Identification of a
            <i>Haemophilus influenzae</i>
            5′-Nucleotidase Protein: Cloning of the
            <i>nucA</i>
            Gene and Immunogenicity and Characterization of the NucA Protein

Zagursky, Robert J.; Ooi, Peggy; Jones, Kevin F.; Fiske, Michael J.; Smith, Robert P.; Green, Bruce A.

doi:10.1128/iai.68.5.2525-2534.2000

Cited by 22 publications

(26 citation statements)

References 45 publications

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“…Recombinant NadN (NucA), originally derived from nontypeable H. influenzae (NTHi) was purified from E. coli INVFЈ(pPX691) as previously described (36). The purified recombinant protein was dialyzed into phosphate-buffered saline (pH 7.…”

Section: Methodsmentioning

confidence: 99%

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (19). After SDS-PAGE, proteins were detected by Western blot analysis, according to the methods of Towbin et al (34), with monoclonal antibodies (MAbs) directed against e (P4) and NadN, respectively, as described earlier (12,36).…”

Section: Methodsmentioning

confidence: 99%

“…The protein product of nucA possessed a 5Ј-nucleotidase activity that acted on 5Ј-phosphorylated nucleosides, e.g., adenosine monophosphate (AMP) (36). Green and coworkers showed that e (P4) and NadN proteins are antigenically highly conserved among both typeable H. influenzae and NTHi isolates (12,13,36), perhaps indicating the physiological importance of these proteins (36).…”

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confidence: 99%

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NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

et al. 2001

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Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5-nucleotidase activity. The e (P4) protein is also shown to have NMN 5-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.Haemophilus influenzae, a gram-negative facultative anaerobic bacterium, is responsible for significant morbidity and mortality in young children (9, 35). In order to cultivate H. influenzae, complex medium is required, and if it is not blood based, it must contain two growth factors: nicotinamide adenine dinucleotide (NAD) and hemin (6). Early biochemical investigations established that nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can substitute for NAD, whereas nicotinamide, niacin, or other nicotine-based intermediates of the Preiss-Handler pathway cannot (10, 20, 31). The NAD dependency of H. influenzae was confirmed by the absence of the genes encoding the enzymes necessary for the de novo biosynthesis of NAD (8). Accumulation of nicotinamide nucleotides derived from NAD or NR has been demonstrated in H. influenzae and Haemophilus parainfluenzae (4, 11). For H. parainfluenzae the K m for transport is about 0.55 M for NAD and 0.14 M for NR, while the V max for NR is about four times that of NAD (4). This implies that NR is the substrate for an as-yet-unidentified inner membrane transporter, a proposal that is supported by the observation that NAD cannot be taken up into the cytosolic compartment as an intact molecule. Limited NAD salvage capacity resides within the H. influenzae cytosol, which can be demonstrated if cell extracts are incubated with NR or NMN, indicating the presence of an NMN adenylyl transferase or an NAD pyrophosphorylase activity (5, 16).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

et al. 2001

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“…The increase in abundance of the prolylpeptidase BA0165, the nucleotidase BA3162, and the SLH protein BA3338 in the ⌬Vollum secretome could suggest that they are not essential for manifestation of B. anthracis virulence, although proteins belonging to the first two families are documented virulence factors in other pathogens (10,102,157,158).…”

Section: Vol 188 2006mentioning

confidence: 99%

Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO₂-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities

et al. 2006

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The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulence plasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomic approach. More than 400 protein spots, representing the products of 64 genes, were identified, and a unique pattern of protein relative abundance with respect to the presence of the virulence plasmids was revealed. In minimal medium under high CO 2 tension, conditions considered to simulate those encountered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carried genes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5 pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2 plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic medium not supplemented with CO 2 , the virulent and avirulent B. anthracis strains manifest very similar protein signatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of gene products attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome. Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulence determinants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification factors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO 2 -responsive chromosome-and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.Bacillus anthracis is a gram-positive spore-forming bacterium that is the etiological agent of anthrax, a lethal disease sporadically affecting humans and animals, in particular herbivores. In its most severe manifestation, B. anthracis infection is initiated by inhalation of spores, which are taken up by alveolar macrophages and germinate into fast-dividing vegetative cells which secrete toxins and virulence factors during growth (81,99). If untreated by prompt antibiotic administration, the bacteria invade the bloodstream, resulting in massive bacteremia and consequently generalized systemic failure and death. B. anthracis is considered to represent a potential biothreat agent, owing to the severity of the anthrax disease, the ease of respiratory contamination, and the perpetual environmental stability of the infective spores. The recent deliberate dissemination of B. anthracis (15) accelerated the efforts to identify new B. anthracis virulence-related determinants for the design of novel diagnostic, preventive, and/or therapeutic strategies.Fully virulent B. anthracis strains harbor two native plasmids, pXO1 and pXO2, which encode critical pathogenicity factors. The absence of either one of the two plasmids results in a pronounce...

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“…Preimmune and week 6 sera were analyzed for anti-P4 antibodies using a P4 enzyme-linked immunosorbent assay (ELISA) (9) with WT rP4 coating the plate. Wholecell ELISAs (16) to determine the reactivity of the sera against surface-exposed epitopes of P4 were performed using NTHi P860295 (2) as the coating antigen. Geometric mean titers (GMT) were determined using ELISA titers of sera from individual mice.…”

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Certain Site-Directed, Nonenzymatically Active Mutants of the Haemophilus influenzae P4 Lipoprotein Are Able To Elicit Bactericidal Antibodies

et al. 2005

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Identification of a Haemophilus influenzae 5′-Nucleotidase Protein: Cloning of the nucA Gene and Immunogenicity and Characterization of the NucA Protein

Cited by 22 publications

References 45 publications

NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO₂-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities

Certain Site-Directed, Nonenzymatically Active Mutants of the Haemophilus influenzae P4 Lipoprotein Are Able To Elicit Bactericidal Antibodies

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Identification of a Haemophilus influenzae 5′-Nucleotidase Protein: Cloning of the nucA Gene and Immunogenicity and Characterization of the NucA Protein

Cited by 22 publications

References 45 publications

NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO2-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities

Certain Site-Directed, Nonenzymatically Active Mutants of the Haemophilus influenzae P4 Lipoprotein Are Able To Elicit Bactericidal Antibodies

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Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO₂-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities