Structural and biochemical analysis of the unique interactions of the Campylobacter jejuni CorA channel protein with divalent cations.

CorA is a Mg2+ channel that plays a key role in the homeostasis of intracellular Mg2+ in bacteria and archaea. CorA consists of a cytoplasmic domain and a transmembrane domain and generates a Mg2+ pathway by forming a pentamer in the cell membrane. CorA gating is regulated via negative feedback by Mg2+, which is accommodated by the pentamerization interface of the CorA cytoplasmic domain (CorACD). The Mg2+-binding sites of CorACD differ depending on the species, suggesting that the Mg2+-binding modes and Mg2+-mediated gating mechanisms of CorA vary across prokaryotes. To define the Mg2+-binding mechanism of CorA in the Campylobacter jejuni pathogen, we structurally and biochemically characterized C. jejuni CorACD (cjCorACD). cjCorACD adopts a three-layered α/ß/α structure as observed in other CorA orthologs. Interestingly, cjCorACD exhibited enhanced thermostability in the presence of Ca2+, Ni2+, Zn2+, or Mn2+ in addition to Mg2+, indicating that cjCorACD interacts with diverse divalent cations. This cjCorACD stabilization is mediated by divalent cation accommodation by negatively charged residues located at the bottom of the cjCorACD structure away from the pentamerization interface. Consistently, cjCorACD exists as a monomer irrespective of the presence of divalent cations. We concluded that cjCorACD binds divalent cations in a unique pentamerization-independent manner.

Proteome profiling of Campylobacter jejuni 81-176 at 37 °C and 42 °C by label-free mass spectrometry.

BACKGROUND: The main natural reservoir for Campylobacter jejuni is the avian intestinal tract. There, C. jejuni multiplies optimally at 42 °C - the avian body temperature. After infecting humans through oral intake, the bacterium encounters the lower temperature of 37 °C in the human intestinal tract. Proteome profiling by label-free mass spectrometry (DIA-MS) was performed to examine the processes which enable C. jejuni 81-176 to thrive at 37 °C in comparison to 42 °C. In total, four states were compared with each other: incubation for 12 h at 37 °C, for 24 h at 37 °C, for 12 h at 42 °C and 24 h at 42 °C. RESULTS: It was shown that the proteomic changes not only according to the different incubation temperature but also to the length of the incubation period were evident when comparing 37 °C and 42 °C as well as 12 h and 24 h of incubation. Altogether, the expression of 957 proteins was quantifiable. 37.1 - 47.3% of the proteins analyzed showed significant differential regulation, with at least a 1.5-fold change in either direction (i.e. log2 FC ≥ 0.585 or log2 FC ≤ -0.585) and an FDR-adjusted p-value of less than 0.05. The significantly differentially expressed proteins could be arranged in 4 different clusters and 16 functional categories. CONCLUSIONS: The C. jejuni proteome at 42 °C is better adapted to high replication rates than that at 37 °C, which was in particular indicated by the up-regulation of proteins belonging to the functional categories "replication" (e.g. Obg, ParABS, and NapL), "DNA synthesis and repair factors" (e.g. DNA-polymerase III, DnaB, and DnaE), "lipid and carbohydrate biosynthesis" (e.g. capsular biosynthesis sugar kinase, PrsA, AccA, and AccP) and "vitamin synthesis, metabolism, cofactor biosynthesis" (e.g. MobB, BioA, and ThiE). The relative up-regulation of proteins with chaperone function (GroL, DnaK, ClpB, HslU, GroS, DnaJ, DnaJ-1, and NapD) at 37 °C in comparison to 42 °C after 12 h incubation indicates a temporary lower-temperature proteomic response. Additionally the up-regulation of factors for DNA uptake (ComEA and RecA) at 37 °C compared to 42 °C indicate a higher competence for the acquisition of extraneous DNA at human body temperature.

Synthesis of 6-deoxy-d-ido-heptopyranose-containing fragments of the Campylobacter jejuni strain CG8486 capsular polysaccharide.

