Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe-S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe-S clusters have been identified in DHADs, i.e. [2Fe-2S] and [4Fe-4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000-fold increase with kcat as high as ∼6.7 s-1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4Fe-4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (Ki =7.8 and 51.6 µM, respectively) and CjDHAD (Ki =32.9 and 35.1 µM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.

Functional Characterization of Two PLP-Dependent Enzymes Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.

Transcription of Cystathionine ß-Lyase (MetC) Is Repressed by HeuR in Campylobacter jejuni, and Methionine Biosynthesis Facilitates Colonocyte Invasion.

A previously identified transcriptional regulator in Campylobacter jejuni, termed HeuR, was found to positively regulate heme utilization. Additionally, transcriptomic work demonstrated that the putative operons CJJ81176_1390 to CJJ81176_1394 (CJJ81176_1390-1394) and CJJ81176_1214-1217 were upregulated in a HeuR mutant, suggesting that HeuR negatively regulates expression of these genes. Because genes within these clusters include a cystathionine ß-lyase (metC) and a methionine synthase (metE), it appeared HeuR negatively regulates C. jejuni methionine biosynthesis. To address this, we confirmed mutation of HeuR reproducibly results in metC overexpression under nutrient-replete conditions but did not affect expression of metE, while metC expression in the wild type increased to heuR mutant levels during iron limitation. We subsequently determined that both gene clusters are operonic and demonstrated the direct interaction of HeuR with the predicted promoter regions of these operons. Using DNase footprinting assays, we were able to show that HeuR specifically binds within the predicted -35 region of the CJJ81176_1390-1394 operon. As predicted based on transcriptional results, the HeuR mutant was able to grow and remain viable in a defined medium with and without methionine, but we identified significant impacts on growth and viability in metC and metE mutants. Additionally, we observed decreased adherence, invasion, and persistence of metC and metE mutants when incubated with human colonocytes, while the heuR mutant exhibited increased invasion. Taken together, these results suggest that HeuR regulates methionine biosynthesis in an iron-responsive manner and that the ability to produce methionine is an important factor for adhering to and invading the gastrointestinal tract of a susceptible host. IMPORTANCE As the leading cause of bacterium-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health. Investigating colonization factors that allow C. jejuni to successfully infect a host furthers our understanding of genes and regulatory elements necessary for virulence. In this study, we have begun to characterize the role of the transcriptional regulatory protein, HeuR, on methionine biosynthesis in C. jejuni. When the ability to synthesize methionine is impaired, detrimental impacts on growth and viability are observed during growth in limited media lacking methionine and/or iron. Additionally, mutations in the methionine biosynthetic pathway result in decreased adhesion, invasion, and intracellular survival of C. jejuni when incubated with human colonocytes, indicating the importance of regulating methionine biosynthesis.

The flavodoxin FldA activates the class Ia ribonucleotide reductase of Campylobacter jejuni.

Campylobacter jejuni is a microaerophilic zoonotic pathogen with an atypical respiratory Complex I that oxidizes a flavodoxin (FldA) instead of NADH. FldA is essential for viability and is reduced via pyruvate and 2-oxoglutarate oxidoreductases (POR/OOR). Here, we show that FldA can also be reduced by FqrB (Cj0559), an NADPH:FldA reductase. An fqrB deletion mutant was viable but displayed a significant growth defect. FqrB is related to flavoprotein reductases from Gram-positive bacteria that can reduce NrdI, a specialized flavodoxin that is needed for tyrosyl radical formation in NrdF, the beta subunit of class 1b-type (Mn) ribonucleotide reductase (RNR). However, C. jejuni possesses a single class Ia-type (Fe) RNR (NrdAB) that would be expected to be ferredoxin dependent. We show that CjFldA is an unusually high potential flavodoxin unrelated to NrdI, yet growth of the fqrB mutant, but not the wild-type or a complemented strain, was stimulated by low deoxyribonucleoside (dRNS) concentrations, suggesting FldA links FqrB and RNR activity. Using purified proteins, we confirmed the NrdB tyrosyl radical could be regenerated in an NADPH, FqrB, and FldA dependent manner, as evidenced by both optical and electron paramagnetic resonance (EPR) spectroscopy. Thus, FldA activates RNR in C. jejuni, partly explaining its essentiality.

