MOLECULAR CHARACTERISATION OF A NOVEL ADP-RIBOSYLATING PUTATIVE TOXIN OF NEISSERIA MENINGITIDIS

Molecular characterisation of a novel ADP-ribosylating putative toxin of Neisseria meningitidis VEGGIi D, *BALDUCCI E, MASIGNANI V, DI MARCELLO F, SAVINO S, ARICO’ B, COMANDUCCI M, PIZZA M, RAPPUOLI R IRIS, Chiron SpA, Via Fiorentina 1, 53100 Siena Italy; *Dipartimento Scienze morfologiche e Biochimiche Comparate, Università degli Studi di Camerino, Camerino, Italy Session: Surface antigens Introduction: By computer analysis on the Neisseria meningitidis (serogroup B, MC 58 strain) genome sequence, a protein with a feature similar to known bacterial ADP-ribosylating toxins (CT produced by Vibrio cholerae, LT by Escherichia coli and PT by Bordetella pertussis) has been identified. Enzymatic assay has shown that this protein (NM-ADPRT) possesses both NAD glycohydrolase and ADP-ribosyltransferase activity. In this study we describe the identification of the putative catalytic residues, their site-directed mutagenesis, and the resulting activity of the mutants. Materials and methods: The novel NM-ADPRT and the correspondent mutants, were expressed in E. coli as C-terminus His-tag protein fusions. Site-directed mutagenesis was performed using the Multi Site-Directed Mutagenesis Kit (QuikChange). Recombinant NM-ADPRT forms were purified from E. coli in their soluble form by metal chelate affinity chromatography. Both the wild-type and the mutants were assayed for their ADP-ribosylation and NAD-glycohydolase activites, using [adenine –U-14C] NAD and agmatine as ADP-ribose acceptor. Antisera against NM-ADPRT and the mutant derivatives were obtained by immunization of CD1 mice. 20μg of each recombinant protein were given i.p. together with CFA for the first dose and IFA for the second (day 21) and the third (day 35) booster doses. Blood sample were taken on days 34 and 49. Immune sera were used in western blot and tested in a bactericidal assay. Results and discussion: On the basis of sequence homology of NM-ADPRT with LT, CT and PT we have identified the putative residues involved in enzymatic activity. These residues have been changed by site-directed mutagenesis and the purified mutant toxins have been tested for both ADP-ribosylating and NAD-glycohydrolase activities. Interestingly, some of the mutants show reduced or abolished enzymatic activity indicating that the identified residues play a role in catalysis. Antisera against the wild-type and mutant toxins have bactericidal activity. The titers induced by two mutants were higher than those induced by the wild-type form. These data suggest that the mutations introduced could influence not only the enzymatic activity but also the in vivo stability of the toxin. Conclusion: A novel ADP-ribosyltransferase has been identified in meningococcus B. Catalytic residues have been predicted by sequence homology and their role in catalysis has been confirmed by site-directed mutagenesis. These molecules are also able to induce a bactericidal response

Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis.

Abstract NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT

The factor H binding protein of Neisseria meningitidis interacts with xenosiderophores in vitro.

The factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis that confers to the bacterium the ability to resist killing by human serum. The determination of its three-dimensional structure revealed that the carboxyl terminus of the protein folds into an eight-stranded ߠbarrel. The structural similarity of this part of the protein to lipocalins provided the rationale for exploring the ability of fHbp to bind siderophores. We found that fHbp was able to bind in vitro siderophores belonging to the cathecolate family and mapped the interaction site by nuclear magnetic resonance. Our results indicated that the enterobactin binding site was distinct from the site involved in binding to human factor H and stimulates new hypotheses about possible multiple activities of fHbp.Full Tex

NarE: a novel ADP-ribosyltransferase from Neisseria meningitidis.

Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co-transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed

Biliary Secretion of Glutathione in Estradiol 17␤-D-Glucuronide- Induced Cholestasis

