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 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 Coxiella burnetii Exhibits Morphological Change and Delays Phagolysosomal Fusion after Internalization by J774A.1 Cells

2000 ◽  
Vol 68 (7) ◽  
pp. 3815-3821 ◽  
Author(s):  
Dale Howe ◽  
Louis P. Mallavia
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Large Cell ◽  
Infection Process ◽  
Thorium Dioxide ◽  
Small Cell Variant ◽  
Morphological Variants ◽  
Cell Variant ◽  
First Time

ABSTRACT Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium proliferating within the harsh environment of the phagolysosome. Mechanisms controlling trafficking to, and survival of pathogens within, the phagolysosome are unknown. Two distinct morphological variants have been implicated as playing a role in C. burnetii survival. The dormant small-cell variant (SCV) is resistant to extracellular stresses and the more metabolically active large-cell variant (LCV) is sensitive to environmental stresses. To document changes in the ratio of SCVs to LCVs in response to environment, a protein specific to SCV, ScvA, was quantitated. During the first 2 h after internalization ofC. burnetii by J774A.1 cells, the level of ScvA decreased, indicating a change from a population containing primarily SCVs to one containing primarily LCVs. In vitro experiments showed that 2 h of incubation at pH 5.5 caused a significant decrease in ScvA in contrast to incubation at pH 4.5. Measuring in vitro internalization of [35S]methionine-[35S]cysteine in response to pH, we found the uptake to be optimal at pH 5.5. To explore the possibility that after uptake C. burnetii was able to delay phagolysosomal fusion, we used thorium dioxide and acid phosphatase to label phagolysosomes during infection of J774A.1 cells. We determined that viable C. burnetii was able to delay phagolysosomal fusion. This is the first time that a delay in phagolysosomal fusion has been shown to be a part of the infection process of this pathogenic microorganism.

scholarly journals A DNA-Binding Peroxiredoxin of Coxiella burnetii Is Involved in Countering Oxidative Stress during Exponential-Phase Growth

2010 ◽  
Vol 192 (8) ◽  
pp. 2077-2084 ◽  
Author(s):  
Linda D. Hicks ◽  
Rahul Raghavan ◽  
James M. Battisti ◽  
Michael F. Minnick
Keyword(s):  
Oxidative Stress ◽  
Dna Binding ◽  
Host Cell ◽  
Coxiella Burnetii ◽  
Binding Protein ◽  
Parasitophorous Vacuole ◽  
Dna Binding Protein ◽  
Heat Denaturation ◽  
Exponential Growth Phase

ABSTRACT Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen's proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100°C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain's ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P < 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen's early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella's metabolism during intracellular replication.

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 The Effect of pH on Antibiotic Efficacy against Coxiella burnetii in Axenic Media

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cody B. Smith ◽  
Charles Evavold ◽  
Gilbert J. Kersh
Keyword(s):  
Coxiella Burnetii ◽  
Antimicrobial Agents ◽  
Q Fever ◽  
Antimicrobial Treatment ◽  
Etiologic Agent ◽  
Growth And Survival ◽  
Intracellular Compartments ◽  
The Impact

AbstractCoxiella burnetii, the etiologic agent of Q fever, replicates in an intracellular phagolysosome with pH between 4 and 5. The impact of this low pH environment on antimicrobial treatment is not well understood. An in vitro system for testing antibiotic susceptibility of C. burnetii in axenic media was set up to evaluate the impact of pH on C. burnetii growth and survival in the presence and absence of antimicrobial agents. The data show that C. burnetii does not grow in axenic media at pH 6.0 or higher, but the organisms remain viable. At pH of 4.75, 5.25, and 5.75 moxifloxacin, doxycycline, and rifampin are effective at preventing growth of C. burnetii in axenic media, with moxifloxacin and doxycycline being bacteriostatic and rifampin having bactericidal activity. The efficacy of doxycycline and moxifloxacin improved at higher pH, whereas rifampin activity was pH independent. Hydroxychloroquine is thought to inhibit growth of C. burnetii in vivo by raising the pH of typically acidic intracellular compartments. It had no direct bactericidal or bacteriostatic activity on C. burnetii in axenic media, suggesting that raising pH of acidic intracellular compartments is its primary mechanism of action in vivo. The data suggest that doxycycline and hydroxychloroquine are primarily independent bacteriostatic agents.