Campylobacters are important causes of gastrointestinal illness and the capsular polysaccharides (CPS) they produce are key virulence factors and targets for vaccine development. We report here the synthesis of two fragments of the Campylobacter jejuni CG8486 strain CPS that contain a rare 6-deoxy-d-ido-heptopyranose residue and, in one target, two O-methyl phosphoramidate (MeOPN) motifs. The synthetic approach features the stereoselective construction of the ß-d-ido-heptopyranoside linkage via glycosylation with a ß-d-galacto-heptopyranoside donor followed by a one-pot sequential C-2 and C-3 inversion. During the syntheses, we uncovered a number of interesting conformational effects with regard to the 6-deoxy-ido-heptopyranose ring, the glycosidic linkage connecting the two monosaccharides, and the MeOPN groups.

Regio- and stereo-controlled synthesis of 6-deoxy-ß-D-ido-heptopyranosides related to Campylobacter jejuni HS:4.

Campylobacter jejuni is a bacterial pathogen that causes hundreds of millions of cases of food-borne gastroenteritis worldwide annually. The infection caused by this bacterium is also associated with several forms of post-infectious autoimmune sequelae that can be very serious, including the life-threatening Guillain-Barré syndrome. The capsular polysaccharides (CPS) of C. jejuni HS:4 consist of a very unique repeating disaccharide unit that is characterized by a ß-1,4-linked 6-deoxy-ß-D-ido-heptopyranose and an N-acetyl-ß-D-glucosamine. Eliciting carbohydrate-specific antibodies against the CPS structures of C. jejuni HS:4 is an attractive strategy. The 6-deoxy-ido-configuration of the heptose combined with its ß-anomeric configuration makes the chemical synthesis of the disaccharide very challenging. Here, we report an efficient synthesis to obtain the key repeating disaccharide and its analog in reverse order plus a trisaccharide. Our synthesis features a highly efficient, one-step stereo- and regioselective conversion of ß-D-galacto-heptopyranosides to 6-deoxy-ß-D-ido-heptopyranosides via the intermediate 2,3-anhydro-ß-D-talo-heptopyranosides.

Identification of pH-specific protein expression responses by Campylobacter jejuni strain NCTC 11168.

In this study a data dependent acquisition label-free based proteomics approach was used to identify pH-dependent proteins that respond in a growth phase independent manner in Campylobacter jejuni reference strain NCTC 11168. NCTC 11168 was grown within its pH physiological normal growth range (pH 5.8, 7.0 and 8.0, µ = âˆ¼0.5 h-1) and exposed to pH 4.0 shock for 2 h. It was discovered that gluconate 2-dehydrogenase GdhAB, NssR-regulated globins Cgb and Ctb, cupin domain protein Cj0761, cytochrome c protein CccC (Cj0037c), and phosphate-binding transporter protein PstB all show acidic pH dependent abundance increases but are not activated by sub-lethal acid shock. Glutamate synthase (GLtBD) and the MfrABC and NapAGL respiratory complexes were induced in cells grown at pH 8.0. The response to pH stress by C. jejuni is to bolster microaerobic respiration and at pH 8.0 this is assisted by accumulation of glutamate the conversion of which could bolster fumarate respiration. The pH dependent proteins linked to growth in C. jejuni NCTC 11168 aids cellular energy conservation maximising growth rate and thus competitiveness and fitness.

Metabolic Labeling of Legionaminic Acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase.

Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC-MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness.

Synthesis of Rare 6-Deoxy-d-/l-Heptopyranosyl Fluorides: Assembly of a Hexasaccharide Corresponding to Campylobacter jejuni Strain CG8486 Capsular Polysaccharide.

Campylobacter jejuni is the leading cause of human diarrheal diseases and has been designated as one of highly resistant pathogens by the World Health Organization. The C. jejuni capsular polysaccharides feature broad existence of uncommon 6dHepp residues and have proven to be potential antigens to develop innovative antibacterial glycoconjugation vaccines. To address the lack of synthetic methods for rare 6dHepp architectures of importance, we herein describe a novel and efficient approach for the preparation of uncommon d-/l-6dHepp fluorides that have power as glycosylating agents. The synthesis is achieved by a C1-to-C5 switch strategy relying on radical decarboxylative fluorination of uronic acids arising from readily available allyl d-C-glycosides. To further showcase the application of this protocol, a structurally unique hexasaccharide composed of →3)-ß-d-6didoHepp-(1→4)-ß-d-GlcpNAc-(1→ units, corresponding to the capsular polysaccharide of C. jejuni strain CG8486 has been assembled for the first time. The assembly is characterized by highly efficient construction of the synthetically challenging ß-(1,2-cis)-d-ido-heptopyranoside by inversion of the C2 configuration of ß-(1,2-trans)-d-gulo-heptopyranoside, which is conveniently obtained by anchimerically assisted stereoselective glycosylation of the orthogonally protected 6dgulHepp fluoride. Ready accessibility of 6dHepp fluorides and the resulting glycans could serve as a rational starting point for the further development of synthetic vaccines fighting Campylobacter infection.

