scholarly journals Sustained Activation of Akt and Erk1/2 Is Required for Coxiella burnetii Antiapoptotic Activity

2008 ◽  
Vol 77 (1) ◽  
pp. 205-213 ◽  
Author(s):  
Daniel E. Voth ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Stress Responses ◽  
Mammalian Cells ◽  
Apoptotic Cell ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Bacterial Pathogen ◽  
Human Macrophage ◽  
Antiapoptotic Activity

ABSTRACT Coxiella burnetii is an obligate intracellular bacterial pathogen that directs biogenesis of a lysosome-like, parasitophorous vacuole in mammalian cells. We recently reported that C. burnetii inhibits apoptotic cell death in macrophages, presumably as a mechanism to sustain the host for completion of its lengthy infectious cycle. In the current study, we further investigated C. burnetii manipulation of host cell signaling and apoptosis by examining the effect of C. burnetii infection on activation of 15 host proteins involved in stress responses, cytokine production, and apoptosis. C. burnetii infection of THP-1 human macrophage-like cells caused increased levels of phosphorylated c-Jun, Hsp27, Jun N-terminal protein kinase, and p38 at 2 h postinfection (hpi), and this activation rapidly decreased to near basal levels by 24 hpi. The prosurvival kinases Akt and Erk1/2 (extracellular signal-regulated kinases 1 and 2) were also activated at 2 to 6 hpi; however, the phosphorylation of these proteins increased coincident with C. burnetii replication through at least 72 hpi. Sustained phosphorylation of Akt and Erk1/2 was abolished by treatment of infected cells with rifampin, indicating their activation is a C. burnetii-directed event requiring pathogen RNA synthesis. Moreover, pharmacological inhibition of Akt or Erk1/2 significantly decreased C. burnetii antiapoptotic activity. Collectively, these results indicate the importance of C. burnetii modulation of host signaling and demonstrate a critical role for Akt and Erk1/2 in successful intracellular parasitism and maintenance of host cell viability.

scholarly journals Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

2005 ◽  
Vol 77 (1) ◽  
pp. 77-94 ◽  
Author(s):  
Renato A. Mortara ◽  
Walter K. Andreoli ◽  
Noemi N. Taniwaki ◽  
Adriana B. Fernandes ◽  
Claudio V. da Silva ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Mammalian Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Mammalian Host ◽  
Target Cells ◽  
Sylvatic Cycle ◽  
Final Destination ◽  
Different Strains

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

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 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 Critical Role for Molecular Iron in Coxiella burnetii Replication and Viability

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Savannah E. Sanchez ◽  
Anders Omsland
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Cultured Cells ◽  
Critical Role ◽  
Host Cells ◽  
Physiological Adaptation ◽  
Iron Starvation ◽  
Iron Storage ◽  
Content Type ◽  
Forms Of Iron

ABSTRACT Coxiella burnetii, the causative agent of Query (Q) fever in humans, is a highly infectious obligate intracellular bacterium. Following uptake into a host cell, C. burnetii replicates within a phagolysosome-derived compartment referred to as the Coxiella-containing vacuole (CCV). During infection, C. burnetii exhibits tropism for tissues related to iron storage and recycling (e.g., the liver and splenic red pulp), suggesting that pathogen physiology is linked to host iron metabolism. Iron has been described to have a limited role in C. burnetii virulence regulation, despite evidence that C. burnetii-infected host cells increase expression of transferrin receptors, thereby suggesting that active iron acquisition by the bacterium occurs upon infection. Through the use of host cell-free culture, C. burnetii was separated from the host cell in order to directly assess the role of different forms of iron in C. burnetii replication and viability, and therefore virulence. Results indicate that C. burnetii tolerates molecular iron over a broad concentration range (i.e., ∼0.001 to 1 mM) and undergoes gross loss of viability upon iron starvation. C. burnetii protein synthesis and energy metabolism, however, occur nearly uninhibited under iron concentrations not permissive to replication. Despite the apparent absence of genes related to acquisition of host-associated iron-containing proteins, C. burnetii replication is supported by hemoglobin, transferrin, and ferritin, likely due to release of iron from such proteins under acidic conditions. Moreover, chelation of host iron pools inhibited pathogen replication during infection of cultured cells. IMPORTANCE Host organisms restrict the availability of iron to invading pathogens in order to reduce pathogen replication. To counteract the host’s response to infection, bacteria can rely on redundant mechanisms to obtain biologically diverse forms of iron during infection. C. burnetii appears specifically dependent on molecular iron for replication and viability and exhibits a response to iron akin to bacteria that colonize iron-rich environments. Physiological adaptation of C. burnetii to the unique acidic and degradative environment of the CCV is consistent with access of this pathogen to molecular iron.

