. burnetii auxotrophies and other potential metabolic deficiencies, we developed a citrate buffer-based medium termed complex Coxiella medium (CCM) that contains a mixture of three complex nutrient sources (neopeptone, fetal bovine serum, and RPMI cell culture medium). Optimal C. burnetii metabolism occurred in CCM with a high chloride concentration (140 mM) while the concentrations of sodium and potassium had little effect on metabolism. CCM supported prolonged de novo protein and ATP synthesis by C. burnetii (>24 h). Moreover, C. burnetii morphological differentiation was induced in CCM as determined by the transition from small-cell variant to large-cell variant. The sustained in vitro metabolic activity of C. burnetii in CCM provides an important tool to investigate the physiology of this organism including developmental transitions and responses to antimicrobial factors associated with the host cell.

scholarly journals The Coxiella burnetii Dot/Icm System Creates a Comfortable Home through Lysosomal Renovation

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Hayley J. Newton ◽  
Craig R. Roy
Keyword(s):  
Coxiella Burnetii ◽  
Mutational Analysis ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Culture Conditions ◽  
Effector Proteins ◽  
Collective Actions ◽  
Content Type ◽  
Genetic Techniques ◽  
Obligate Intracellular Bacteria

ABSTRACTUnderstanding the molecular pathogenesis ofCoxiella burnetii, the causative agent of human Q fever, has historically been hindered by the technical difficulties of genetically manipulating obligate intracellular bacteria. The recent development of culture conditions suitable for axenic propagation ofC. burnetiihas paved the way for the application of a range of genetic techniques to address key questions within the field. Recent studies using mutational analysis have revealed that theC. burnetiiDot/Icm type 4 secretion system (T4SS) is an important virulence determinant that is essential for renovation of a lysosome into a matureCoxiella-containing vacuole (CCV) permissive of intracellular replication. Interestingly, a mutant ofC. burnetiideficient in Dot/Icm function was found to be capable of replicating within the parasitophorous vacuole created byLeishmania amazonensis, which indicates thatC. burnetiireplication is not dependent on the cohort of Dot/Icm effector proteinsper sebut rather that the collective actions of effectors are required to create the specialized niche supportive of replication. Thus, a role for the Dot/Icm T4SS during the intracellular life cycle ofC. burnetiihas been more clearly defined by these studies, which demonstrate that advances in genetic analysis should allow future studies to focus on the intricacies of Dot/Icm effector functions that facilitate development of the unique CCV.

scholarly journals Coxiella burnetiiSubverts p62/Sequestosome 1 and Activates Nrf2 Signaling in Human Macrophages

2018 ◽  
Vol 86 (5) ◽  
Author(s):  
Caylin G. Winchell ◽  
Amanda L. Dragan ◽  
Katelynn R. Brann ◽  
Frances I. Onyilagha ◽  
Richard C. Kurten ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Content Type ◽  
Sequestosome 1 ◽  
Type Iv ◽  
Host Signaling ◽  
Infected Cells ◽  
Pv Generation ◽  
Interfering Rna

ABSTRACTCoxiella burnetiiis the causative agent of human Q fever, a debilitating flu-like illness that can progress to chronic disease presenting as endocarditis. Following inhalation,C. burnetiiis phagocytosed by alveolar macrophages and generates a lysosome-like replication compartment termed the parasitophorous vacuole (PV). A type IV secretion system (T4SS) is required for PV generation and is one of the pathogen's few known virulence factors. We previously showed thatC. burnetiiactively recruits autophagosomes to the PV using the T4SS but does not alter macroautophagy. In the current study, we confirmed that the cargo receptor p62/sequestosome 1 (SQSTM-1) localizes near the PV in primary human alveolar macrophages infected with virulentC. burnetii. p62 and LC3 typically interact to select cargo for autophagy-mediated degradation, resulting in p62 degradation and LC3 recycling. However, inC. burnetii-infected macrophages, p62 was not degraded when cells were starved, suggesting that the pathogen stabilizes the protein. In addition, phosphorylated p62 levels increased, indicative of activation, during infection. Small interfering RNA experiments indicated that p62 is not absolutely required for intracellular growth, suggesting that the protein serves a signaling role during infection. Indeed, the Nrf2-Keap1 cytoprotective pathway was activated during infection, as evidenced by sustained maintenance of Nrf2 levels and translocation of the protein to the nucleus inC. burnetii-infected cells. Collectively, our studies identify a new p62-regulated host signaling pathway exploited byC. burnetiiduring intramacrophage growth.

Sign in / Sign up

Export Citation Format

Share Document