Turnover Chemistry and Structural Characterization of the Cj0843c Lytic Transglycosylase of Campylobacter jejuni.

The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its nonhydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure-function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference for substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by molecular dynamics simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

Functional and Structural Characterization of the UDP-Glucose Dehydrogenase Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni.

Campylobacter jejuni is a pathogenic organism that can cause campylobacteriosis in children and adults. Most commonly, campylobacter infection is brought on by consumption of raw or undercooked poultry, unsanitary drinking water, or pet feces. Surrounding the C. jejuni bacterium is a coat of sugar molecules known as the capsular polysaccharide (CPS). The capsular polysaccharide can be very diverse among the different strains of C. jejuni, and this diversity is considered important for evading the host immune system. Modifications to the CPS of C. jejuni NCTC 11168 include O-methylation, phosphoramidylation, and amidation of glucuronate with either serinol or ethanolamine. The enzymes responsible for amidation of glucuronate are currently unknown. In this study, Cj1441, an enzyme expressed from the CPS biosynthetic gene cluster in C. jejuni NCTC 11168, was shown to catalyze the oxidation of UDP-α-d-glucose into UDP-α-d-glucuronic acid with NAD+ as the cofactor. No amide products were found in an attempt to determine whether the putative thioester intermediate formed during the oxidation of UDP-glucose by Cj1441 could be captured in the presence of added amines. The three-dimensional crystal structure of Cj1441 was determined in the presence of NAD+ and UDP-glucose bound in the active site of the enzyme (Protein Data Bank entry 7KWS). A more thorough bioinformatic analysis of the CPS gene cluster suggests that the amidation activity is localized to the t-terminal half of Cj1438, a bifunctional enzyme that is currently annotated as a sugar transferase.

Reconstitution and optimisation of the biosynthesis of bacterial sugar pseudaminic acid (Pse5Ac7Ac) enables preparative enzymatic synthesis of CMP-Pse5Ac7Ac.

Pseudaminic acids present on the surface of pathogenic bacteria, including gut pathogens Campylobacter jejuni and Helicobacter pylori, are postulated to play influential roles in the etiology of associated infectious diseases through modulating flagella assembly and recognition of bacteria by the human immune system. Yet they are underexplored compared to other areas of glycoscience, in particular enzymes responsible for the glycosyltransfer of these sugars in bacteria are still to be unambiguously characterised. This can be largely attributed to a lack of access to nucleotide-activated pseudaminic acid glycosyl donors, such as CMP-Pse5Ac7Ac. Herein we reconstitute the biosynthesis of Pse5Ac7Ac in vitro using enzymes from C. jejuni (PseBCHGI) in the process optimising coupled turnover with PseBC using deuterium wash in experiments, and establishing a method for co-factor regeneration in PseH tunover. Furthermore we establish conditions for purification of a soluble CMP-Pse5Ac7Ac synthetase enzyme PseF from Aeromonas caviae and utilise it in combination with the C. jejuni enzymes to achieve practical preparative synthesis of CMP-Pse5Ac7Ac in vitro, facilitating future biological studies.

Antimicrobial Resistance Profiles and Phylogenetic Analysis of Campylobacter jejuni Strains Isolated in Brazil by Whole Genome Sequencing.

