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• W2073921237 abstract "The ygdP and apaH genes of Salmonella enterica serovar Typhimurium (S. Typhimurium) encode two unrelated dinucleoside polyphosphate (Np n N) hydrolases. For example, YgdP cleaves diadenosine tetraphosphate (Ap4A) producing AMP and ATP, while ApaH cleaves Ap4A producing 2ADP. Disruption of ygdP, apaH individually, and disruption of both genes together reduced intracellular invasion of human HEp-2 epithelial cells by S. Typhimurium by 9-, 250-, and 3000-fold, respectively. Adhesion of the mutants was also greatly reduced compared with the wild type. Invasive capacity of both single mutants was restored by transcomplementation with the ygdP gene, suggesting that loss of invasion was due to increased intracellular Np n N. The normal level of 3 μm adenylated Np n N (Ap n N) was increased 1.5-, 3.5-, and 10-fold in the ygdP, apaH and double mutants, respectively. Expression of the putative ptsP virulence gene downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences between the mutants and wild type. The increase in intracellular Np n N in the mutants appears to cause changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian cells. The ygdP and apaH genes of Salmonella enterica serovar Typhimurium (S. Typhimurium) encode two unrelated dinucleoside polyphosphate (Np n N) hydrolases. For example, YgdP cleaves diadenosine tetraphosphate (Ap4A) producing AMP and ATP, while ApaH cleaves Ap4A producing 2ADP. Disruption of ygdP, apaH individually, and disruption of both genes together reduced intracellular invasion of human HEp-2 epithelial cells by S. Typhimurium by 9-, 250-, and 3000-fold, respectively. Adhesion of the mutants was also greatly reduced compared with the wild type. Invasive capacity of both single mutants was restored by transcomplementation with the ygdP gene, suggesting that loss of invasion was due to increased intracellular Np n N. The normal level of 3 μm adenylated Np n N (Ap n N) was increased 1.5-, 3.5-, and 10-fold in the ygdP, apaH and double mutants, respectively. Expression of the putative ptsP virulence gene downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences between the mutants and wild type. The increase in intracellular Np n N in the mutants appears to cause changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian cells. The dinucleoside polyphosphates (Np n N) 1The abbreviations used are: Np n N, dinucleoside 5′,5‴-P 1,P n-polyphosphate; Ap4A, diadenosine 5′,5‴-P 1,P 4-tetraphosphate (other compounds are abbreviated similarly); Ap n N, adenosine(5′)-polyphospho(5′)nucleoside; S. Typhimurium, Salmonella enterica serovar Typhimurium; WT, wild type. are a ubiquitous family of nucleotides found at micromolar to submicromolar concentrations in which two nucleoside moieties are linked 5′-5′ through a polyphosphate chain containing from two to seven phosphoryl groups. The most widely studied are diadenosine 5′,5‴-P 1,P 3-triphosphate (Ap3A) and diadenosine 5′,5‴-P 1,P 4-tetraphosphate (Ap4A), for which several functions have been suggested, although none yet conclusively proved (1McLennan A.G. Ap4 A and Other Dinucleoside Polyphosphates. CRC Press Inc., Boca Raton, FL1992Google Scholar, 2McLennan A.G. Pharmacol. Ther. 2000; 87: 73-89Crossref PubMed Scopus (162) Google Scholar, 3McLennan A.G. Barnes L.D. Blackburn G.M. Brenner C. Guranowski A. Miller A.D. Rovira J.M. Rotllán P. Soria B. Tanner J.A. Sillero A. Drug Dev. Res. 2001; 52: 249-259Crossref Scopus (34) Google Scholar). In Escherichia coli, Ap4A has been proposed to couple DNA replication to cell division (4Nishimura A. Moriya S. Ukai H. Nagai K. Wachi M. Yamada Y. Genes Cells. 