Coxiella burnetii encodes an LvgA-related protein important for intracellular replication.

Coxiella burnetii is a bacterial pathogen that replicates in a specialised lysosome-derived organelle called the Coxiella-containing vacuole (CCV). Establishment of the CCV requires the Dot/Icm type IVB secretion system. A previous transposon mutagenesis screen identified the gene cbu1754 as being important for the intracellular replication of C. burnetii. To understand the function of the protein encoded by cbu1754, CCV maturation and intracellular replication phenotypes of a cbu1754 mutant were analysed. In contrast to vacuoles containing wild-type C. burnetii Nine Mile phase II, vacuoles containing the isogenic cbu1754 mutant were smaller and did not display detectible amounts of the autophagy protein LC3, which indicated a CCV biogenesis defect. The Cbu1754 protein was not efficiently delivered into the host cell cytosol during infection, which indicated this protein is not a Dot/Icm-translocated effector protein. Secondary structure predictions suggested that Cbu1754 could be similar to the Legionella pneumophila LvgA protein, which is a component of the Dot/Icm apparatus. Consistent with this hypothesis, production of Cbu1754 in an L. pneumophila ∆lvgA mutant restored LvgA-dependent activities. The L. pneumophila proteins LvgA, IcmS and IcmW are interacting partners that comprise a subassembly of the coupling protein complex that mediates Dot/Icm-dependent effector translocation. Similarly, the Cbu1754 protein was found to be a component of the chaperone complex containing the C. burnetii proteins IcmS and IcmW. Thus, the Cbu1754 protein is an LvgA-related protein important for Dot/Icm function and intracellular replication of C. burnetii.

The Coxiella burnetii effector protein CaeB modulates endoplasmatic reticulum (ER) stress signalling and is required for efficient replication in Galleria mellonella.

The obligate intracellular pathogen Coxiella burnetii is the causative agent of the zoonosis Q fever. C. burnetii infection can have severe outcomes due to the development of chronic infection. To establish and maintain an infection, C. burnetii depends on a functional type IVB secretion system (T4BSS) and, thus, on the translocation of effector proteins into the host cell. Here, we showed that the C. burnetii T4BSS effector protein CaeB targets the conserved endoplasmatic reticulum (ER) stress sensor IRE1 during ER stress in mammalian and plant cells. CaeB-induced upregulation of IRE1 RNase activity was essential for CaeB-mediated inhibition of ER stress-induced cell death. Our data reveal a novel role for CaeB in ER stress signalling modulation and demonstrate that CaeB is involved in pathogenicity in vivo. Furthermore, we provide evidence that C. burnetii infection leads to modulation of the ER stress sensors IRE1 and PERK, but not ATF6 during ER stress. While the upregulation of the RNase activity of IRE1 during ER stress depends on CaeB, modulation of PERK is CaeB independent, suggesting that C. burnetii encodes several factors influencing ER stress during infection.

Anion-π interactions in active centers of superoxide dismutases.

We investigated 1060 possible anion-π interactions in a data set of 41 superoxide dismutase active centers. Our observations indicate that majority of the aromatic residues are capable to form anion-π interactions, mainly by long-range contacts, and that there is preference of Trp over other aromatic residues in these interactions. Furthermore, 68% of total predicted interactions in the dataset are multiple anion-π interactions. Anion-π interactions are distance and orientation dependent. We analyzed the energy contribution resulting from anion-π interactions using ab initio calculations. The results showed that, while most of their interaction energies lay in the range from -0 to -4kcalmol-1, those energies can be up to -9kcalmol-1 and about 34% of interactions were found to be repulsive. Majority of the suggested anion-π interacting residues in ternary complexes are metal-assisted. Stabilization centers for these proteins showed that all the six residues found in predicted anion-π interactions are important in locating one or more of such centers. The anion-π interacting residues in these proteins were found to be highly conserved. We hope that these studies might contribute useful information regarding structural stability and its interaction in future designs of novel metalloproteins.

Crystal structure of Rv1220c, a SAM-dependent O-methyltransferase from Mycobacterium tuberculosis.