ABSTRACT Estradiol-17␤-D-glucuronide (E2-17G) induces an acute but reversible inhibition of bile flow after its intravenous administration to rats, due in part to the endocytic retrieval of the canalicular multidrug resistance-associated transporter protein 2 and the bile salt export pump, transporters that contribute to bile flow. Decreased bile salt-independent bile flow (BSIF) is also involved and persists during the phase of recovery from cholestasis. Because glutathione and HCO 3 Ϫ are major contributors to BSIF, we evaluated changes in their biliary excretion and the hepatic content of total glutathione during E2-17G-induced cholestasis. E2-17G acutely decreased bile flow and biliary excretion of total glutathione by about 80%; glutathione excretion was still inhibited at 80 min and 120 min, even though bile flow was partially and totally restored, respectively. Neither liver glutathione content nor the proportions of oxidized glutathione in bile and liver were affected by E2-17G at any time. HCO 3 Ϫ concentrations in bile were unchanged, so that secretion paralleled variations in bile flow. In the isolated perfused liver, addition of E2-17G decreased both bile flow and the biliary concentration of glutathione, whereas addition of its noncholestatic isomer estradiol-3-D-glucuronide (E2-3G) did not inhibit bile flow, but significantly reduced the concentration of glutathione in bile. The bile:liver concentration ratios of glutathione were significantly decreased in vivo by E2-17G and in the perfused liver by E2-17G and E2-3G. These data indicate that E2-17G cis-inhibits the canalicular transport of glutathione and thus contributes to the cholestatic effect by inhibiting BSIF

1H, 13C and 15N assignment of the C-terminal domain of GNA2132 from Neisseria meningitidis

GNA2132 (Genome-derived Neisseria Antigen 2132) is a surface-exposed lipoprotein discovered by reverse vaccinology and expressed by genetically diverse Neisseria meningitidis strains (Pizza et al. 2000). The protein induces bactericidal antibodies against most strains of Meningococccus and has been included in a multivalent recombinant vaccine against N. meningitidis serogroup B. Structure determination of GNA2132 is important for understanding the antigenic properties of the protein in view of increased efficiency vaccine development. We report practically complete 1H, 13C and 15N assignment of the detectable spectrum of a highly conserved C-terminal region of GNA2132 (residues 245–427) in micellar solution, a medium used to improve the spectral quality. The first 32 residues of our construct up to residue 277 were not visible in the spectrum, presumably because of line broadening due to solvent and/or conformational exchange. Secondary structure predictions based on chemical shift information indicate the presence of an all β-protein with eight β strands

Distinct Binding and Immunogenic Properties of the Gonococcal Homologue of Meningococcal Factor H Binding Protein

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups

Dvojni lijekovi primakina i nesteroidnih protuupalnih lijekova: Sinteza, hvatanje slobodnih radikala, antioksidativno djelovanje i keliranje Fe2+ iona

Novel primaquine conjugates with non-steroidal anti-inflammatory drugs (PQ-NSAIDs, 4a-h) were prepared, fully chemically characterized and screened for radical scavenging and antioxidant activities. The synthetic procedure leading to twin drugs 4a-h involved two steps: i) preparation of NSAID benzotriazolides 3a-h from the corresponding NSAID (ibuprofen, ketoprofen, fenoprofen, ketoprofen hydroxy and methylene analogues, diclofenac or indomethacin) and benzotriazole carboxylic acid chloride (BtCOCl, 1), ii) reaction of intermediates 3a-h with PQ. The prepared PQ-NSAIDs exerted moderate activities in the DPPH free radical test and -carotene-linoleic acid assay. Moreover, ketoprofen derivatives 4d and 4b demonstrated a notable Fe2+ chelating ability as well. On the other hand, negligible antiproliferative and antituberculotic effects of conjugates 4a-h were observed.U radu je opisana sinteza novih konjugata primakina s nesteroidnim protuupalnim lijekovima (PQ-NSAIDs, 4a-h), njihova potpuna karakterizacija te testiranje sposobnosti hvatanja slobodnih radikala i antioksidativnog djelovanja. Sintetski postupak za pripravu dvojnih lijekova 4a-h uključuje dva koraka: i) pripravu NSAID-benzotriazolida 3a-h iz odgovarajućih nesteroidnih protuupalnih lijekova (ibuprofena, ketoprofena, fenoprofena, hidroksi i metilenskih analoga ketoprofena, diklofenaka i indometacina) i klorida 1-benzotriazol karboksilne kiseline (BtCOCl, 1), ii) reakciju intermedijera 3a-h s primakinom. Novi PQ-NSAID konjugati pokazuju umjerenu sposobnost hvatanja slobodnih radikala u DPPH testu te umjereno antioksidativno djelovanje u pokusu s -karotenom i linoleinskom kiselinom. Osim toga, derivati ketoprofena 4d i 4b imaju primjetnu sposobnost keliranja Fe2+ iona. Svi konjugati 4a-h pokazuju vrlo slabo antiproliferativno i antituberkulotsko djelovanje