1997; 2: 401-413Crossref PubMed Scopus (45) Google Scholar, 5Nishimura A. Trends Biochem. Sci. 1998; 23: 157-159Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and to participate in stress responses by modulating protein refolding by chaperones (3McLennan A.G. Barnes L.D. Blackburn G.M. Brenner C. Guranowski A. Miller A.D. Rovira J.M. Rotllán P. Soria B. Tanner J.A. Sillero A. Drug Dev. Res. 2001; 52: 249-259Crossref Scopus (34) Google Scholar, 6Johnstone D.B. Farr S.B. EMBO J. 1991; 10: 3897-3904Crossref PubMed Scopus (64) Google Scholar, 7Fuge E.K. Farr S.B. J. Bacteriol. 1993; 175: 2321-2326Crossref PubMed Google Scholar). Ap4A and related adenylated dinucleotides (e.g. Ap3N and Ap4N, where N = any nucleoside) are synthesized mainly by aminoacyl-tRNA synthetases, although other ligases have been shown to synthesize them in vitro (8Brevet A. Chen J. Leveque F. Plateau P. Blanquet S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8275-8279Crossref PubMed Scopus (89) Google Scholar, 9Sillero A. Sillero M.A.G. Pharmacol. Ther. 2000; 87: 91-102Crossref PubMed Scopus (59) Google Scholar). In Gram-negative bacteria, the predominant enzyme believed to be responsible for Ap4N hydrolysis is the symmetrically cleaving diadenosine tetraphosphatase, ApaH. This enzyme, which is active toward many Np n N nucleotides (n ≥ 3), degrades Ap4A to two moles of ADP and Ap5A to ADP and ATP (10Plateau P. Fromant M. Brevet A. Gesquière A. Blanquet S. Biochemistry. 1985; 24: 914-922Crossref PubMed Scopus (46) Google Scholar, 11Guranowski A. Jakubowski H. Holler E. J. Biol. Chem. 1983; 258: 14784-14789Abstract Full Text PDF PubMed Google Scholar, 12Guranowski A. Pharmacol. Ther. 2000; 87: 117-139Crossref PubMed Scopus (94) Google Scholar) and is structurally related to serine/threonine protein phosphatases (13Barton G.J. Cohen P.T.W. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (154) Google Scholar, 14Lohse D.L. Denu J.M. Dixon J.E. Structure (Lond.). 1995; 3: 987-990Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Deletion of the E. coli apaH gene leads to a 10 to 100-fold increase in intracellular Ap4N (15Lévêque F. Blanchin-Roland S. Fayat G. Plateau P. Blanquet S. J. Mol. Biol. 1990; 212: 319-329Crossref PubMed Scopus (26) Google Scholar, 16Farr S.B. Arnosti D.N. Chamberlin M.J. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5010-5014Crossref PubMed Scopus (59) Google Scholar). Recently, a second prokaryotic dinucleoside polyphosphate hydrolase was discovered. The IalA protein from the invasive pathogen Bartonella bacilliformis is a member of the Nudix (nucleoside diphosphate linked to X) hydrolase family and hydrolyzes Ap4A asymmetrically to AMP and ATP and Ap5A to ADP and ATP (17Conyers G.B. Bessman M.J. J. Biol. Chem. 1999; 274: 1203-1206Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18Cartwright J.L. Britton P. Minnick M.F. McLennan A.G. Biochem. Biophys. Res. Commun. 1999; 256: 474-479Crossref PubMed Scopus (73) Google Scholar). It is closely related to the eukaryotic Ap4A hydrolases, particularly those from plants, which also hydrolyze many Np n N species, where n ≥ 4 (12Guranowski A. Pharmacol. Ther. 2000; 87: 117-139Crossref PubMed Scopus (94) Google Scholar, 18Cartwright J.L. Britton P. Minnick M.F. McLennan A.G. Biochem. Biophys. Res. Commun. 1999; 256: 474-479Crossref PubMed Scopus (73) Google Scholar). The ialA gene has been implicated indirectly in the process of cellular invasion by this bacterium; expression of B. bacilliformis ialA in non-invasive E. coli renders it invasive (19Mitchell S.J. Minnick M.F. Infect. Immun. 