A Survey of Two-Component Systems in Coxiella burnetii Reveals Redundant Regulatory Schemes and a Requirement for an Atypical PhoBR System in Mammalian Cell Infection.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever in humans. C. burnetii transitions between a replicative, metabolically active large-cell variant (LCV) and a spore-like, quiescent small-cell variant (SCV) as a likely mechanism to ensure survival between host cells and mammalian hosts. C. burnetii encodes three canonical two-component systems, four orphan hybrid histidine kinases, five orphan response regulators, and a histidine phosphotransfer protein, which have been speculated to play roles in the signaling required for C. burnetii morphogenesis and virulence. However, very few of these systems have been characterized. By employing a CRISPR interference system for genetic manipulation of C. burnetii, we created single- and multigene transcriptional knockdown strains targeting most of these signaling genes. Through this, we revealed a role for the C. burnetii PhoBR canonical two-component system in virulence, regulation of [Pi] maintenance, and Pi transport. We also outline a novel mechanism by which PhoBR function may be regulated by an atypical PhoU-like protein. We also determined that the GacA.2/GacA.3/GacA.4/GacS orphan response regulators coordinately and disparately regulate expression of SCV-associated genes in C. burnetii LCVs. These foundational results will inform future studies on the role of C. burnetii two-component systems in virulence and morphogenesis. IMPORTANCE C. burnetii is an obligate intracellular bacterium with a spore-like stability allowing it to survive long periods of time in the environment. This stability is likely due to its biphasic developmental cycle, whereby it can transition from an environmentally stable small-cell variant (SCV) to a metabolically active large-cell variant (LCV). Here, we define the role of two-component phosphorelay systems (TCS) in C. burnetii's ability to survive within the harsh environment contained in the phagolysosome of host cells. We show that the canonical PhoBR TCS has an important role in C. burnetii virulence and phosphate sensing. Further examination of the regulons controlled by orphan regulators indicated a role in modulating gene expression of SCV-associated genes, including genes essential for cell wall remodeling.

The endogenous Coxiella burnetii plasmid encodes a functional toxin-antitoxin system.

Coxiella burnetii is the causative agent of Q fever. All C. burnetii isolates encode either an autonomously replicating plasmid (QpH1, QpDG, QpRS, or QpDV) or QpRS-like chromosomally integrated plasmid sequences. The role of the ORFs present in these sequences is unknown. Here, the role of the ORFs encoded on QpH1 was investigated. Using a new C. burnetii shuttle vector (pB-TyrB-QpH1ori), we cured the C. burnetii Nine Mile Phase II strain of QpH1. The ΔQpH1 strain grew normally in axenic media but had a significant growth defect in Vero cells, indicating QpH1 was important for C. burnetii virulence. We developed an inducible CRISPR interference system to examine the role of individual QpH1 plasmid genes. CRISPRi of cbuA0027 resulted in significant growth defects in axenic media and THP-1 cells. The cbuA0028/cbuA0027 operon encodes CBUA0028 (ToxP) and CBUA0027 (AntitoxP), which are homologous to the HigB2 toxin and HigA2 antitoxin, respectively, from Vibrio cholerae. Consistent with toxin-antitoxin systems, overexpression of toxP resulted in a severe intracellular growth defect that was rescued by co-expression of antitoxP. ToxP inhibited protein translation. AntitoxP bound the toxP promoter (PtoxP) and ToxP, with the resulting complex binding also PtoxP. In summary, our data indicate that C. burnetii maintains an autonomously replicating plasmid because of a plasmid-based toxin-antitoxin system.

Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.

Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV) and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1) and cationic-independent mannose-6-phosphate receptor (CI-M6PR), respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the endosomal system is required for CCV generation. These findings delineate which of the many actin related events that shape the endosomal compartment are important for CCV formation.

Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation.

Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.

Dependency of Coxiella burnetii Type 4B Secretion on the Chaperone IcmS.