Rv1220c from Mycobacterium tuberculosis is annotated as an O-methyltransferase (MtbOMT). Currently, no structural information is available for this protein. Here, the crystal structure of MtbOMT refined to 2.0 Šresolution is described. The structure reveals the presence of a methyltransferase fold and shows clear electron density for one molecule of S-adenosylmethionine (SAM), which was apparently bound by the protein during its production in Escherichia coli. Although the overall structure of MtbOMT resembles the structures of O-methyltransferases from Cornybacterium glutamicum, Coxiella burnetti and Alfa alfa, differences are observed in the residues that make up the active site. Notably, substitution of Asp by His164 seems to abrogate metal binding by MtbOMT. A putative catalytic His-Asp pair located in the vicinity of SAM is absolutely conserved in MtbOMT homologues from all species of Mycobacterium, suggesting a conserved function for this protein.

Identification of Coxiella burnetii CD8+ T-Cell Epitopes and Delivery by Attenuated Listeria monocytogenes as a Vaccine Vector in a C57BL/6 Mouse Model.

Coxiella burnetii is a gram-negative bacterium that causes acute and chronic Q fever. Because of the severe adverse effect of whole-cell vaccination, identification of immunodominant antigens of C. burnetii has become a major focus of Q fever vaccine development. We hypothesized that secreted C. burnetii type IV secretion system (T4SS) effectors may represent a major class of CD8+ T-cell antigens, owing to their cytosolic localization. Twenty-nine peptides were identified that elicited robust CD8+ T-cell interferon γ (IFN-γ) recall responses from mice infected with C. burnetii. Interestingly, 22 of 29 epitopes were derived from 17 T4SS-related proteins, none of which were identified as immunodominant antigens by using previous antibody-guided approaches. These epitopes were expressed in an attenuated Listeria monocytogenes vaccine strain. Immunization with recombinant L. monocytogenes vaccines induced a robust CD8+ T-cell response and conferred measurable protection against C. burnetii infection in mice. These data suggested that T4SS effectors represent an important class of C. burnetii antigens that can induce CD8+ T-cell responses. We also showed that attenuated L. monocytogenes vaccine vectors are an efficient antigen-delivery platform that can be used to induce robust protective CD8+ T-cell immune responses against C. burnetii infection.

Modifications in the glycerophospholipid composition between the Coxiella burnetii phase I and phase II cells suggest an association with phase variation of the bacterium.

Glycerophospholipids (GP) extracted from the Coxiella burnetii strain Nine Mile in virulent phase I (NM I) and low virulent phase II (NM II) were analyzed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) that gave a superior mass resolution and mass accuracy allowing unambiguous peak recognition and precise assignment of ions. We showed that GP present in the pathogen's outer membrane underwent considerable modifications during the phase variation that might be related to impact of various environmental factors. It was found that GP from phase I cells were much more complex than those from phase II cells. While glycerophosphoethanolamines (PE), glycerophosphocholines (PC) and glycerophosphoglycerols (PG) were present in both phases of C. burnetii, major differences were observed in the presence of glycerophosphates (PA) and glycerophosphoserines (PS). Thus, PA but no PS were detected in NM I variant in contrast with NM II cells where PS but no PA were identified. It is suggested that enzymes for PA head group modifications to form PS, PE, and PG become active during the phase variation of the bacterium.

An Unusual Cation-Binding Site and Distinct Domain-Domain Interactions Distinguish Class II Enolpyruvylshikimate-3-phosphate Synthases.

Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes a critical step in the biosynthesis of a number of aromatic metabolites. An essential prokaryotic enzyme and the molecular target of the herbicide glyphosate, EPSPSs are the subject of both pharmaceutical and commercial interest. Two EPSPS classes that exhibit low sequence homology, differing substrate/glyphosate affinities, and distinct cation activation properties have previously been described. Here, we report structural studies of the monovalent cation-binding class II Coxiella burnetii EPSPS (cbEPSPS). Three cbEPSPS crystal structures reveal that the enzyme undergoes substantial conformational changes that alter the electrostatic potential of the active site. A complex with shikimate-3-phosphate, inorganic phosphate (Pi), and K(+) reveals that ligand induced domain closure produces an unusual cation-binding site bordered on three sides by the N-terminal domain, C-terminal domain, and the product Pi. A crystal structure of the class I Vibrio cholerae EPSPS (vcEPSPS) clarifies the basis of differential class I and class II cation responsiveness, showing that in class I EPSPSs a lysine side chain occupies the would-be cation-binding site. Finally, we identify distinct patterns of sequence conservation at the domain-domain interface and propose that the two EPSPS classes have evolved to differently optimize domain opening-closing dynamics.

The inhibition of the apoptosis pathway by the Coxiella burnetii effector protein CaeA requires the EK repetition motif, but is independent of survivin.