Structure-function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways.

Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes-C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC-are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.

Stress resistance associated with multi-host transmission and enhanced biofilm formation at 42 °C among hyper-aerotolerant generalist Campylobacter jejuni.

One of the emerging conundrums of Campylobacter food-borne illness is the bacterial ability to survive stressful environmental conditions. We evaluated the heterogeneity among 90 C. jejuni and 21 C. coli isolates from different sources in Egypt with respect to biofilm formation capabilities (under microaerobic and aerobic atmosphere) and resistance to a range of stressors encountered along the food chain (aerobic stress, refrigeration, freeze-thaw, heat, peracetic acid, and osmotic stress). High prevalence (63%) of hyper-aerotolerant (HAT) isolates was observed, exhibiting also a significantly high tolerance to heat, osmotic stress, refrigeration, and freeze-thaw stress, coupled with high biofilm formation ability which was clearly enhanced under aerobic conditions, suggesting a potential link between stress adaptation and biofilm formation. Most HAT multi-stress resistant and strong biofilm producing C. jejuni isolates belonged to host generalist clonal complexes (ST-21, ST-45, ST-48 and ST-206). These findings highlight the potential role of oxidative stress response systems in providing cross-protection (resistance to other multiple stress conditions) and enhancing biofilm formation in Campylobacter and suggest that selective pressures encountered in hostile environments have shaped the epidemiology of C. jejuni in Egypt by selecting the transmission of highly adapted isolates, thus promoting the colonization of multiple host species by important disease-causing lineages.

Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviously, detecting reagents such as a specific antibody is essential for the development of immune-based detection methods for C. jejuni or C. coli. In this study, in silico techniques were used to design a chimeric recombinant antigen, named multiepitope antigen (MEA), for the production of specific polyclonal antibody. To design MEA polypeptide based on C. jejuni fibronectin-binding protein or CadF, four conserved and unique antigenic peptides were identified and fused together directly. The C. jejuni CadF-based MEA polypeptide fused with two single six-histidine tags at both C- and N-terminal ends was expressed under Escherichia coli expression system. The recombinant MEA was successfully produced and purified by Ni-NTA resin with a high satisfactory yield. Indirect ELISA results showed that anti-MEA polyclonal antibody derived from rabbit serum had a titer of 16,000, indicating high antigenicity of MEA polypeptide. Dot blot results also confirmed that the produced anti-MEA antibody could specifically recognize both C. jejuni and C. coli whole cells as expected while there was no cross-reactivity to non-Campylobacter spp. tested in this study.

A copper site is required for iron transport by the periplasmic proteins P19 and FetP.

Campylobacter jejuni is a leading cause of food-borne gastrointestinal disease in humans and uropathogenic Escherichia coli is a leading cause of urinary tract infections. Both human pathogens harbour a homologous iron uptake system (termed cjFetM-P19 in C. jejuni and ecFetM-FetP in E. coli). Although these systems are important for growth under iron limitation, the mechanisms by which these systems function during iron transport remain undefined. The copper ions bound to P19 and FetP, the homologous periplasmic proteins, are coordinated in an uncommon penta-dentate manner involving a Met-Glu-His3 motif and exhibit positional plasticity. Here we demonstrate the function of the Met and Glu residues in modulating copper binding and controlling copper positioning through site-directed variants, binding assays, and crystal structures. Growth of C. jejuni strains with these p19 variants is impaired under iron limited conditions as compared to the wild-type strain. Additionally, an acidic residue-rich secondary site is required for binding iron and function in vivo. Finally, western blot analyses demonstrate direct and specific interactions between periplasmic P19 and FetP with the large periplasmic domain of their respective inner membrane transporters cjFetM and ecFetM.

Inhibition studies of ketol-acid reductoisomerases from pathogenic microorganisms.