Macrophage parasitism by Coxiella burnetii, the cause of human Q fever, requires the translocation of proteins with effector functions directly into the host cell cytosol via a Dot/Icm type 4B secretion system (T4BSS). Secretion by the analogous Legionella pneumophila T4BSS involves signal sequences within the C-terminal and internal domains of effector proteins. The cytoplasmic chaperone pair IcmSW promotes secretion and binds internal sites distinct from signal sequences. In the present study, we investigated requirements of C. burnetii IcmS for host cell parasitism and effector translocation. A C. burnetiiicmS deletion mutant (ΔicmS) exhibited impaired replication in Vero epithelial cells, deficient formation of the Coxiella-containing vacuole, and aberrant T4BSS secretion. Three secretion phenotypes were identified from a screen of 50 Dot/Icm substrates: IcmS dependent (secreted by only wild-type bacteria), IcmS independent (secreted by both wild-type and ΔicmS bacteria), or IcmS inhibited (secreted by only ΔicmS bacteria). Secretion was assessed for N-terminal or C-terminal truncated forms of CBU0794 and CBU1525. IcmS-inhibited secretion of CBU1525 required a C-terminal secretion signal whereas IcmS-dependent secretion of CBU0794 was directed by C-terminal and internal signals. Interchange of the C-terminal 50 amino acids of CBU0794 and CBU1525 revealed that sites within the C terminus regulate IcmS dependency. Glutathione S-transferase-tagged IcmSW bound internal sequences of IcmS-dependent and -inhibited substrates. Thus, the growth defect of the C. burnetii ΔicmS strain is associated with a loss of T4BSS chaperone activity that both positively and negatively regulates effector translocation.IMPORTANCE The intracellular pathogen Coxiella burnetii employs a type 4B secretion system (T4BSS) that promotes growth by translocating effectors of eukaryotic pathways into host cells. T4BSS regulation modeled in Legionella pneumophila indicates IcmS facilitates effector translocation. Here, we characterized type 4B secretion by a Coxiella ΔicmS mutant that exhibits intracellular growth defects. T4BSS substrates demonstrated increased, equivalent, or decreased secretion by the ΔicmS mutant relative to wild-type Coxiella Similar to the Legionella T4BSS, IcmS dependency in Coxiella was determined by C-terminal and/or internal secretion signals. However, IcmS inhibited secretion of some effectors by Coxiella that were previously shown to be translocated by Legionella Thus, Coxiella has a unique IcmS regulatory mechanism that both positively and negatively regulates T4BSS export.

Coxiella burnetii RpoS Regulates Genes Involved in Morphological Differentiation and Intracellular Growth.

Coxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponential-phase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, including Legionella pneumophila, which has a developmental cycle superficially similar to that of C. burnetii Here, we used a C. burnetii ΔrpoS mutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with the rpoS mutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of the C. burnetii ΔrpoS mutant revealed that a substantial portion of the C. burnetii genome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCV-specific protein ScvA. Genes comprising the dot/icm locus, responsible for production of the Dot/Icm type 4B secretion system, were also dysregulated in the rpoS mutant. These data were corroborated with independent assays demonstrating that the C. burnetii ΔrpoS strain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved in C. burnetii SCV development and intracellular growth.IMPORTANCE The Q fever bacterium Coxiella burnetii has spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in the rpoS mutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of the Coxiella developmental cycle and identify developmentally regulated genes involved in morphological differentiation.

Replication of Coxiella burnetii in a Lysosome-Like Vacuole Does Not Require Lysosomal Hydrolases.

Coxiella burnetii is an intracellular bacterium that causes query, or Q fever, a disease that typically manifests as a severe flu-like illness. The initial target of C. burnetii is the alveolar macrophage. Here, it regulates vesicle trafficking pathways and fusion events to establish a large replication vacuole called the Coxiella-containing vacuole (CCV). Similar to a phagolysosome, the CCV has an acidic pH and contains lysosomal hydrolases obtained via fusion with late endocytic vesicles. Lysosomal hydrolases break down various lipids, carbohydrates, and proteins; thus, it is assumed C. burnetii derives nutrients for growth from these degradation products. To investigate this possibility, we utilized a GNPTAB-/- HeLa cell line that lacks lysosomal hydrolases in endocytic compartments. Unexpectedly, examination of C. burnetii growth in GNPTAB-/- HeLa cells revealed replication and viability are not impaired, indicating C. burnetii does not require by-products of hydrolase degradation to survive and grow in the CCV. However, although bacterial growth was normal, CCVs were abnormal, appearing dark and condensed rather than clear and spacious. Lack of degradation within CCVs allowed waste products to accumulate, including intraluminal vesicles, autophagy protein LC3, and cholesterol. The build-up of waste products coincided with an altered CCV membrane, where LAMP1 was decreased and CD63 and LAMP1 redistributed from a punctate to uniform localization. This disruption of CCV membrane organization may account for the decreased CCV size due to impaired fusion with late endocytic vesicles. Collectively, these results demonstrate lysosomal hydrolases are not required for C. burnetii survival and growth but are needed for normal CCV development. These data provide insight into mechanisms of CCV biogenesis while raising the important question of how C. burnetii obtains essential nutrients from its host.

Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages.

Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.

A Coxiella burnetii phospholipase A homolog pldA is required for optimal growth in macrophages and developmental form lipid remodeling.

BACKGROUND: Many gram-negative bacteria produce an outer membrane phospholipase A (PldA) that plays an important role in outer membrane function and is associated with virulence. RESULTS: In the current study, we characterized a pldA mutant of Coxiella burnetii, an intracellular gram-negative pathogen and the agent of human Q fever. The C. burnetti pldA open reading frame directs synthesis of a protein with conserved PldA active site residues. A C. burnetii ΔpldA deletion mutant had a significant growth defect in THP-1 macrophages, but not axenic medium, that was rescued by complementation. Thin layer chromatography was employed to assess whether pldA plays a role in remodeling membrane lipids during C. burnetii morphological differentiation. Extracted lipids were analyzed from replicating, logarithmic phase large cell variants (LCVs), non-replicating, stationary phase small cell variants (SCVs), and a mixture of LCVs and SCVs. Similar to Escherichia coli, all three forms contained cardiolipin (CL), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). However, PE and PG were present in lower quantities in the SCV while three additional lipid species were present in higher quantities. Co-migration with standards tentatively identified two of the three SCV-enriched lipids as lyso-phosphatidylethanolamine, a breakdown product of PE, and free fatty acids, which are generally toxic to bacteria. Developmental form lipid modifications required the activity of PldA. CONCLUSIONS: Collectively, these results indicate developmentally-regulated lipid synthesis by C. burnetii contributes to colonization of macrophages and may contribute to the environmental stability and the distinct biological properties of the SCV.

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L.

Coxiella burnetii is a gram-negative intracellular bacterium that forms a large, lysosome-like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol-binding protein-related protein 1 long (ORP1L) is a mammalian lipid-binding protein that plays a dual role in cholesterol-dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N-terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co-localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L-depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.

Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium.

UNLABELLED: Host cell-free (axenic) culture of Coxiella burnetii in acidified citrate cysteine medium-2 (ACCM-2) has provided important opportunities for investigating the biology of this naturally obligate intracellular pathogen and enabled the development of tools for genetic manipulation. However, ACCM-2 has complex nutrient sources that preclude a detailed study of nutritional factors required for C. burnetii growth. Metabolic reconstruction of C. burnetii predicts that the bacterium cannot synthesize all amino acids and therefore must sequester some from the host. To examine C. burnetii amino acid auxotrophies, we developed a nutritionally defined medium with known amino acid concentrations, termed ACCM-D. Compared to ACCM-2, ACCM-D supported longer logarithmic growth, a more gradual transition to stationary phase, and approximately 5- to 10-fold greater overall replication. Small-cell-variant morphological forms generated in ACCM-D also showed increased viability relative to that generated in ACCM-2. Lack of growth in amino acid-deficient formulations of ACCM-D revealed C. burnetii auxotrophy for 11 amino acids, including arginine. Heterologous expression of Legionella pneumophila argGH in C. burnetii permitted growth in ACCM-D missing arginine and supplemented with citrulline, thereby providing a nonantibiotic means of selection of C. burnetii genetic transformants. Consistent with bioinformatic predictions, the elimination of glucose did not impair C. burnetii replication. Together, these results highlight the advantages of a nutritionally defined medium in investigations of C. burnetii metabolism and the development of genetic tools. IMPORTANCE: Host cell-free growth and genetic manipulation of Coxiella burnetii have revolutionized research of this intracellular bacterial pathogen. Nonetheless, undefined components of growth medium have made studies of C. burnetii physiology difficult and have precluded the development of selectable markers for genetic transformation based on nutritional deficiencies. Here, we describe a medium, containing only amino acids as the sole source of carbon and energy, which supports robust growth and improved viability of C. burnetii Growth studies confirmed that C. burnetii cannot replicate in medium lacking arginine. However, genetic transformation of the bacterium with constructs containing the last two genes in the L. pneumophila arginine biosynthesis pathway (argGH) allowed growth on defined medium missing arginine but supplemented with the arginine precursor citrulline. Our results advance the field by facilitating studies of C. burnetii metabolism and allowing non-antibiotic-based selection of C. burnetii genetic transformants, an important achievement considering that selectable makers based on antibiotic resistance are limited.