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 Salmonella Effector PipB2 Affects Late Endosome/Lysosome Distribution to Mediate Sif Extension

2005 ◽  
Vol 16 (9) ◽  
pp. 4108-4123 ◽  
Author(s):  
Leigh A. Knodler ◽  
Olivia Steele-Mortimer
Keyword(s):  
Host Cell ◽  
Mammalian Cells ◽  
Ectopic Expression ◽  
Bacterial Pathogen ◽  
Sequence Divergence ◽  
Cell Periphery ◽  
Host Cell Proteins ◽  
Late Endosomes ◽  
Membrane Bound ◽  
Endocytic Compartments

After internalization into mammalian cells, the bacterial pathogen Salmonella enterica resides within a membrane-bound compartment, the Salmonella-containing vacuole (SCV). During its maturation process, the SCV interacts extensively with host cell endocytic compartments, especially late endosomes/lysosomes (LE/Lys) at later stages. These interactions are mediated by the activities of multiple bacterial and host cell proteins. Here, we show that the Salmonella type III effector PipB2 reorganizes LE/Lys compartments in mammalian cells. This activity results in the centrifugal extension of lysosomal glycoprotein-rich membrane tubules, known as Salmonella-induced filaments, away from the SCV along microtubules. Salmonella overexpressing pipB2 induce the peripheral accumulation of LE/Lys compartments, reducing the frequency of LE/Lys tubulation. Furthermore, ectopic expression of pipB2 redistributes LE/Lys, but not other cellular organelles, to the cell periphery. In coexpression studies, PipB2 can overcome the effects of dominant-active Rab7 or Rab34 on LE/Lys positioning. Deletion of a C-terminal pentapeptide motif of PipB2, LFNEF, prevents its peripheral targeting and effect on organelle positioning. The PipB2 homologue PipB does not possess this motif or the same biological activity as PipB2. Therefore, it seems that a divergence in the biological functions of these two effectors can be accounted for by sequence divergence in their C termini.

scholarly journals Antiapoptotic Activity of Coxiella burnetii Effector Protein AnkG Is Controlled by p32-Dependent Trafficking

2014 ◽  
Vol 82 (7) ◽  
pp. 2763-2771 ◽  
Author(s):  
Rita A. Eckart ◽  
Stephanie Bisle ◽  
Jan Schulze-Luehrmann ◽  
Irene Wittmann ◽  
Jonathan Jantsch ◽  
...  
Keyword(s):  
Host Cell ◽  
Nuclear Localization ◽  
Coxiella Burnetii ◽  
Intracellular Localization ◽  
Mitochondrial Protein ◽  
Effector Proteins ◽  
Content Type ◽  
Type Iv ◽  
Antiapoptotic Activity ◽  
Induced Apoptosis

ABSTRACTIntracellular bacterial pathogens frequently inhibit host cell apoptosis to ensure survival of their host, thereby allowing bacterial propagation. The obligate intracellular pathogenCoxiella burnetiidisplays antiapoptotic activity which depends on a functional type IV secretion system (T4SS). Accordingly, antiapoptotic T4SS effector proteins, like AnkG, have been identified. AnkG inhibits pathogen-induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, the molecular mechanism of AnkG activity remains unknown. Here, we demonstrate that ectopically expressed AnkG associates with mitochondria and traffics into the nucleus after apoptosis induction, although AnkG lacks a predicted nuclear localization signal. We identified the p32 interaction region in AnkG and constructed an AnkG mutant (AnkGR22/23S) unable to bind to p32. By using this mutant, we found that intracellular localization and trafficking of AnkG into the nucleus are dependent on binding to p32. Furthermore, we demonstrated that nuclear localization of AnkG but not binding to p32 is required for apoptosis inhibition. Thus, the antiapoptotic activity of AnkG is controlled by p32-mediated intracellular trafficking, which, in turn, seems to be regulated by host cell processes that sense stress.