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway, is an emerging target for the discovery of biocides. Here, we demonstrate that cyclopropane-1,1-dicarboxylate (CPD) inhibits KARIs from the pathogens Mycobacterium tuberculosis (Mt) and Campylobacter jejuni (Cj) reversibly with Ki values of 3.03 µM and 0.59 µM, respectively. Another reversible inhibitor of both KARIs, Hoe 704, is more potent than CPD with Ki values of 300 nM and 110 nM for MtKARI and CjKARI, respectively. The most potent inhibitor tested here is N-hydroxy-N-isopropyloxamate (IpOHA). It has a Ki of ~26 nM for MtKARI, but binds rather slowly (kon ~900 M-1s-1). In contrast, IpOHA binds more rapidly (kon ~7000 M-1s-1) to CjKARI and irreversibly.

The Unique Phospholipidome of the Enteric Pathogen Campylobacter jejuni: Lysophosholipids Are Required for Motility at Low Oxygen Availability.

In response to changes in their environment bacteria need to change both their protein and phospholipid repertoire to match environmental requirements, but the dynamics of bacterial phospholipid composition under different growth conditions is still largely unknown. In the present study, we investigated the phospholipidome of the bacterial pathogen Campylobacter jejuni. Transcription profiling on logarithmic and stationary phase grown cells of the microaerophilic human pathogen C. jejuni using RNA-seq revealed differential expression of putative phospholipid biosynthesis genes. By applying high-performance liquid chromatography tandem-mass spectrometry, we identified 203 phospholipid species representing the first determination of the phospholipidome of this pathogen. We identified nine different phospholipid classes carrying between one and three acyl chains. Phospholipidome analysis on bacteria of different ages (0-5 days) showed rapid changes in the ratio of phospholipids containing ethanolamine, or glycerol as phospholipid head group and in the number of cyclopropane bond containing fatty acids. Oxygen concentration influenced the percentage of lysophospholipids, and cyclo-propane bonds containing acyl chains. We show that large amounts of the phospholipids are lysophospholipids (30-45%), which mutant studies reveal are needed for normal C. jejuni motility at low oxygen conditions. C. jejuni possesses an unusual phospholipidome that is highly dynamic in response to environmental changes.

Structural analysis of a Simpl-like protein from Campylobacter jejuni.

Signaling molecule that interacts with mouse pelle-like kinase (Simpl) is an animal protein that contributes to the regulation of inflammatory responses. Although Simpl-like proteins (SLPs) are mainly found in bacteria, functional and structural studies on bacterial SLPs are limited to BP26, a periplasmic protein from Brucella species. We identified a group of bacterial SLPs, including Campylobacter jejuni SLP (cjSLP) and Shewanella putrefaciens SLP (spSLP), that exhibit significant sequence variation from Simpl and BP26. To address the structural and oligomeric diversities of SLPs, we determined the crystal structure of cjSLP and performed a comparative analysis of SLP structures. cjSLP adopts a boomerang-shaped, two-domain structure, and each domain of cjSLP adopts an α-helix-decorated ß-sheet structure as observed in BP26. This observation suggests that the duplicated α/ß structure would be the canonical fold of the Simpl family. Despite the fold similarity, cjSLP exhibits a more open interdomain organization than BP26 and displays unique local structural features that are not observed in BP26. Furthermore, cjSLP and its ortholog spSLP are monomeric in solution in contrast to the hexadecameric assembly of BP26. Therefore, we conclude that cjSLP represents a unique bacterial SLP group that is distinct from BP26 in both structures and oligomeric states.

Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli.

The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (ß-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (ß-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli-preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75-78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside-related oligosaccharides.

Functional Characterization of Cj1427, a Unique Ping-Pong Dehydrogenase Responsible for the Oxidation of GDP-d-glycero-α-d-manno-heptose in Campylobacter jejuni.

The capsular polysaccharides (CPS) of Campylobacter jejuni contain multiple heptose residues with variable stereochemical arrangements at C3-C6. The immediate precursor to all of these possible variations is currently believed to be GDP-d-glycero-α-d-manno-heptose. Oxidation of this substrate at C4 enables subsequent epimerization reactions at C3-C5 that can be coupled to the dehydration/reduction at C5/C6. However, the enzyme responsible for the critical oxidation of C4 from GDP-d-glycero-α-d-manno-heptose has remained elusive. The enzyme Cj1427 from C. jejuni NCTC 11168 was shown to catalyze the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose in the presence of α-ketoglutarate using mass spectrometry and nuclear magnetic resonance spectroscopy. At pH 7.4, the apparent kcat is 0.6 s-1, with a value of kcat/Km of 1.0 × 104 M-1 s-1 for GDP-d-glycero-α-d-manno-heptose. α-Ketoglutarate is required to recycle the tightly bound NADH nucleotide in the active site of Cj1427, which does not dissociate from the enzyme during catalysis.