Life on the outside: the rescue of Coxiella burnetii from its host cell.

For over seven decades, Coxiella burnetii, the causative agent of human Q fever, has been considered a prototypical obligate intracellular bacterium that relies exclusively on a eukaryotic cell for growth. Intracellularly, the organism prospers in an acidified, phagolysosome-like vacuole. C. burnetii has evolved to replicate in this harsh compartment by a mechanism involving acid activation of metabolism. The ?2 Mb genome of C. burnetii is about twice the size of genomes of most obligate intracellular bacteria, and the organism's central metabolic pathways are largely intact. The absence of extensive genome reduction suggests the adaptation of C. burnetii to an obligate intracellular lifestyle is a recent evolutionary event. Indeed, insight from early work on C. burnetii metabolism, along with new information gained from metabolic pathway reconstructions, nutrient typing, and expression profiling, allowed the rescue of C. burnetii from its host cell to regain the axenic growth capacity of its ancestors. This advance removes the extensive experimental obstacles associated with intracellular obligatism and opens the door for a renaissance in C. burnetii research.

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis.

Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL[LI] and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ΔcvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms.

Coxiella burnetii effector proteins that localize to the parasitophorous vacuole membrane promote intracellular replication.

The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication 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 promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS 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 by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpD and ΔcvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ΔcvpB and ΔcvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.

Bacterial colonization of host cells in the absence of cholesterol.

Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/-) mouse embryonic fibroblasts (MEFs). DHCR24(-/-) MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/-) MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/-) MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(V)ß(3) integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/-) MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

Essential role for the response regulator PmrA in Coxiella burnetii type 4B secretion and colonization of mammalian host cells.

Successful host cell colonization by the Q fever pathogen, Coxiella burnetii, requires translocation of effector proteins into the host cytosol by a Dot/Icm type 4B secretion system (T4BSS). In Legionella pneumophila, the two-component system (TCS) PmrAB regulates the Dot/Icm T4BSS and several additional physiological processes associated with pathogenesis. Because PmrA consensus regulatory elements are associated with some dot/icm and substrate genes, a similar role for PmrA in regulation of the C. burnetii T4BSS has been proposed. Here, we constructed a C. burnetii pmrA deletion mutant to directly probe PmrA-mediated gene regulation. Compared to wild-type bacteria, C. burnetii ΔpmrA exhibited severe intracellular growth defects that coincided with failed secretion of effector proteins. Luciferase gene reporter assays demonstrated PmrA-dependent expression of 5 of 7 dot/icm operons and 9 of 11 effector-encoding genes with a predicted upstream PmrA regulatory element. Mutational analysis verified consensus sequence nucleotides required for PmrA-directed transcription. RNA sequencing and whole bacterial cell mass spectrometry of wild-type C. burnetii and the ΔpmrA mutant uncovered new components of the PmrA regulon, including several genes lacking PmrA motifs that encoded Dot/Icm substrates. Collectively, our results indicate that the PmrAB TCS is a critical virulence factor that regulates C. burnetii Dot/Icm secretion. The presence of PmrA-responsive genes lacking PmrA regulatory elements also suggests that the PmrAB TCS controls expression of regulatory systems associated with the production of additional C. burnetii proteins involved in host cell parasitism.

Natural reversion promotes LPS elongation in an attenuated Coxiella burnetii strain.