1995; 63: 1552-1562Crossref PubMed Google Scholar). Furthermore, the orthologous ygdP gene from E. coli K1 may be required for the invasion of human brain microvascular endothelial cells as its expression is up-regulated by invasion-enhancing growth conditions and down-regulated by invasion-repressing conditions (20Badger J.L. Wass C.A. Kim K.S. Mol. Microbiol. 2000; 36: 174-182Crossref PubMed Scopus (87) Google Scholar). YgdP and the related InvA protein from Rickettsia prowazekii preferentially hydrolyze Ap5A (21Bessman M.J. Walsh J.D. Dunn C.A. Swaminathan J. Weldon J.E. Shen J.Y. J. Biol. Chem. 2001; 276: 37834-37838Abstract Full Text Full Text PDF PubMed Google Scholar, 22Gaywee J. Xu W. Radulovic S. Bessman M.J. Azad A.F. Mol. Cell. Proteom. 2002; 1: 179-185Abstract Full Text Full Text PDF PubMed Google Scholar). Since the intracellular levels of several Ap n N species are known to increase substantially under conditions of oxidative stress (23Bochner B.R. Lee P.C. Wilson S.W. Cutler C.W. Ames B.N. Cell. 1984; 37: 225-232Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 24Lee P.C. Bochner B.R. Ames B.N. J. Biol. Chem. 1983; 258: 6827-6834Abstract Full Text PDF PubMed Google Scholar), we previously suggested that the ability to metabolize Np n N may be necessary for invasion in the face of an oxidative attack by the invaded cell (18Cartwright J.L. Britton P. Minnick M.F. McLennan A.G. Biochem. Biophys. Res. Commun. 1999; 256: 474-479Crossref PubMed Scopus (73) Google Scholar). If that were so, then the apaH gene would be expected to be essential for invasion as well. Indeed, a DNA fragment from the oral pathogen Actinobacillus actinomycetemcomitans that confers an invasive ability on E. coli contains the apaH gene (25Saarela M. Asikainen S. Alaluusua S. Fives-Taylor P. Anaerobe. 1998; 4: 139-144Crossref PubMed Scopus (7) Google Scholar). We have, therefore, examined the effects of deleting the ygdP and apaH genes both singly and doubly on the invasive ability of Salmonella enterica serovar Typhimurium (S. Typhimurium), a facultative intracellular parasite that can invade and multiply within various cell types, including phagocytes and epithelial cells, and so establish a chronic infection. Our results provide the first direct evidence of the involvement of these bacterial genes in intracellular invasion. Reagents, Bacterial Strains, and Plasmids—Recombinant human Ap4A hydrolase was prepared as described for the Caenorhabditis elegans enzyme (26Abdelghany H.M. Gasmi L. Cartwright J.L. Bailey S. Rafferty J.B. McLennan A.G. Biochim. Biophys. Acta. 2001; 1550: 27-36Crossref PubMed Scopus (34) Google Scholar). Strains and plasmids used and produced in this study are listed in Table I. Plasmids were introduced into E. coli by transformation and into S. Typhimurium strain LT2 by electroporation using a Bio-Rad Gene Pulser II.Table IBacterial strains and plasmids usedStrain or plasmidDescriptionSource or Ref.E. coli strainsBL21(DE3)F- ompT hsdSB (rB-mB-) gal dcm (DE3)NovagenCC118 (λpir)araD139 Δ(ara-leu)7697 ΔlacX74 phoAD20 galE galK thi rpsE rpoB argE(Am) recA1λ[ρ]pir27Herrero M. de Lorenzo V. Timmis K.N. J. Bacteriol. 1990; 172: 6557-6567Crossref PubMed Scopus (1304) Google ScholarTOP10F- mcrA Δ(mrr-hsdMRS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL(StrRR) endA1 nupGInvitrogenS. typhimurium strainsLT2Wild typeSTYA201LT2; ΔapaH::kan (KanR)This studySTYY202LT2; ΔygdP::kan (KanR)This studySTYAY203LT2; ΔapaH::cat ΔygdP::kan (CamR KanR)This studyPlasmidsplysSCamRNovagenpUC4KKanRAmershampET-ApaHVector pET15b (Novagen) carrying the S. typhimurium apaH gene; (AmpR)This studypET-YgdPVector pET32b (Novagen) carrying the S. typhimurium ygdP gene; (AmpR)This studypGEM-AKVector pGEM®-T Easy (Promega) carrying the apaH::kan disruption cassetteThis