Eimeria canadensis (Protozoa, Sporozoea): ultrastructure of the host–parasite interface during development of first-generation schizonts in vitro

1980 ◽  
Vol 58 (11) ◽  
pp. 2018-2025 ◽  
Author(s):  
Bodo E. G. Mueller
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Eimeria Species ◽  
Outer Zone ◽  
Ultrastructural Observation ◽  
Cell Cytoplasm ◽  
Cell Organelles ◽  
Host Cell Cytoplasm ◽  
Host Parasite

Eimeria canadensis sporozoites were inoculated into monolayer cultures of Madin–Darby bovine kidney and primary bovine embryonic kidney cells. Sporozoites retained their shape for at least 9 days. At that time, the nucleus was enlarged and contained a prominent nucleolus, and amylopectin granules were no longer apparent. The width of the parasitophorous vacuole (pv) between host cell cytoplasm and parasite pellicle widened during transformation of sporozoites into multinucleate schizonts. Areas of altered host cell cytoplasm immediately adjacent to the pv membrane increased in size and became confluent, resulting in the formation of two distinct layers of cytoplasm. The outer zone contained the host cell nucleus, mitochondria, Golgi stacks, and ER, whereas the inner layer appeared granular and was void of all cell organelles except structures resembling ribosomes. Microfilaments were abundant at the border between inner and outer zone. In the most advanced stages observed, host cell organelles persisted only in the perinuclear region. The remaining, attenuated cytoplasm resembled the former inner zone.The novel ultrastructural observation of a bilayered cytoplasm of cells harbouring E. canadensis schizonts is compared with light microscope reports of similar effects caused by other Eimeria species of ruminants and with electron microscope findings of altered intestinal and abomasal cells of sheep harbouring "globidial" schizonts.

scholarly journals Bacteriostatic and Bactericidal Activities of Tigecycline against Coxiella burnetii and Comparison with Those of Six Other Antibiotics

2009 ◽  
Vol 53 (6) ◽  
pp. 2690-2692 ◽  
Author(s):  
Ioanna Spyridaki ◽  
Anna Psaroulaki ◽  
Iosif Vranakis ◽  
Yannis Tselentis ◽  
Achilleas Gikas
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Vero Cells ◽  
In Vitro Susceptibility ◽  
Shell Vial ◽  
Bactericidal Activities ◽  
Acute Q Fever ◽  
Reference Strains ◽  
Shell Vial Assay

ABSTRACT The present article is a study of the in vitro susceptibility of eight Greek Coxiella burnetii isolates, derived from patients with acute Q fever, and two reference strains of Coxiella burnetii to tigecycline. The bacteriostatic activity of tigecycline was compared with those of six other antibiotics using a shell vial assay. The MICs of the examined antibiotics were as follows: tigecycline ranged from 0.25 to 0.5 μg/ml; doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 μg/ml; linezolid and clarithromycin ranged from 2 to 4 μg/ml; and ciprofloxacin ranged from 4 to 8 μg/ml. Tigecycline was effective in inhibiting the infection of Vero cells by C. burnetii. No bactericidal activity was observed against C. burnetii at 4 μg/ml.

scholarly journals Coxiella burnetii Interaction with Neutrophils and MacrophagesIn Vitroand in SCID Mice following Aerosol Infection

2013 ◽  
Vol 81 (12) ◽  
pp. 4604-4614 ◽  
Author(s):  
Alexandra Elliott ◽  
Ying Peng ◽  
Guoquan Zhang
Keyword(s):  
Immune Response ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Natural Infection ◽  
Scid Mice ◽  
Innate Immune ◽  
Content Type ◽  
Aerosol Infection

ABSTRACTCoxiella burnetiiis an obligate intracellular bacterium that causes acute and chronic Q fever in humans. Human Q fever is mainly transmitted by aerosol infection. However, there is a fundamental gap in the knowledge regarding the mechanisms of pulmonary immunity againstC. burnetiiinfection. This study focused on understanding the interaction betweenC. burnetiiand innate immune cellsin vitroandin vivo. Both virulentC. burnetiiNine Mile phase I (NMI) and avirulent Nine Mile phase II (NMII) were able to infect neutrophils, while the infection rates were lower than 29%, suggesting thatC. burnetiican infect neutrophils, but infection is limited. Interestingly,C. burnetiiinside neutrophils can infect and replicate within macrophages, suggesting that neutrophils cannot killC. burnetiiandC. burnetiimay be using infection of neutrophils as an evasive strategy to infect macrophages. To elucidate the mechanisms of the innate immune response toC. burnetiinatural infection, SCID mice were exposed to aerosolizedC. burnetii. Surprisingly, neutrophil influx into the lungs was delayed until day 7 postinfection in both NMI- and NMII-infected mice. This result suggests that neutrophils may play a unique role in the early immune response against aerosolizedC. burnetii. Studying the interaction betweenC. burnetiiand the innate immune system can provide a model system for understanding how the bacteria evade early immune responses to cause infection.

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