Aims: The objectives of this work were to use whole genome sequencing (WGS) to determine the antimicrobial resistance genotypes of 116 Campylobacter jejuni strains isolated in Brazil and to compare it with the results obtained by antimicrobial susceptibility testing (AST). In addition, WGS was used to uncover the phylogenetic relationship among those strains. Results: By AST, the C. jejuni strains resistant to ciprofloxacin, tetracycline, doxycycline, and erythromycin were 51 (44%), 41 (35.3%), 41 (35.3%), and 6 (5.2%), respectively. By WGS, the genes aph(3')III, aadE, blaOXA-449, blaOXA-184, blaOXA-61, and tet(O) were detected in 6 (5.2%), 3 (2.6%), 1 (0.9%), 10 (8.6%), 55 (47.4%), and 44 (38%) strains, respectively. Fifty-four (46.6%) strains showed the mutation T86I in the gyrA gene, and four (3.4%) strains presented the mutation A2075G in the 23S rRNA gene. The correlation between AST and WGS was 100% for ciprofloxacin, 97.5% for tetracyclines, and 66.7% for erythromycin. The whole genome single nucleotide polymorphism (SNP) tree clustered the C. jejuni strains into two clades comprising strains that were highly related from different sources, places, and years. Conclusion: The high rates of C. jejuni strains resistant to ciprofloxacin and tetracyclines are of concern and may represent a public health problem. WGS has a potential to be a powerful tool for the prediction of resistance of antibiotics used to treat campylobacteriosis. The results obtained by whole genome SNP analysis suggested the potential for transmission between clinical and nonclinical sources and between human and animal sources over the course of 20 years in Brazil.

Kinetic consequences of the endogenous ligand to molybdenum in the DMSO reductase family: a case study with periplasmic nitrate reductase.

The molybdopterin enzyme family catalyzes a variety of substrates and plays a critical role in the cycling of carbon, nitrogen, arsenic, and selenium. The dimethyl sulfoxide reductase (DMSOR) subfamily is the most diverse family of molybdopterin enzymes and the members of this family catalyze a myriad of reactions that are important in microbial life processes. Enzymes in the DMSOR family can transform multiple substrates; however, quantitative information about the substrate preference is sparse, and, more importantly, the reasons for the substrate selectivity are not clear. Molybdenum coordination has long been proposed to impact the catalytic activity of the enzyme. Specifically, the molybdenum-coordinating residue may tune substrate preference. As such, molybdopterin enzyme periplasmic nitrate reductase (Nap) is utilized as a vehicle to understand the substrate preference and delineate the kinetic underpinning of the differences imposed by exchanging the molybdenum ligands. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic properties of these variants are discussed and compared with those of the native enzyme, providing quantitative information to understand the function of the molybdenum-coordinating residue.

Chemoenzymatic Synthesis of Campylobacter jejuni Lipo-oligosaccharide Core Domains to Examine Guillain-Barré Syndrome Serum Antibody Specificities.

Guillain-Barré syndrome is often caused by Campylobacter jejuni infection that has induced antibodies to the lipo-oligosaccharide (LOS) that cross-react with gangliosides at peripheral nerves causing polyneuropathy. To examine fine specificities of anti-ganglioside antibodies and develop a more robust platform for diagnosis and disease monitoring, we developed a chemoenzymatic approach that provided an unprecedented panel of oligosaccharides composed of the inner-core of the LOS of C. jejuni extended by various ganglioside mimics. The compounds and corresponding ganglio-oligosaccharides were printed as a microarray to examine binding specificities of lectins, anti-ganglioside antibodies, and serum antibodies of GBS patients. Although lectins and anti-ganglioside antibodies did not differentiate the ganglio-oligosaccharides and mimics, the patient serum samples bound much more strongly to the ganglioside mimics. The data indicate that antibodies have been elicited to a foreign epitope that includes a heptosyl residue unique of bacterial LOS and that these antibodies subsequently cross-react with lower affinity to gangliosides. The microarray detected anti-GM1a antibodies with high sensitivity and will be attractive for diagnosis, disease monitoring, and immunological research.

Functional analysis and cryo-electron microscopy of Campylobacterjejuni serine protease HtrA.

