Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic

Coxiella burnetii is an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascent Coxiella phagosome fuses with the host endocytic pathway to form a large lysosome-like vacuole called the parasitophorous vacuole (PV). The PV membrane is rich in sterols, and drugs perturbing host cell cholesterol homeostasis inhibit PV formation and bacterial growth. Using cholesterol supplementation of a cholesterol-free cell model system, we found smaller PVs and reduced Coxiella growth as cellular cholesterol concentration increased. Further, we observed in cells with cholesterol a significant number of nonfusogenic PVs that contained degraded bacteria, a phenotype not observed in cholesterol-free cells. Cholesterol had no effect on axenic Coxiella cultures, indicating that only intracellular bacteria are sensitive to cholesterol. Live-cell microscopy revealed that both plasma membrane-derived cholesterol and the exogenous cholesterol carrier protein low-density lipoprotein (LDL) traffic to the PV. To test the possibility that increasing PV cholesterol levels affects bacterial survival, infected cells were treated with U18666A, a drug that traps cholesterol in lysosomes and PVs. U18666A treatment led to PVs containing degraded bacteria and a significant loss in bacterial viability. The PV pH was significantly more acidic in cells with cholesterol or cells treated with U18666A, and the vacuolar ATPase inhibitor bafilomycin blocked cholesterol-induced PV acidification and bacterial death. Additionally, treatment of infected HeLa cells with several FDA-approved cholesterol-altering drugs led to a loss of bacterial viability, a phenotype also rescued by bafilomycin. Collectively, these data suggest that increasing PV cholesterol further acidifies the PV, leading to Coxiella death.IMPORTANCE The intracellular Gram-negative bacterium Coxiella burnetii is a significant cause of culture-negative infectious endocarditis, which can be fatal if untreated. The existing treatment strategy requires prolonged antibiotic treatment, with a 10-year mortality rate of 19% in treated patients. Therefore, new clinical therapies are needed and can be achieved by better understanding C. burnetii pathogenesis. Upon infection of host cells, C. burnetii grows within a specialized replication niche, the parasitophorous vacuole (PV). Recent data have linked cholesterol to intracellular C. burnetii growth and PV formation, leading us to further decipher the role of cholesterol during C. burnetii-host interaction. We observed that increasing PV cholesterol concentration leads to increased acidification of the PV and bacterial death. Further, treatment with FDA-approved drugs that alter host cholesterol homeostasis also killed C. burnetii through PV acidification. Our findings suggest that targeting host cholesterol metabolism might prove clinically efficacious in controlling C. burnetii infection

Antibody-mediated immunity to the obligate intracellular bacterial pathogen Coxiella burnetii is Fc receptor- and complement-independent

<p>Abstract</p> <p>Background</p> <p>The obligate intracellular bacterial pathogen <it>Coxiella burnetii </it>causes the zoonosis Q fever. The intracellular niche of <it>C. burnetii </it>has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect naïve animals from challenge with virulent <it>C. burnetii</it>, indicating a role for antibody (Ab) in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR) or complement contribute to Ab-mediated immunity (AMI) to <it>C. burnetii</it>.</p> <p>Results</p> <p>Virulent <it>C. burnetii </it>infects and replicates within human dendritic cells (DC) without inducing their maturation or activation. We investigated the effects of Ab opsonized <it>C. burnetii </it>on human monocyte-derived and murine bone marrow-derived DC. Infection of DC with Ab-opsonized <it>C. burnetii </it>resulted in increased expression of maturation markers and inflammatory cytokine production. Bacteria that had been incubated with naïve serum had minimal effect on DC, similar to virulent <it>C. burnetii </it>alone. The effect of Ab opsonized <it>C. burnetii </it>on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o) compared to C57Bl/6 (B6) mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to <it>C. burnetii </it>in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to <it>C. burnetii </it>is also complement-independent.</p> <p>Conclusion</p> <p>Despite our data showing FcR-dependent stimulation of DC in vitro, Ab-mediated immunity to <it>C. burnetii </it>in vivo is FcR-independent. We also found that passive immunity to this pathogen is independent of complement.</p

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

Identification of Anaplasma marginale Type IV Secretion System Effector Proteins

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.Published copyLockwood, S., D. E. Voth, K. A. Brayton, P. A. Beare, W. C. Brown, R. A. Heinzen, and S. L. Broschat, Identification of Anaplasma marginale type IV secretion system effector proteins, PLoS ONE, Vol. 6, No. 11, e7724, Nov. 2011. DOI: 10.1371/journal.pone.0027724

Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

Scrub typhus is an understudied, potentially fatal infection that threatens one billion persons in the Asia-Pacific region. How the causative obligate intracellular bacterium, Orientia tsutsugamushi, facilitates its intracellular survival and pathogenesis is poorly understood. Many intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) that traffic to distinct subcellular locations and modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank repertoires plus T1SS and T4SS components. Whether these potential virulence factors are expressed during infection, how the Anks are potentially secreted, and to where they localize in the host cell are not known. We determined that O. tsutsugamushi transcriptionally expresses 20 unique ank genes as well as genes for both T1SS and T4SS during infection of mammalian host cells. Examination of the Anks’ C-termini revealed that the majority of them resemble T1SS substrates. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks in an HlyBD-dependent manner. Thus, O. tsutsugamushi Anks C-termini are T1SS-compatible. Conversely, Coxiella burnetii could not secrete heterologously expressed Anks in a T4SS-dependent manner. Analysis of the subcellular distribution patterns of 20 ectopically expressed Anks revealed that, while 6 remained cytosolic or trafficked to the nucleus, 14 localized to, and in some cases, altered the morphology of the endoplasmic reticulum. This study identifies O. tsutsugamushi Anks as T1SS substrates and indicates that many display a tropism for the host cell secretory pathway

Rapid typing of Coxiella burnetii

Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels) among multispacer sequence typing (MST) loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies

Quantum correlated twin atomic beams via photo-dissociation of a molecular Bose-Einstein condensate

We study the process of photo-dissociation of a molecular Bose-Einstein condensate as a potential source of strongly correlated twin atomic beams. We show that the two beams can possess nearly perfect quantum squeezing in their relative numbers.Comment: Corrected LaTeX file layou

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

CPT and Lorentz Tests in Penning Traps

A theoretical analysis is performed of Penning-trap experiments testing CPT and Lorentz symmetry through measurements of anomalous magnetic moments and charge-to-mass ratios. Possible CPT and Lorentz violations arising from spontaneous symmetry breaking at a fundamental level are treated in the context of a general extension of the SU(3) x SU(2) x U(1) standard model and its restriction to quantum electrodynamics. We describe signals that might appear in principle, introduce suitable figures of merit, and estimate CPT and Lorentz bounds attainable in present and future Penning-trap experiments. Experiments measuring anomaly frequencies are found to provide the sharpest tests of CPT symmetry. Bounds are attainable of approximately $10^{-20}$ in the electron-positron case and of $10^{-23}$ for a suggested experiment with protons and antiprotons. Searches for diurnal frequency variations in these experiments could also limit certain types of Lorentz violation to the level of $10^{-18}$ in the electron-positron system and others at the level of $10^{-21}$ in the proton-antiproton system. In contrast, measurements comparing cyclotron frequencies are sensitive within the present theoretical framework to different kinds of Lorentz violation that preserve CPT. Constraints could be obtained on one figure of merit in the electron-positron system at the level of $10^{-16}$, on another in the proton-antiproton system at $10^{-24}$, and on a third at $10^{-25}$ using comparisons of $H^-$ ions with antiprotons.Comment: 31 pages, published in Physical Review

Proteome and Antigen Profiling of Coxiella burnetii Developmental Forms

A biphasic developmental cycle whereby highly resistant small-cell variants (SCVs) are generated from large-cell variants (LCVs) is considered fundamental to the virulence of Coxiella burnetii, the causative agent of human Q fever. In this study a proteome analysis of C. burnetii developmental forms was conducted to provide insight into their unique biological and immunological properties. Silver-stained gels of SCV and LCV lysates separated by two-dimensional (2-D) gel electrophoresis resolved over 675 proteins in both developmental forms. Forty-eight proteins were greater than twofold more abundant in LCVs than in SCVs, with six proteins greater than twofold more abundant in SCVs than in LCVs. Four and 15 upregulated proteins of SCVs and LCVs, respectively, were identified by mass spectrometry, and their predicted functional roles are consistent with a metabolically active LCV and a structurally resistant SCV. One-dimensional and 2-D immunoblots of cell form lysates probed with sera from infected/vaccinated guinea pigs and convalescent-phase serum from human patients who had recovered from acute Q fever, respectively, revealed both unique SCV/LCV antigens and common SCV/LCV antigens that were often differentially synthesized. Antigens recognized during human infection were identified by mass spectroscopy and included both previously described immunodominant proteins of C. burnetii and novel immunogenic proteins that may be important in the pathophysiology of clinical Q fever and/or the induction of protective immunity