studypGEM-YKVector pGEM®-T Easy (Promega) carrying the ygdP::kan disruption cassetteThis studypGEM-ACVector pGEM®-T Easy (Promega) carrying the apaH::cat disruption cassetteThis studypMRS101oriR6K oriE1 mobRK2 bla(AmpR) strAB(StrR) sacBRBCCM/LMBPpTI201pMRS101 carrying the apaH::kan disruption cassetteThis studypTI202pMRS101 carrying the ygdP::kan disruption cassetteThis studypTI203pMRS101 carrying the ygdP::cat disruption cassetteThis studypTrc-ApaHVector pTrcHis2-TOPO (Invitrogen) carrying the S. typhimurium apaH gene; (AmpR)This studypTrc-YgdPVector pTrcHis2-TOPO (Invitrogen) carrying the S. typhimurium ygdP gene; (AmpR)This study Open table in a new tab DNA Manipulation and Analysis—DNA ligation, restriction analysis, and gel electrophoresis were carried out as described by Sambrook et al. (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Cloning, Expression, and Purification of ApaH and YgdP—The coding regions of the apaH and ygdP genes were PCR-amplified from S. Typhimurium genomic DNA. ApaH was amplified using the 5′ primer (5′-ATTATACATATGGCAACTTATCTCATC-3′) containing an NdeI site and the 3′ primer (5′-TTTCGGGATCCTGGAGCGTC-3′) containing a BamHI site and inserted after restriction digestion between the NdeI and BamHI sites of pET15b (Novagen) to give pET-ApaH. YgdP was amplified using the 5′ primer (5′-AAGGTTTATCCATGGGTAGTCCGGTG-3′) containing an NcoI site and the 3′ primer (5′-AATATTCAGCGCCTCGAGCAGAC-3′) containing a XhoI site and inserted after restriction digestion between the NcoI and XhoI sites of pET32b (Novagen) to give pET-YgdP. For expression, plasmids were transformed into E. coli BL21(DE3). The His-tagged recombinant proteins were expressed and purified on NiCAM™-HC resin (Sigma) as described previously (29AbdelRaheim, S., and McLennan, A. G. (2002) BMC Biochemistry http://www.biomedcentral.com/1471-2091/3/5.Google Scholar). Construction of apaH and ygdP Disruption Cassettes—Gene disruption cassettes were generated by PCR according to Wach et al. (30Wach A. Brachat A. Pöhlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar). An apaH::kan cassette was constructed as follows. First, a 300-bp 5′ segment of the apaH gene was amplified from S. Typhimurium genomic DNA using the 5′ primer HP1 (5′-TCCCCCGGGATGAACGTTACGTTTTTGC-3′) and the 3′ primer HP2K (5′-TGCAGGTCGACGGATCCGGCGATCAGTTCGTCGTAG-3′), where HP1 corresponds to part of the 5′ non-coding region of apaH and HP2K contains the 5′ end of the coding region of apaH and the 5′ end of the non-coding region of the kanamycin resistance gene from pUC4K. A 285-bp 3′ segment of the apaH gene was also amplified using the 5′ primer HP3K (5′-GCTCGATGAGTTTTTCTAATGCGCTGGGAAGATAAACAG-3′) and the 3′ primer HP4 (5′-CATGTCTAGACGCAGAATTCTCACAGCTATTG-3′), where HP3K contains the 3′ end of the coding region of apaH and the 3′ end of the non-coding region of the kanamycin-resistance gene and HP4 corresponds to part of the 3′ non-coding region of apaH. In the second step, the complete kanamycin resistance gene was amplified from pUC4K using HP1, HP4, and the two PCR products from the first step as primers to give the apaH::kan cassette in which the kanamycin resistance gene is flanked by >250 bp segments of the apaH gene. A ygdP::kan disruption cassette was produced in a similar manner using primers YP1 (5′-CATGTCTAGACTTAGATGTGATGCTGGTCA-3′), YP2K (5′-TGCAGGTCGACGGATC ATCATCAATCACCGGAC-3′), YP3K (5′-GCTCGATGAGTTTTTCTAAGCTCAG GATAATCC-3′), and YP4 (5′-CATGTCTAGAATGATCGGCACACCGAG-3′). Finally, an apaH::cat cassette containing the chloramphenicol resistance gene from pLysS was produced using the primers HP1, HP2C (5′-GGGACACCAGGATTTATGGCGATCAGTTCGTCGTAG-3′), HP3C (5′-GTGGCAGGGCGGGGCGTAATGCGCTGGGAAGATAAACAG-3′), and