Lipopolysaccharide (LPS) phase variation is a critical aspect of virulence in many Gram-negative bacteria. It is of particular importance to Coxiella burnetii, the biothreat pathogen that causes Q fever, as in vitro propagation of this organism leads to LPS truncation, which is associated with an attenuated and exempted from select agent status (Nine Mile II, NMII). Here, we demonstrate that NMII was recovered from the spleens of infected guinea pigs. Moreover, these strains exhibit a previously unrecognized form of elongated LPS and display increased virulence in comparison with the initial NMII strain. The reversion of a 3-bp mutation in the gene cbu0533 directly leads to LPS elongation. To address potential safety concerns, we introduce a modified NMII strain unable to produce elongated LPS.

Sec-mediated secretion by Coxiella burnetii.

BACKGROUND: Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication. RESULTS: To assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase. CONCLUSIONS: C. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed.

Identification of Type 4B Secretion System Substrates That Are Conserved among Coxiella burnetii Genomes and Promote Intracellular Growth.

Coxiella burnetii is a Gram-negative pathogen that infects a variety of mammalian hosts. Infection of domesticated ewes can cause fetal abortion, whereas acute human infection normally manifests as the flu-like illness Q fever. Successful host infection requires replication of the pathogen within the lysosomal Coxiella-containing vacuole (CCV). The bacterium encodes a type 4B secretion system (T4BSS) that delivers effector proteins into the host cell. Disruption of C. burnetii T4BSS effector export abrogates CCV biogenesis and bacterial replication. Over 150 C. burnetii T4BSS substrates have been designated often based on heterologous protein translocation by the Legionella pneumophila T4BSS. Cross-genome comparisons predict that many of these T4BSS substrates are truncated or absent in the acute-disease reference strain C. burnetii Nine Mile. This study investigated the function of 32 proteins conserved among diverse C. burnetii genomes that are reported to be T4BSS substrates. Despite being previously designated T4BSS substrates, many of the proteins were not translocated by C. burnetii when expressed fused to the CyaA or BlaM reporter tags. CRISPR interference (CRISPRi) indicated that of the validated C. burnetii T4BSS substrates, CBU0122, CBU1752, CBU1825, and CBU2007 promote C. burnetii replication in THP-1 cells and CCV biogenesis in Vero cells. When expressed in HeLa cells tagged at its C or N terminus with mCherry, CBU0122 localized to the CCV membrane and the mitochondria, respectively. Collectively, these data further define the repertoire of bona fide C. burnetii T4BSS substrates. IMPORTANCE Coxiella burnetii secretes effector proteins via a T4BSS that are required for successful infection. Over 150 C. burnetii proteins are reported to be T4BSS substrates and often by default considered putative effectors, but few have assigned functions. Many C. burnetii proteins were designated T4BSS substrates using heterologous secretion assays in L. pneumophila and/or have coding sequences that are absent or pseudogenized in clinically relevant C. burnetii strains. This study examined 32 previously reported T4BSS substrates that are conserved among C. burnetii genomes. Of the proteins tested that were previously designated T4BSS substrates using L. pneumophila, most were not exported by C. burnetii. Several T4BSS substrates that were validated in C. burnetii also promoted pathogen intracellular replication and one trafficked to late endosomes and the mitochondria in a manner suggestive of effector activity. This study identified several bona fide C. burnetii T4BSS substrates and further refined the methodological criteria for their designation.

A simple method for enrichment of phase I Coxiella burnetii.

Coxiella burnetii is the bacterial causative agent of the zoonosis Q fever. This bacterium undergoes lipopolysaccharide (LPS) phase transition similar to Enterobacteriaciae upon in vitro passage. Full-length, phase I C. burnetii LPS is a critical virulence factor and profoundly impacts vaccine-induced immunogenicity; thus, LPS phase is an important consideration in C. burnetii experimentation and Q fever vaccine design. Typically, phase I LPS-expressing organisms are obtained from the tissues of infected experimental animals. In this process, residual phase II LPS-expressing organisms are thought to be cleared by the host immune system. Here, we propose an efficient and non-animal-based method for the enrichment of C. burnetii phase I LPS-expressing bacteria in vitro. We utilize both Vero cell culture to selectively enrich solutions with phase I and intermediate phase LPS-expressing bacteria. This simple and quick method decreases reliance on experimental animals and is a sustainable solution for Q fever diagnostic and vaccine development hurdles.