ABSRTACT Coxiella burnetii is an obligate intracellular bacterium that causes Query (Q) fever, a zoonotic disease. It requires a functional type IV secretion system (T4SS) which translocate bacterial effector proteins into the host cell cytoplasm and thereby facilitates bacterial replication. To date, more than 130 effector proteins have been identified, but their functions remain largely unknown. Recently, we demonstrated that one of these proteins, CaeA (CBU1524) localized to the host cell nucleus and inhibited intrinsic apoptosis of HEK293 or CHO cells. In the present study we addressed the question whether CaeA also affects the extrinsic apoptosis pathway. Ectopic expression of CaeA reduced extrinsic apoptosis and prevented the cleavage of the executioner caspase 7, but did not impair the activation of initiator caspase 9. CaeA expression resulted in an up-regulation of survivin (an inhibitor of activated caspases), which, however, was not causal for the anti-apoptotic effect of CaeA. Comparing the sequence of CaeA from 25 different C. burnetii isolates we identified an EK (glutamic acid/ lysine) repetition motif as a site of high genetic variability. The EK motif of CaeA was essential for the anti-apoptotic activity of CaeA. From these data, we conclude that the C. burnetii effector protein CaeA interferes with the intrinsic and extrinsic apoptosis pathway. The process requires the EK repetition motif of CaeA, but is independent of the upregulated expression of survivin.

Identification of ElpA, a Coxiella burnetii pathotype-specific Dot/Icm type IV secretion system substrate.

Coxiella burnetii causes 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. burnetii uses 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 100 C. burnetii Dot/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 all C. burnetii isolates 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-localizing protein A). 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 during C. burnetii infection.

Serological characterization of surface-exposed proteins of Coxiella burnetii.

The obligate intracellular Gram-negative bacterium Coxiella burnetii causes Q fever, a worldwide zoonosis. Here we labelled Cox. burnetii with biotin and used biotin-streptavidin affinity chromatography to isolate surface-exposed proteins (SEPs). Using two-dimensional electrophoresis combined with mass spectrometry, we identified 37 proteins through bioinformatics analysis. Thirty SEPs expressed in Escherichia coli (recombinant SEPs, rSEPs) were used to generate microarrays, which were probed with sera from mice experimentally infected with Cox. burnetii or sera from Q fever patients. Thirteen rSEPs were recognized as seroreactive, and the majority reacted with at least 50 % of the sera from mice infected with Cox. burnetii but not with sera from mice infected with Rickettsia rickettsii, R. heilongjiangensis, or R. typhi. Further, 13 proteins that reacted with sera from patients with Q fever did not react with sera from patients with brucellosis or mycoplasma pneumonia. Our results suggest that these seroreactive SEPs have potential as serodiagnostic antigens or as subunit vaccine antigens against Q fever.

Identification of Coxiella burnetii surface-exposed and cell envelope associated proteins using a combined bioinformatics plus proteomics strategy.

The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity. Herein, we propose a complementary strategy consisting of (i) in silico prediction and (ii) inventory of the proteomic composition using three enrichment approaches coupled with protein identification. The efficiency of classical Triton X-114 phase partitioning was compared with two novel procedures; isolation of alkaline proteins by liquid-phase IEF, and cell surface enzymatic shaving using biofunctional magnetic beads. Of the 2026 protein sequences analyzed using seven distinct bioinformatic algorithms, 157 were predicted to be outer membrane proteins (OMP) and/or lipoproteins (LP). Using the three enrichment protocols, we identified 196 nonredundant proteins, including 39 predicted OMP and/or LP, 32 unknown or poorly characterized proteins, and 17 effectors of the Type IV secretion system. We additionally identified eight proteins with moonlighting activities, and several proteins apparently peripherally associated with integral or anchored OMP and/or LP.

Unraveling persistent host cell infection with Coxiella burnetii by quantitative proteomics.

The interaction between the immune system and invading bacteria is sufficient to eradicate microorganisms for the majority of bacterial infections, but suppression of the microbicidal response leads to reactivation or chronic evolution of infections and to bacterial persistence. To identify the cellular pathways affected by bacterial persistence, we applied the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique for a comparative study of protein expression in the C. burnetii strains Nine Mile (NM) and its respective strain (NMper) isolated from 18 months persistently infected cell cultures. In total, three different proteome comparisons were performed with the total bacterial proteome, potentially secreted bacterial proteins, and the eukaryotic infected proteome being assessed. Our results revealed that among the 547 identified bacterial proteins, 53 had significantly altered levels of expression and indicated potential metabolic differences between the two strains. Regarding differences in the secreted proteins between both strains and different modulation of the host cell, machineries reflect at least large rearrangements of both bacterial and eukaryotic proteomes during the persistent model of infection when compared to the acute one, which emphasizes that C. burnetii orchestrates a vast number of different bacterial and eukaryotic host cell processes to persist within its host.