HP4. Construction of Single and Double apaH and ygdP Deletion Mutants of S. Typhimurium—ApaH::kan and ygdP::kan disruption cassettes were first cloned into the pGEM®-T Easy vector (Promega) to give plasmids pGEM-AK and pGEM-YK respectively and the plasmids transformed into TOP10 E. coli for propagation. Recovered plasmids were digested with ApaI and SpeI and the cassettes purified after gel electrophoresis using a Qiagen purification kit. Cassettes were then ligated into the cut suicide vector pMRS101 (31Sarker M.R. Cornelis G.R. Mol. Microbiol. 1997; 23: 409-411Crossref PubMed Scopus (41) Google Scholar). The ligation mixture was transformed into E. coli K12 CC118 (λpir) and the resulting plasmids, pTI201 and pTI202, electroporated into S. Typhimurium LT2. The transformants were isolated on LB medium supplemented with 50 μg/ml kanamycin and 50 μg/ml streptomycin and subcultured again on LB medium containing 10% sucrose to select deletants resulting from a double crossover event. Surviving colonies were then subcultured again on kanamycin-containing medium to give strains STYA201 (ΔapaH::kan) and STYY202 (ΔygdP::kan). The apaH::cat chloramphenicol disruption cassette was cloned into pGEM®-T Easy in a similar manner to give pGEM-AC then transferred to pMRS101 to give pTI203, which was introduced into STYY202. Colonies of the ΔapaH::cat ΔygdP::kan double mutant STYAY203 were selected as above on sucrose and on medium containing 25 μg/ml chloramphenicol and 50 μg/ml kanamycin. All deletions were confirmed by PCR analysis of three independent isolates. Construction of Expression Plasmids for Complementation—The apaH gene was PCR-amplified from genomic DNA using Taq polymerase and the 5′ primer (5′-ATGGCAACTTATCTCATCGGCGAC-3′) and 3′ primer (5′-CATGTCTAGACGCAGAATTCTCACAGCTATTG-3′) and the ygdP gene using the 5′ primer (5′-GGTAGTCCGGTGATTGATGACGATG-3′) and the 3′ primer (5′-TCGACTATTTCGCGCAGGCGAGTG-3′). Both PCR products were cloned into the pTrcHis2-TOPO vector (Invitrogen) and propagated in TOP10 E. coli to yield the plasmids pTrc-ApaH and pTrc-YgdP. Purified plasmids were then electroporated into S. Typhimurium strains STYA201 and STYY202 and transformants selected on LB agar plates containing 75 μg/ml ampicillin and 50 μg/ml kanamycin. Invasion and Adhesion Assays—HEp-2 epithelial cells and U937 macrophage-like cells were maintained in Eagle's minimal essential medium (Invitrogen) supplemented with 5% (v/v) fetal calf serum and 0.15% Na2HCO3. HEp-2 cells were seeded on coverslips in vials at 1 × 105 cells/coverslip and grown overnight at 37 °C in 1 ml of medium. U937 cells were split into 1-ml cultures at a density of 2 × 105 cells/ml on the day of use. After inoculation with 50 μl of overnight bacterial cultures (4 × 109/ml), cells were incubated at 37 °C in a 5% CO2 for 3 h. For invasion assays, cells were incubated for 1 h with 25 μg/ml gentamicin, washed five times with 1 ml of phosphate-buffered saline, then lysed in 0.5 ml 0.5% sodium deoxycholate. Lysates were diluted in phosphate-buffered saline and the viable count determined on LB agar plates (containing 50 μg/ml kanamycin for mutants). For adhesion assays, incubation with gentamicin was omitted. In addition, HEp-2 monolayers were washed with phosphate-buffered saline after adhesion, fixed with methanol, and stained with a 10% solution of Giemsa prior to examination by light microscopy. Metabolic Phenotype—Nineteen different enzyme activities of the wild type (WT) S. Typhimurium and its apaH, ygdP, and apaH ygdP double null mutants were examined using the API ZYM kit (bioMérieux, Basingstoke, UK). The ability of the WT and mutant strains to metabolize a variety of different carbohydrates was determined by using the API 50 CH system (bioMérieux). This system tests assimilation, oxidation, and fermentation of the carbohydrate sources. Extraction and Assay of ApnN—Cells from 50-ml cultures of S. Typhimurium (WT and mutants) in mid log phase (OD 0.6–0.7) were collected by rapid centrifugation (5000 × g for 5 min). Pellets were resuspended in 5 ml of ice-cold 0.4 m trichloroacetic acid and shaken for 15 min. Neutralization, alkaline phosphatase digestion, and purification of the dinucleotide-containing fraction were as described previously (32Murphy G.A. Halliday D. McLennan A.G. Cancer Res. 2000; 60: 2342-2344PubMed Google Scholar). The freeze-dried extract was dissolved in 0.1 ml 30 mm Hepes-NaOH, pH 7.7, 5 mm magnesium acetate and triplicate 25-μl samples each mixed with 25 μl of luciferin/luciferase ATP-monitoring reagent (Bio-Orbit). After measuring the background luminescence, 1 ng of recombinant human Ap4A hydrolase was added to generate ATP and the increase in luminescence determined. This generalized assay measures all ATP-generating nucleotides of the form Ap n N, where n ≥ 4. Samples (10 μl) of the neutralized acid extract were also retained for luminometric ATP determination before alkaline phosphatase digestion. These were mixed with 90 μl of 30 mm Hepes-NaOH, pH 7.7, 5 mm magnesium acetate, and triplicate 25-μl samples then each added to 25 μl of luciferin/luciferase ATP-monitoring reagent and the luminescence determined. Previous ATP determinations had revealed no significant differences in intracellular ATP between any of the strains (values ± S.E. were 10.1 ± 1.5, 9.5 ± 0.6, 10.9 ± 1.6, and 9.7 ± 1.3 nmol/mg of protein (n = 3) in wild type LT, STYA201, STYY202, and STYAY203, respectively). Therefore, the ATP content of the extracts acts as an internal standard for the extraction process. The intracellular concentration of Ap n N (n ≥ 4) was then estimated from the Ap n N/ATP ratio making the following assumptions: (i) that, for all strains, the intracellular ATP concentration is 3 mm (33Bochner B.R. Ames B.N. J. Biol. Chem. 1982; 257: 9759-9769Abstract Full Text PDF PubMed Google Scholar); (ii) that Ap n A is 50% of the Ap n N pool (34Plateau P. Fromant M. Kepes F. Blanquet S. J. Bacteriol. 1987; 169: 419-424Crossref PubMed Scopus (21) Google Scholar); (iii) that the human Ap4A hydrolase used in the assay cleaves equally efficiently at either end of each Ap n N and so 1 mol of Ap n A yields 1 mol of ATP, while 1 mol of Ap n N yields 0.5 mol of ATP. The latter two assumptions require the figure for Ap n N concentration to be multiplied by 1.33 to compensate for the 50% efficiency in ATP production from Ap n N (N ≠ A). Assay of ApaH and YgdP Activity—Activity of YgdP with Ap4A, Ap5A, and Ap6A was measured luminometrically by direct continuous assay of the ATP product and kinetic parameters determined by non-linear regression analysis (26Abdelghany H.M. Gasmi L. Cartwright J.L. Bailey S. Rafferty J.B. McLennan A.G. Biochim. Biophys. Acta. 2001; 1550: 27-36Crossref PubMed Scopus (34) Google Scholar). Activity of ApaH with Ap5A was measured in the same way, while activity with Ap4A required the inclusion of 2 mm phosphoenolpyruvate and 5 μg of pyruvate kinase in the assay to convert the ADP product to ATP (35Prescott M. Thorne N.M.H. Milne A.D. McLennan A.G. Int. J. Biochem. 1992; 24: 565-571Crossref PubMed Scopus (13) Google Scholar). In addition, all ApaH assays contained 100 μm CoCl2 (10Plateau P. Fromant M. Brevet A. Gesquière A. Blanquet S. Biochemistry. 1985; 24: 914-922Crossref PubMed Scopus (46) Google Scholar, 11Guranowski A. Jakubowski H. Holler E. J. Biol. Chem. 1983; 258: 14784-14789Abstract Full Text PDF PubMed Google Scholar). Product identification was by high performance liquid chromatography as described previously (26Abdelghany H.M. Gasmi L. Cartwright J.L. Bailey S. Rafferty J.B. McLennan A.G. Biochim. Biophys. Acta. 2001; 1550: 27-36Crossref PubMed Scopus (34) Google Scholar). Reverse Transcription-PCR—Expression of the ptsP gene in WT and mutant strains was determined by reverse transcription-PCR. Total S. Typhimurium RNA (1 μg, DNase-treated) was incubated at 70 °C for 5 min with 20 pmol of reverse primer (5′-CGCGACCAGAATAAAACGTTCC-3′) in 11 μl of water, then incubated at 37 °C for 5 min in a final volume of 19 μl containing 4 μl of Moloney murine leukemia virus buffer (MBI Fermentas), 1 mm concentration of each dNTP, and 20 units RNase inhibitor. First strand cDNA was synthesized by adding 1 μl (200 units) of Moloney murine leukemia virus reverse transcriptase (MBI Fermentas) and incubating at 42 °C for 60 min. One μl of this was amplified in a final volume of 20 μl containing 20 pmol of forward (5′-GATCATTCAGCGTCGCCAAC-3′) and reverse primers, 0.1 mm concentration of each dNTP, 2.5 mm MgCl2, 2.5 units of Taq polymerase, and 2 μl of Taq buffer (MBI Fermentas). Properties of the ApaH and YgdP Proteins—To confirm that the S. Typhimurium ApaH and YgdP proteins had the enzymic activities predicted from their sequences, they were cloned and expressed in E. coli BL21(DE3) cells: ApaH in pET15b as a His-tagged 33.6-kDa protein and YgdP in pET32b as a His-tagged thioredoxin fusion protein of total mass 39.2 kDa. When purified to homogeneity (Fig. 1A), the enzymes had the expected activities. ApaH efficiently hydrolyzed Ap4A, Ap5A, and Ap6A, always producing ADP as one product, while YgdP hydrolyzed the same nucleotides, with a preference for Ap5A, like the E. coli and R. prowazekii enzymes (21Bessman M.J. Walsh J.D. Dunn C.A. Swaminathan J. Weldon J.E. Shen J.Y. J. Biol. Chem. 2001; 276: 37834-37838Abstract Full Text Full Text PDF PubMed Google Scholar, 22Gaywee J. Xu W. Radulovic S. Bessman M.J. Azad A.F. Mol. Cell. Proteom. 2002; 1: 179-185Abstract Full Text Full Text PDF PubMed Google Scholar), and always producing ATP as one product. Both enzymes followed Michaelis-Menten kinetics with all substrates tested; representative plots for the hydrolysis of Ap4A by both enzymes are shown in Fig. 1B. Kinetic constants were calculated by non-linear regression. Km and k cat values for ApaH for Ap4A and Ap5A were 37 μm and 37 s–1 and 14 μm and 33 s–1, respectively, similar to the E. coli enzyme when assayed under the same conditions (10Plateau P. Fromant M. Brevet A. Gesquière A. Blanquet S. Biochemistry. 1985; 24: 914-922Crossref PubMed Scopus (46) Google Scholar, 11Guranowski A. Jakubowski H. Holler E. J. Biol. Chem. 1983; 258: 14784-14789Abstract Full Text PDF PubMed Google Scholar). Km and k cat values for YgdP for Ap4A, Ap5A, and Ap6A were 18 μm and 18 s–1, 22 μm and 32 s–1, and 54 μm and 0.8 s–1, respectively. ApaH, YgdP, and ApnN in WT and Mutant Cells—ΔapaH (STYA201) and ΔygdP (STYY202) null mutants and a ΔapaH ΔygdP double null mutant (STYAY203) were generated by replacement of the genes with antibiotic resistance cassettes (Table I). Gene deletion was confirmed by PCR and by measurement of Ap4A hydrolytic activities in cell extracts. ApaH and YgdP have predicted pI values of 4.8 and 10.0, respectively. Thus, they can be measured independently after separation by batch anion-exchange chromatography at pH 7.5. Table II confirms the absence of the enzymes in the appropriate mutants. The total concentration of nucleotides of general structure Ap n N(n ≥ 4) was also measured using a luciferase-based assay in which human Ap4A