Biochemical characterization of the Campylobacter jejuni Cj1294, a novel UDP-4-keto-6-deoxy-GlcNAc aminotransferase that generates UDP-4-amino-4,6-dideoxy-GalNAc

Campylobacter jejuni produces multiple glycoproteins whose glycans contain 4-amino 6-deoxy sugars or their derivatives, such as diacetamidobacillosamine or pseudaminic acid. Because the proteoglycans contribute to bacterial virulence and their constitutive sugars are not commonly found in humans, inhibitors developed against the enzymes that are responsible for their biosynthesis could be novel therapeutic targets to fight this important food-borne pathogen. The biosynthesis of diacetamidobacillosamine is anticipated to involve a sugar nucleotide C6 dehydratase, a C4 aminotransferase and an acetyltransferase. We have identified a set of genes (cj1293, cj1294, and cj1298) potentially encoding one of each enzymatic activity, and demonstrated earlier that Cj1293 was a UDP-GlcNAc-specific C6 dehydratase. Others have shown that Cj1293 was involved in protein glycosylation. Here, we report on our investigation of the potential activity of Cj1294 as a sugar nucleotide C4 aminotransferase. Our biochemical characterization of overexpressed and purified protein shows that Cj1294 is a pyridoxal phosphate-dependent aminotransferase specific for UDP-4-keto-6-deoxy-GlcNAc that uses preferentially glutamic acid as an amino donor. A detailed physicokinetic study of Cj1294 was performed to determine the K(m) of 1.28 +/- 0.2 mm and k(cat) of 11.5 +/- 1.3 min(-1). Also, two residues essential for protein stability and activity, Arg(228) and Lys(181), respectively, were identified by site-directed mutagenesis. Finally, we demonstrated by NMR analysis of purified reaction product that Cj1294 produces UDP-4-amino-4,6-dideoxy-GalNAc. These results indicate that Cj1294 is involved in the biosynthesis of diacetamidofucosamine, a C4 epimer of diacetamidobacillosamine not yet described in C. jejuni proteoglycans, suggesting that the composition of C. jejuni proteoglycans is more variable than anticipated.     

Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine biosynthetic pathways

Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-alpha-D-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His(6)-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-alpha-D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose.     

Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.     

Giant DNA virus mimivirus encodes pathway for biosynthesis of unusual sugar 4-amino-4,6-dideoxy-D-glucose (Viosamine)

Mimivirus is one the largest DNA virus identified so far, infecting several Acanthamoeba species. Analysis of its genome revealed the presence of a nine-gene cluster containing genes potentially involved in glycan formation. All of these genes are co-expressed at late stages of infection, suggesting their role in the formation of the long fibers covering the viral surface. Among them, we identified the L136 gene as a pyridoxal phosphate-dependent sugar aminotransferase. This enzyme was shown to catalyze the formation of UDP-4-amino-4,6-dideoxy-D-glucose (UDP-viosamine) from UDP-4-keto-6-deoxy-D-glucose, a key compound involved also in the biosynthesis of L-rhamnose. This finding further supports the hypothesis that Mimivirus encodes a glycosylation system that is completely independent of the amoebal host. Viosamine, together with rhamnose, (N-acetyl)glucosamine, and glucose, was found as a major component of the viral glycans. Most of the sugars were associated with the fibers, confirming a capsular-like nature of the viral surface. Phylogenetic analysis clearly indicated that L136 was not a recent acquisition from bacteria through horizontal gene transfer, but it was acquired very early during evolution. Implications for the origin of the glycosylation machinery in giant DNA virus are also discussed.     

Functional characterization of the flagellar glycosylation locus in Campylobacter jejuni 81-176 using a focused metabolomics approach

Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81-176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81-176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-L-glycero-alpha-L-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways.     

Structural and functional characterization of PseC, an aminotransferase involved in the biosynthesis of pseudaminic acid, an essential flagellar modification in Helicobacter pylori

Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-alpha-D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-beta-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the beta-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.     

Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene

Flagellins from Campylobacter jejuni 81-176 and Campylobacter coli VC167 are heavily glycosylated. The major modifications on both flagellins are pseudaminic acid (Pse5Ac7Ac), a nine carbon sugar that is similar to sialic acid, and an acetamidino-substituted analogue of pseudaminic acid (PseAm). Previous data have indicated that PseAm is synthesized via Pse5Ac7Ac in C. jejuni 81-176, but that the two sugars are synthesized using independent pathways in C. coli VC167. The Cj1293 gene of C. jejuni encodes a putative UDP-GlcNAc C6-dehydratase/C4-reductase that is similar to a protein required for glycosylation of Caulobacter crescentus flagellin. The Cj1293 gene is expressed either under the control of a sigma 54 promoter that overlaps the coding region of Cj1292 or as a polycistronic message under the control of a sigma 70 promoter upstream of Cj1292. A mutant in gene Cj1293 in C. jejuni 81-176 was non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. This mutant was complemented in trans with the homologous C. jejuni gene, as well as the Helicobacter pylori homologue, HP0840, which has been shown to encode a protein with UDP-GlcNAc C6-dehydratase/C4-reductase activity. Mutation of Cj1293 in C. coli VC167 resulted in a fully motile strain that synthesized a flagella filament composed of flagellin in which Pse5Ac7Ac was replaced by PseAm. The filament from the C. coli Cj1293 mutant displayed increased solubility in SDS compared with the wild-type filament. A double mutant in C. coli VC167, defective in both Cj1293 and ptmD, encoding part of the independent PseAm pathway, was also non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. Collectively, the data indicate that Cj1293 is essential for Pse5Ac7Ac biosynthesis from UDP-GlcNAc, and that glycosylation is required for flagella biogenesis in campylobacters.     

Two respiratory enzyme systems in Campylobacter jejuni NCTC 11168 contribute to growth on l‐lactate

Campylobacter jejuni, a major food‐borne intestinal pathogen, preferentially utilizes a few specific amino acids and some organic acids such as pyruvate and l ‐ and d ‐lactate as carbon sources, which may be important for growth in the avian and mammalian gut. Here, we identify the enzymatic basis for C. jejuni growth on l ‐lactate. Despite the presence of an annotated gene for a fermentative lactate dehydrogenase (cj1167), no evidence for lactate excretion could be obtained in C. jejuni NCTC 11168, and inactivation of the cj1167 gene did not affect growth on lactate as carbon source. Instead, l ‐lactate utilization in C. jejuni NCTC 11168 was found to proceed via two novel NAD‐independent l ‐LDHs; a non‐flavin iron–sulfur containing three subunit membrane‐associated enzyme (Cj0075c‐73c), and a flavin and iron–sulfur containing membrane‐associated oxidoreductase (Cj1585c). Both enzymes contribute to growth on l ‐lactate, as single mutants in each system grew as well as wild‐type on this substrate, while a cj0075c cj1585c double mutant showed no l ‐lactate oxidase activity and did not utilize or grow on l ‐lactate; d ‐lactate‐dependent growth was unaffected. Orthologues of Cj0075c‐73c (LldEFG/LutABC) and Cj1585c (Dld‐II) were recently shown to represent two novel families of l ‐ and d ‐lactate oxidases; this is the first report of a bacterium where both enzymes are involved in l ‐lactate utilization only. The cj0075c‐73c genes are located directly downstream of a putative lactate transporter gene (cj0076c, lctP), which was also shown to be specific for l ‐lactate. The avian and mammalian gut environment contains dense populations of obligate anaerobes that excrete lactate; our data indicate that C. jejuni is well equipped to use l ‐ and d ‐lactate as both electron‐donor and carbon source.     

Biosynthesis of UDP-N-acetyl-L-fucosamine, a precursor to the biosynthesis of lipopolysaccharide in Pseudomonas aeruginosa serotype O11

UDP-N-acetyl-L-fucosamine is a precursor to l-fucosamine in the lipopolysaccharide of Pseudomonas aeruginosa serotype O11 and the capsule of Staphylococcus aureus type 5. We have demonstrated previously the involvement of three enzymes, WbjB, WbjC, and WbjD, in the biosynthesis of UDP-2-acetamido-2,6-dideoxy-L-galactose or UDP-N-acetyl-L-fucosamine (UDP-l-FucNAc). An intermediate compound from the coupled-reaction of WbjB-WbjC with the initial substrate UDP-2-acetamido-2-deoxy-alpha-D-glucose or UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) was purified, and the structure was determined by NMR spectroscopy to be UDP-2-acetamido-2,6-dideoxy-L-talose (UDP-L-PneNAc). WbjD could then convert this intermediate into a new product with the same mass, consistent with a C-2 epimerization reaction. Those results led us to propose a pathway for the biosynthesis of UDP-L-FucNAc; however, the exact enzymatic activity of each of these proteins has not been defined. Here, we describe a fast protein liquid chromatography (FPLC)-based anion-exchange procedure, which allowed the separation and purification of the products of C-2 epimerization due to WbjD. Also, the application of a cryogenically cooled probe in NMR spectrometry offers the greatest sensitivity for determining the structures of minute quantities of materials, allowing the identification of the final product of the pathway. Our results showed that WbjB is bifunctional, catalyzing firstly C-4, C-6 dehydration and secondly C-5 epimerization in the reaction with the substrate UDP-D-GlcNAc, producing two intermediates. WbjC is also bifunctional, catalyzing C-3 epimerization of the second intermediate followed by reduction at C-4. The FPLC-based procedure provided good resolution of the final product of WbjD reaction from its epimer/substrate UDP-l-PneNAc, and the use of the cryogenically cooled probe in NMR revealed unequivocally that the final product is UDP-L-FucNAc.     

Functional Analysis of the Campylobacter jejuni cj0183 and cj0588 Genes

The cj0183 and cj0588 genes identified in the Campylobacter jejuni NCTC 11168 genome encode proteins with amino acid sequences predicted to be homologous to other bacterial hemolysins. The Cj0183 protein exhibits homology to Brachyspira hyodysenteriae TlyC protein, whereas the cj0588 gene product is homologous to TlyA proteins Brachyspira hyodysenteriae, Helicobacter pylori, and Mycobacterium tuberculosis, which play a crucial role in bacterial virulence. The aim of our work was to examine the hemolytic activity and determine the role of cj0183- and cj0588-encoded proteins on the adherence of chosen C. jejuni strains to the Caco-2 cell line by constructing deletion mutants in the mentioned genes. We found out there is no difference in hemolytic activity between both mutants in gene cj0183 and cj0588 and the wild strains. However, Cj0588 protein but not Cj0183 is involved in adherence to the Caco-2 cells.     

Structural and functional analysis of a new subfamily of glycosyltransferases required for glycosylation of serine-rich streptococcal adhesins

Serine-rich repeat glycoproteins (SRRPs) are a growing family of bacterial adhesins found in many streptococci and staphylococci; they play important roles in bacterial biofilm formation and pathogenesis. Glycosylation of this family of adhesins is essential for their biogenesis. A glucosyltransferase (Gtf3) catalyzes the second step of glycosylation of a SRRP (Fap1) from an oral streptococcus, Streptococcus parasanguinis. Although Gtf3 homologs are highly conserved in SRRP-containing streptococci, they share minimal homology with functionally known glycosyltransferases. We report here the 2.3 ? crystal structure of Gtf3. The structural analysis indicates that Gtf3 forms a tetramer and shares significant structural homology with glycosyltransferases from GT4, GT5, and GT20 subfamilies. Combining crystal structural analysis with site-directed mutagenesis and in vitro glycosyltransferase assays, we identified residues that are required for UDP- or UDP-glucose binding and for oligomerization of Gtf3 and determined their contribution to the enzymatic activity of Gtf3. Further in vivo studies revealed that the critical amino acid residues identified by the structural analysis are crucial for Fap1 glycosylation in S. parasanguinis in vivo. Moreover, Gtf3 homologs from other streptococci were able to rescue the gtf3 knock-out mutant of S. parasanguinis in vivo and catalyze the sugar transfer to the modified SRRP substrate in vitro, demonstrating the importance and conservation of the Gtf3 homologs in glycosylation of SRRPs. As the Gtf3 homologs only exist in SRRP-containing streptococci, we conclude that the Gtf3 homologs represent a unique subfamily of glycosyltransferases.     