Structural Analysis of Cj1427, an Essential NAD-Dependent Dehydrogenase for the Biosynthesis of the Heptose Residues in the Capsular Polysaccharides of Campylobacter jejuni.

Many strains of Campylobacter jejuni display modified heptose residues in their capsular polysaccharides (CPS). The precursor heptose was previously shown to be GDP-d-glycero-α-d-manno-heptose, from which a variety of modifications of the sugar moiety have been observed. These modifications include the generation of 6-deoxy derivatives and alterations of the stereochemistry at C3-C6. Previous work has focused on the enzymes responsible for the generation of the 6-deoxy derivatives and those involved in altering the stereochemistry at C3 and C5. However, the generation of the 6-hydroxyl heptose residues remains uncertain due to the lack of a specific enzyme to catalyze the initial oxidation at C4 of GDP-d-glycero-α-d-manno-heptose. Here we reexamine the previously reported role of Cj1427, a dehydrogenase found in C. jejuni NTCC 11168 (HS:2). We show that Cj1427 is co-purified with bound NADH, thus hindering catalysis of oxidation reactions. However, addition of a co-substrate, α-ketoglutarate, converts the bound NADH to NAD+. In this form, Cj1427 catalyzes the oxidation of l-2-hydroxyglutarate back to α-ketoglutarate. The crystal structure of Cj1427 with bound GDP-d-glycero-α-d-manno-heptose shows that the NAD(H) cofactor is ideally positioned to catalyze the oxidation at C4 of the sugar substrate. Additionally, the overall fold of the Cj1427 subunit places it into the well-defined short-chain dehydrogenase/reductase superfamily. The observed quaternary structure of the tetrameric enzyme, however, is highly unusual for members of this superfamily.

Influence of Environmental and Genetic Factors on Proteomic Profiling of Outer Membrane Vesicles from Campylobacter jejuni.

The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81-176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains - ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81­176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains ­ ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.

Structure of Outward-Facing PglK and Molecular Dynamics of Lipid-Linked Oligosaccharide Recognition and Translocation.

PglK is a lipid-linked oligosaccharide (LLO) flippase essential for asparagine-linked protein glycosylation in Campylobacter jejuni. Previously we have proposed a non-alternating-access LLO translocation mechanism, where postulated outward-facing states play a primary role. To investigate this unusual mechanistic proposal, we have determined a high-resolution structure of PglK that displays an outward semi-occluded state with the two nucleotide binding domains forming an asymmetric closed dimer with two bound ATPγS molecules. Based on this structure, we performed extensive molecular dynamics simulations to investigate LLO recognition and flipping. Our results suggest that PglK may employ a "substrate-hunting" mechanism to locally increase the LLO concentration and facilitate its jump into the translocation pathway, for which sugars from the LLO head group are essential. We further conclude that the release of LLO to the outside occurs before ATP hydrolysis and is followed by the closing of the periplasmic cavity of PglK.

Purification and characterization of Campylobacter jejuni ferric uptake regulator.

The ferric uptake regulator (Fur) is a superfamily of transcription factors found in bacteria which control the expression of a myriad of genes. In this study, we report a simple protocol for the purification of recombinant untagged Campylobacter jejuni Fur (CjFur). CjFur was isolated using a combination of three ion exchange chromatography steps followed by size exclusion chromatography on a Superdex 75. ESI-MS analysis shows that our method yields pure CjFur and that this tag-free version incorporates metal more efficiently than recombinant CjFur harboring a tag or tag remnants. Finally, electrophoretic mobility shift assays show that this new purification method yields a CjFur preparation that binds DNA more efficiently. These results suggest that adding a N-terminus tag onto CjFur is detrimental to its activity. Overall, the approaches detailed in this study offer an alternative strategy for the purification of CjFur, and likely other metalloregulators, for future biochemical and biophysical studies.