Identification of potentially involved proteins in levofloxacin resistance mechanisms in Coxiella burnetii.

The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a phagosome-like parasitophorous vacuole. Fluoroquinolones have been used as an alternative therapy for Q fever. Resistance to fluoroquinolones can arise via several mechanisms utilized by pathogens to avoid killing. Until today, genome-based studies have shown that the main mechanism of C. burnetii to resist inhibition by fluoroquinolones is based on mutations in quinolone-resistance-determining region (QRDR). In this study, in a broader search at the protein level for C. burnetii mechanisms that confer resistance to fluoroquinolones, the proteomes of in vitro developed fluoroquinolone resistant bacteria and susceptible bacteria were compared using the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique. Quantitative comparison of the 381 proteins identified in both strains indicated the different expression of 15 bacterial proteins. These proteins are involved in different cellular processes indicating that the antibiotic resistance mechanism of the bacterium is a multifaceted process.

Polar localization of the Coxiella burnetii type IVB secretion system.

Coxiella burnetii is a Gram-negative pleomorphic bacterium and the causative agent of Q fever. During infection, the pathogen survives and replicates within a phagosome-like parasitophorous vacuole while influencing cellular functions throughout the host cell, indicating a capacity for effector protein secretion. Analysis of the C. burnetii (RSA 493 strain) genome sequence indicates that C. burnetii contains genes with homology to the Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS). T4BSSs have only been described in L. pneumophila and C. burnetii, marking it a unique virulence determinate. Characterization of bacterial virulence determinants ranging from autotransporter proteins to diverse secretion systems suggests that polar localization may be a virulence mechanism hallmark. To characterize T4BSS subcellular localization in C. burnetii, we analyzed C. burnetii-infected Vero cells by indirect immunofluorescent antibody (IFA) and immunoelectron microscopy (IEM). Using antibodies against the C. burnetii T4BSS homologs IcmT, IcmV, and DotH, IFA show that these proteins are localized to the poles of the bacterium. IEM supports this finding, showing that antibodies against C. burnetii IcmT and DotH preferentially localize to the bacterial cell pole(s). Together, these data demonstrate that the C. burnetii T4BSS localizes to the pole(s) of the bacterium during infection of host cells.

Laboratory maintenance of Coxiella burnetii.

Coxiella burnetii, an obligate intracellular Gram-negative bacterium, is the agent of Q fever, a self-limited flu-like illness that may also present as chronic endocarditis. The ability to persist in the environment at a low infectious dose in aerosols resulted in the classification of C. burnetii as a BSL-3 select agent. Routine propagation of this agent is in embryonated eggs or tissue culture. Purification from host tissues includes multiple differential centrifugations to separate bacteria from host material. Infection of host cells is routinely verified microscopically by using Gimenez stain, fluorescent dyes, or immunofluorescence antibody (IFA) staining. Identification of C. burnetii DNA in host material is measured by PCR. Quantification of purified C. burnetii is accomplished through conversion of optical density to dry weight or, more precisely, by RT-PCR to determine genome equivalents. Serum antibody titer to C. burnetii is determined by microagglutination assay or ELISA.

Coxiella burnetii glycomics and proteomics--tools for linking structure to function.

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacterium and a highly infectious pathogen. The disease is a widespread zoonosis and is endemic throughout the world. An easy aerosol dissemination, environmental persistence, and high infectivity make the bacterium a serious threat for humans and animals. Lipopolysaccharide is considered one of the major factors of virulence expression and infection of the bacterium. Detailed glycomic studies enabled to better understand structural and functional peculiarities of this biopolymer and its role in pathogenesis and immunity of Q fever. Recent proteomic studies of C. burnetii have brought new approaches in accurate detection of the infectious agent and offered new insights into the inter- or intra-species relatedness. Thus, structure/function relationship studies are currently of utmost importance in the field. This paper will focus on glycomic and proteomic approaches providing information on unique glycan and protein species of the microorganism as the candidate molecules for the use in detection/diagnosis, therapy, and prophylaxis.