Biochemical characterization of the O-linked glycosylation pathway in Neisseria gonorrhoeae responsible for biosynthesis of protein glycans containing N,N'-diacetylbacillosamine

The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607-624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447-4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N'-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets.     

Structure and function of both domains of ArnA, a dual function decarboxylase and a formyltransferase, involved in 4-amino-4-deoxy-L-arabinose biosynthesis

Modification of the lipid A moiety of lipopolysaccharide by the addition of the sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N) is a strategy adopted by pathogenic Gram-negative bacteria to evade cationic antimicrobial peptides produced by the innate immune system. L-Ara4N biosynthesis is therefore a potential anti-infective target, because inhibiting its synthesis would render certain pathogens more sensitive to the immune system. The bifunctional enzyme ArnA, which is required for L-Ara4N biosynthesis, catalyzes the NAD(+)-dependent oxidative decarboxylation of UDP-glucuronic acid to generate a UDP-4'-keto-pentose sugar and also catalyzes transfer of a formyl group from N-10-formyltetrahydrofolate to the 4'-amine of UDP-L-Ara4N. We now report the crystal structure of the N-terminal formyltransferase domain in a complex with uridine monophosphate and N-5-formyltetrahydrofolate. Using this structure, we identify the active site of formyltransfer in ArnA, including the key catalytic residues Asn(102), His(104), and Asp(140). Additionally, we have shown that residues Ser(433) and Glu(434) of the decarboxylase domain are required for the oxidative decarboxylation of UDP-GlcUA. An E434Q mutant is inactive, suggesting that chemical rather than steric properties of this residue are crucial in the decarboxylation reaction. Our data suggest that the decarboxylase domain catalyzes both hydride abstraction (oxidation) from the C-4' position and the subsequent decarboxylation.     

The Campylobacter jejuni Cj0268c Protein Is Required for Adhesion and Invasion In Vitro

Adherence of Campylobacter jejuni to its particular host cells is mediated by several pathogen proteins. We screened a transposon-based mutant library of C. jejuni in order to identify clones with an invasion deficient phenotype towards Caco2 cells and detected a mutant with the transposon insertion in gene cj0268c. In vitro characterization of a generated non-random mutant, the mutant complemented with an intact copy of cj0268c and parental strain NCTC 11168 confirmed the relevance of Cj0268c in the invasion process, in particular regarding adherence to host cells. Whereas Cj0268c does not impact autoagglutination or motility of C. jejuni, heterologous expression in E. coli strain DH5α enhanced the potential of the complemented E. coli strain to adhere to Caco2 cells significantly and, thus, indicates that Cj0268c does not need to interact with other C. jejuni proteins to develop its adherence-mediating phenotype. Flow cytometric measurements of E. coli expressing Cj0268c indicate a localization of the protein in the periplasmic space with no access of its C-terminus to the bacterial surface. Since a respective knockout mutant possesses clearly reduced resistance to Triton X-100 treatment, Cj0268c contributes to the stability of the bacterial cell wall. Finally, we could show that the presence of cj0268c seems to be ubiquitous in isolates of C. jejuni and does not correlate with specific clonal groups regarding pathogenicity or pathogen metabolism.     

Disruption of mannose activation in Leishmania mexicana: GDP-mannose pyrophosphorylase is required for virulence, but not for viability.