hydrolase is used to generate ATP from Ap n N (n ≥ 4) compounds. WT cells had 3.6 μm Ap n N, which compares favorably with previous figures of 3 μm in both S. Typhimurium (23Bochner B.R. Lee P.C. Wilson S.W. Cutler C.W. Ames B.N. Cell. 1984; 37: 225-232Abstract Full Text PDF PubMed Scopus (206) Google Scholar) and E. coli (15Lévêque F. Blanchin-Roland S. Fayat G. Plateau P. Blanquet S. J. Mol. Biol. 1990; 212: 319-329Crossref PubMed Scopus (26) Google Scholar). Deletion of ygdP led to a slight, 1.5-fold increase in Ap n N and deletion of apaH to a 3.5-fold increase, while deletion of both genes led to a 10-fold increase. These results show that both YgdP and ApaH contribute to control of the Ap n N pool in S. Typhimurium. As non-adenylated Np n N, which are not detected by the assay, are also substrates for these two hydrolases, it is likely that their levels also increase. Hence the Ap n N pool measurements provide a rough indication of the effects of deleting the hydrolase genes but do not yet convey the detailed picture.Table IIApaH, YgdP, and ApnN in cell extractsStrainApaH activityYgdP activityApnNunits/mg proteinunits/mg proteinμ mWT7.17.53.6 ± 0.4STYY202 (ΔygdP)6.205.1 ± 0.3STYA201 (ΔapaH)06.312.6 ± 1.2STYAY203 (ΔapaH ΔygdP)0034.0 ± 1.6 Open table in a new tab Invasion—The ability of the mutants to invade HEp-2 epithelial cells was determined using a gentamicin protection assay (36Fletcher J.N. Embaye H.E. Getty B. Batt R.M. Hart C.A. Saunders J.R. Infect. Immun. 1992; 60: 2229-2236Crossref PubMed Google Scholar). Deletion of ygdP (STYY202) reduced invasion by 9-fold compared with the WT, while deletion of apaH (STYA201) reduced invasion by 250-fold. Deletion of both genes (STYAY203) produced a dramatic 3000-fold reduction (Fig. 2). Importantly, transformation of both STYY202 and STYA201 with the YgdP expression plasmid pTrc-YgdP restored full invasive capacity. This strongly suggests that loss of invasion is primarily related to a common property of YgdP and ApaH, i.e. the hydrolysis of Np n N, rather than to some protein-specific function. Transcomplementation of apaH in STYA201 partially restored invasion (18-fold) but appeared to have no effect on STYY202 (Fig. 2). This incomplete restoration may indicate poor expression of the cloned apaH gene or that the specific Ap n N or Np n N responsible for creating the non-invasive phenotype are better substrates for YgdP. It also suggests that the full phenotypes of the single mutants have elements that are distinct, in addition to the common feature and consequences of increased Ap n N and Np n N. Similar data were obtained with U-937 macrophage-like cells. Adhesion of the single mutants to both HEp-2 and U-937 cells was measured by recovery of colony-forming units from washed cell cultures. Both STYA201 and STYY202 showed a dramatically reduced ability to adhere to either cell type (Fig. 3). This was confirmed by microscopic examination after Giemsa staining (36Fletcher J.N. Embaye H.E. Getty B. Batt R.M. Hart C.A. Saunders J.R. Infect. Immun. 1992; 60: 2229-2236Crossref PubMed Google Scholar). Cell Growth, Morphology, and Motility—Apparent doubling times in LB determined from optical density measurements were similar for WT, STYA201, and STYY202 (20–22 min). However, STYA201 showed a much longer lag-phase after inoculation compared with the others (4.5 versus" @default.
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• W2073921237 title "Regulation of Dinucleoside Polyphosphate Pools by the YgdP and ApaH Hydrolases Is Essential for the Ability of Salmonella enterica serovar Typhimurium to Invade Cultured Mammalian Cells" @default.
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