scholarly journals Coxiella burnetii Inhibits Activation of Host Cell Apoptosis through a Mechanism That Involves Preventing Cytochrome c Release from Mitochondria

2007 ◽  
Vol 75 (11) ◽  
pp. 5282-5289 ◽  
Author(s):  
Anja Lührmann ◽  
Craig R. Roy
Keyword(s):  
Cell Death ◽  
Host Cell ◽  
Coxiella Burnetii ◽  
Mammalian Cells ◽  
Q Fever ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Obligate Intracellular ◽  
Cell Death Pathway ◽  
Obligate Intracellular Pathogen

ABSTRACT Coxiella burnetii is an obligate intracellular pathogen and the etiological agent of the human disease Q fever. C. burnetii infects mammalian cells and then remodels the membrane-bound compartment in which it resides into a unique lysosome-derived organelle that supports bacterial multiplication. To gain insight into the mechanisms by which C. burnetii is able to multiply intracellularly, we examined the ability of host cells to respond to signals that normally induce apoptosis. Our data show that mammalian cells infected with C. burnetii are resistant to apoptosis induced by staurosporine and UV light. C. burnetii infection prevented caspase 3/7 activation and limited fragmentation of the host cell nucleus in response to agonists that induce apoptosis. Inhibition of bacterial protein synthesis reduced the antiapoptotic effect that C. burnetii exerted on infected host cells. Inhibition of apoptosis in C. burnetii-infected cells did not correlate with the degradation of proapoptotic BH3-only proteins involved in activation of the intrinsic cell death pathway; however, cytochrome c release from mitochondria was diminished in cells infected with C. burnetii upon induction of apoptosis. These data indicate that C. burnetii can interfere with the intrinsic cell death pathway during infection by producing proteins that either directly or indirectly prevent release of cytochrome c from mitochondria. It is likely that inhibition of apoptosis by C. burnetii represents an important virulence property that allows this obligate intracellular pathogen to maintain host cell viability despite inducing stress that would normally activate the intrinsic death pathway.

scholarly journals Evolution and Adaptation of Legionella pneumophila to Manipulate the Ubiquitination Machinery of Its Amoebae and Mammalian Hosts

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 112
Author(s):  
Christopher T.D. Price ◽  
Yousef Abu Kwaik
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Bacterial Pathogen ◽  
Structural Similarity ◽  
Ubiquitin Pathway ◽  
Diverse Range ◽  
E3 Ligases ◽  
Environmental Bacterium ◽  
Natural Hosts

The ubiquitin pathway is highly conserved across the eukaryotic domain of life and plays an essential role in a plethora of cellular processes. It is not surprising that many intracellular bacterial pathogens often target the essential host ubiquitin pathway. The intracellular bacterial pathogen Legionella pneumophila injects into the host cell cytosol multiple classes of classical and novel ubiquitin-modifying enzymes that modulate diverse ubiquitin-related processes in the host cell. Most of these pathogen-injected proteins, designated as effectors, mimic known E3-ubiquitin ligases through harboring F-box or U-box domains. The classical F-box effector, AnkB targets host proteins for K48-linked polyubiquitination, which leads to excessive proteasomal degradation that is required to generate adequate supplies of amino acids for metabolism of the pathogen. In contrast, the SidC and SdcA effectors share no structural similarity to known eukaryotic ligases despite having E3-ubiquitin ligase activity, suggesting that the number of E3-ligases in eukaryotes is under-represented. L. pneumophila also injects into the host many novel ubiquitin-modifying enzymes, which are the SidE family of effectors that catalyze phosphoribosyl-ubiquitination of serine residue of target proteins, independently of the canonical E1-2-3 enzymatic cascade. Interestingly, the environmental bacterium, L. pneumophila, has evolved within a diverse range of amoebal species, which serve as the natural hosts, while accidental transmission through contaminated aerosols can cause pneumonia in humans. Therefore, it is likely that the novel ubiquitin-modifying enzymes of L. pneumophila were acquired by the pathogen through interkingdom gene transfer from the diverse natural amoebal hosts. Furthermore, conservation of the ubiquitin pathway across eukaryotes has enabled these novel ubiquitin-modifying enzymes to function similarly in mammalian cells. Studies on the biological functions of these effectors are likely to reveal further novel ubiquitin biology and shed further lights on the evolution of ubiquitin.

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