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDPMP) is essential for the formation of GDP-mannose, the central activated mannose donor in glycosylation reactions. Deletion of its gene is lethal in fungi, most likely as a consequence of disrupted glycoconjugate biosynthesis. Furthermore, absence of GDPMP enzyme activity and the expected loss of all mannose-containing glycoconjugates have so far not been observed in any eukaryotic organism. In this study we have cloned and characterized the gene encoding GDPMP from the eukaryotic protozoan parasite Leishmania mexicana. We report the generation of GDPMP gene deletion mutants of this human pathogen that are devoid of detectable GDPMP activity and completely lack mannose-containing glycoproteins and glycolipids, such as lipophosphoglycan, proteophosphoglycans, glycosylphosphatidylinositol protein membrane anchors, glycoinositolphospholipids and N-glycans. The loss of GDPMP renders the parasites unable to infect macrophages or mice, while gene addback restores virulence. Our study demonstrates that GDP-mannose biosynthesis is not essential for Leishmania viability in culture, but constitutes a virulence pathway in these human pathogens.     

Cj1386, an Atypical Hemin-Binding Protein,Mediates Hemin Trafficking to KatA in Campylobacter jejuni

Catalase enzymes detoxify H₂O₂ by the dismutation of H₂O₂ into O₂ and H₂O through the use of hemin cofactors. While the structure and biochemical properties of catalase enzymes have been well characterized over many decades of research, it remained unclear how catalases acquire hemin. We have previously reported that Cj1386 is essential for ensuring proper hemin content in Campylobacter jejuni catalase (KatA) (A. Flint, Y. Q. Sun, and A. Stintzi, J Bacteriol 194:334–345, 2012). In this report, an in-depth molecular characterization of Cj1386 was performed to elucidate the mechanistic details of this association. Coimmunoprecipitation assays revealed that KatA-Cj1386 transiently interact in vivo, and UV-visible spectroscopy demonstrated that purified Cj1386 protein binds hemin. Furthermore, hemin titration experiments determined that hemin binds to Cj1386 in a 1:1 ratio with hexacoordinate hemin binding. Mutagenesis of potential hemin-coordinating residues in Cj1386 showed that tyrosine 57 was essential for hemin coordination when Cj1386 was overexpressed in Escherichia coli. The importance of tyrosine 57 in hemin trafficking in vivo was confirmed by introducing the cj1386^Y57A allele into a C. jejuni Δcj1386 mutant background. The cj1386^Y57A mutation resulted in increased sensitivity toward H₂O₂ relative to the wild type, suggesting that KatA was not functional in this strain. In support of this finding, KatA immunoprecipitated from the Δcj1386+cj1386^Y57A mutant had significantly reduced hemin content compared to that of the cj1386^WT background. Overall, these findings indicate that Cj1386 is involved in directly trafficking hemin to KatA and that tyrosine 57 plays a key role in this function.     

Cj0011c, a periplasmic single- and double-stranded DNA-binding protein, contributes to natural transformation in Campylobacter jejuni

Campylobacter jejuni is an important bacterial pathogen causing gastroenteritis in humans. C. jejuni is capable of natural transformation, which is considered a major mechanism mediating horizontal gene transfer and generating genetic diversity. Despite recent efforts to elucidate the transformation mechanisms of C. jejuni, the process of DNA binding and uptake in this organism is still not well understood. In this study, we report a previously unrecognized DNA-binding protein (Cj0011c) in C. jejuni that contributes to natural transformation. Cj0011c is a small protein (79 amino acids) with a partial sequence homology to the C-terminal region of ComEA in Bacillus subtilis. Cj0011c bound to both single- and double-stranded DNA. The DNA-binding activity of Cj0011c was demonstrated with a variety of DNAs prepared from C. jejuni or Escherichia coli, suggesting that the DNA binding of Cj0011c is not sequence dependent. Deletion of the cj0011c gene from C. jejuni resulted in 10- to 50-fold reductions in the natural transformation frequency. Different from the B. subtilis ComEA, which is an integral membrane protein, Cj0011c is localized in the periplasmic space of C. jejuni. These results indicate that Cj0011c functions as a periplasmic DNA receptor contributing to the natural transformation of C. jejuni.