Campylobacter jejuni is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA Cj ), which targets tight and adherens junctional proteins in the gut epithelium. Here we have investigated the function and structure of HtrA Cj using biochemical assays and cryo-electron microscopy. Mass spectrometry analysis identified differences and similarities in the cleavage site specificity for HtrA Cj by comparison to the HtrA counterparts from Helicobacter pylori and Escherichia coli. We defined the architecture of HtrA Cj at 5.8 Å resolution as a dodecamer, built of four trimers. The contacts between the trimers are quite loose, a fact that explains the flexibility and mobility of the dodecameric assembly. This flexibility has also been studied through molecular dynamics simulation, which revealed opening of the dodecamer to expose the proteolytically active site of the protease. Moreover, we examined the rearrangements at the level of oligomerization in the presence or absence of substrate using size exclusion chromatography, which revealed hexamers, dodecamers and larger oligomeric forms, as well as remarkable stability of higher oligomeric forms (> 12-mers) compared to previously tested homologs from other bacteria. Extremely dynamic decay of the higher oligomeric forms into lower forms was observed after full cleavage of the substrate by the proteolytically active variant of HtrA Cj . Together, this is the first report on the in-depth functional and structural analysis of HtrA Cj , which may allow the construction of therapeutically relevant HtrA Cj inhibitors in the near future.

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.

Identifying Sialylation Linkages at the Glycopeptide Level by Glycosyltransferase Labeling Assisted Mass Spectrometry (GLAMS).

Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of α2,3/α2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of α2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of α2,6-sailoglycopeptide isomers. With this strategy, a total of 1236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.

C8J_1298, a bifunctional thiol oxidoreductase of Campylobacter jejuni, affects Dsb (disulfide bond) network functioning.

Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner.

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.

Activity of fructose-1,6-bisphosphatase from Campylobacter jejuni.

The glycolytic pathway of the enteric pathogen Campylobacter jejuni is incomplete; the absence of phosphofructokinase means that the suppression of futile cycling at this point in the glycolytic-gluconeogenic pathway might not be required, and therefore the mechanism for controlling pathway flux is likely to be quite different or absent. In this study, the characteristics of fructose-1,6-bisphosphatase (FBPase) of C.jejuni are described and the regulation of this enzyme is compared with the equivalent enzymes from organisms capable of glycolysis. The enzyme is insensitive to AMP inhibition, unlike other type I FBPases. Campylobacterjejuni FBPase also shows limited sensitivity to other glycolytic and gluconeogenic intermediates. The allosteric cooperative control of the enzyme's activity found in type I FBPases appears to have been lost.

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.

HtrA-dependent adherence and invasion of Campylobacter jejuni in human vs avian cells.

The aim of this study was to investigate whether HtrA is responsible for differences in adherence and invasion of Campylobacter jejuni towards human and chicken cell lines. Gentamicin protection assays were performed with either human Caco-2 or chicken 2G4 cells using C. jejuni strain NCTC11168 to compare the adhesion and invasion rates towards these two cell types. The results revealed significant differences in the adhesion and invasion rates between the human and avian cells. Deletion of the Campylobacter htrA gene, coding for the dual function of serine protease and chaperonin with a role in pathogenesis, led to a reduction of the rates in both cell lines. Using a single-amino acid substitution mutant (ΔhtrA/htrAS197A ) that lacked protease activity, but retained chaperonin activity, we show that the first is involved in the invasion of human Caco-2 and chicken 2G4 cells, whereas the latter mutant invaded at lower levels. Adherence towards the chicken cells is higher than towards Caco-2 cells and this is also dependent on HtrA. Together, these data suggest that the proteolytic activity of HtrA is involved in the difference in host response of C. jejuni towards human and chicken-derived cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter jejuni is the main cause for bacterial foodborne enterocolitis worldwide. While colonization of the human intestine can lead to severe problems, avian hosts - as the major source of infection - remain unaffected by the bacteria. We showed that the bacterial serine protease and chaperonin HtrA are involved in adhesion and invasion in both species and not responsible for the discrepancy of virulence between the different hosts. In future, HtrA might act as a target for inhibitors to avoid or eradicate colonization in chickens as a less problematic alternative to antibiotics in commercial livestock breeding.

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria.

The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown "route I" nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni.