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Molecular Pathogenesis

Ferric Uptake Regulator and Its Role in the Pathogenesis of Nontypeable Haemophilus influenzae

Alistair Harrison, Estevan A. Santana, Blake R. Szelestey, David E. Newsom, Peter White, Kevin M. Mason
S. M. Payne, Editor
Alistair Harrison
The Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, The Center for Microbial Interface Biology, and Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
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Estevan A. Santana
The Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, The Center for Microbial Interface Biology, and Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
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Blake R. Szelestey
The Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, The Center for Microbial Interface Biology, and Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
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David E. Newsom
The Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, The Center for Microbial Interface Biology, and Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
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Peter White
The Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, The Center for Microbial Interface Biology, and Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
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Kevin M. Mason
The Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, The Center for Microbial Interface Biology, and Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
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S. M. Payne
Roles: Editor
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DOI: 10.1128/IAI.01227-12
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  • Fig 1
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    Fig 1

    CLUSTAL W alignment of the deduced amino acid sequence of Fur from NTHi strain 86-028NP, E. coli K-12, P. aeruginosa, H. pylori, and V. cholerae. Amino acids determined to be important in zinc coordination are marked. Open triangles indicate aspartate, arginine (P. aeruginosa), and tyrosine (E. coli) residues. Stars indicate cysteines (H. pylori and E. coli). Open squares show glutamic acids (P. aeruginosa, H. pylori, and V. cholerae). The open circle shows a zinc-coordinating histidine (P. aeruginosa, H. pylori, and V. cholerae). The zinc coordinating HHDH motif is identified using a square bracket (P. aeruginosa, H. pylori, and V. cholerae).

  • Fig 2
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    Fig 2

    The expression of hemoglobin-haptoglobin binding protein B is regulated by both Fur and phase variation. (A) The coding sequence for HgpB (red) contains 12 tetranucleotide repeats (TR) at the 5′ end. In the fur mutant, hgpB contains an extra tetranucleotide repeat, which generated a frameshift. These data are derived from three independent sequencing experiments. (B) qRT-PCR analyses using primers that hybridized upstream of the tetranucleotide repeats (5′) showed that expression of hgpB is classically Fur repressed. Conversely, qRT-PCR analyses using primers that hybridized downstream of the tetranucleotide repeats (3′) showed that the frameshift generated by phase variation reduced the transcription of hgpB in the fur mutant, relative to the parent. **, P < 0.05, while the P value for the 3′ data was 0.08. The data was generated using a Student paired t test, with a two-tailed distribution.

  • Fig 3
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    Fig 3

    Regulation of protein expression by Fur as demonstrated by PAGE analyses. Dysregulation of protein expression in sarcosyl-insoluble membrane fractions of strain 86-028NP (P) and a strain 86-028NP fur mutant (M) when grown in either sBHI (A) or DIS medium supplemented with human hemoglobin (B) was demonstrated.

  • Fig 4
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    Fig 4

    Characterization of strain 86-028NP-specific consensus Fur DNA binding site. (A) The model shows the frequencies of bases at each position (relative heights of the letters) and the degree of sequence conservation (total height of the letter stack). (B) The strain 86-028NP predicted Fur box is highly homologous to that derived for E. coli by Escolar et al. (63). Like the E. coli Fur box, the strain 86-028NP predicted Fur box can be considered to be two 9-bp inverted repeats (B, upper panel) or three repeats of 6 bp (two directed and one inverted) of the invariable sequence (B, lower panel). (C) The promoter regions of Fur-regulated genes bound Fur. Biotin-labeled DNA that contained predicted Fur boxes were mixed with cytoplasmic enriched extracts from either 86-028NPΔfur::Tn903 (−) or 86-Δfur::Tn903(pTS-fur) in which Fur expression was induced (+). Biotinylated DNA was subsequently isolated with streptavidin beads and resolved by PAGE. Fur that copurified with DNA was identified by Western blotting with an anti-Pseudomonas Fur antibody. Three replicate experiments were carried out, and a representative example is shown. An asterisk (*) indicates a nonspecific band that was used as a loading control. (D) To confirm that the binding of Fur to promoter regions was dependent on a predicted Fur box, the Fur binding assay was repeated with the hfeA promoter region with its predicted Fur box, as well as the hfeA promoter region in which the predicted Fur box had been scrambled.

  • Fig 5
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    Fig 5

    Fur is required for the persistence of NTHi strain 86-028NP when in competition with its parent in a chinchilla model of otitis media. (A) Five chinchillas were infected with a 1:1 mix of NTHi strain 86-028NP and NTHi strain 86-028NPΔfur::Tn903. Middle ear fluids were recovered by epitympanic tap on days 4, 7, and 10 postinfection, and the CFU were quantified by plating on selective media. By day 7, the fur mutant could not be recovered from any animal, while the parent persisted for the course of the experiment. (B) Ten days after infection, the fur mutant could not be recovered from middle ear mucosae. The data are expressed as the number of CFU/mg of homogenized mucosa. Two asterisks (**) denote P < 0.05, while one asterisk (*) denotes P < 0.001 as determined by a two-tailed Mann-Whitney U test.

Tables

  • Figures
  • Table 1

    Bacterial strains and plasmids

    Strain or plasmidRelevant genotypeSource or reference
    Strains
        H. influenzae 86-028NPNTHi strain from a child with chronic OM20
        H. influenzae 86-028NPΔfur::Tn903Derivative of 86-028NP with an insertion/deletion mutation of furThis study
        H. influenzae 86-028NPΔfur::Tn903(pTS-fur)86-028NPΔfur::Tn903 transformed with pTS-furThis study
        H. influenzae strain RdA rough derivative of H. influenzae serotype d strain KW2021
        E. coli strain DY380DH10B derivative containing a defective λ prophage; red, bet, and gam genes are controlled by the temperature-sensitive λcI857 repressor95
        E. cloniStrain used for general cloningLucigen
        E. coli strain DH10BStrain used for general cloningInvitrogen
    Plasmids
        pKD3Template plasmid used for mutant construction in E. coli; contains R6Kγ origin of replication30
        pKD3kanpKD3 derivative in which the chloramphenicol resistance gene was replaced with the kanamycin resistance gene from Tn903This study
        pSPECRpCR-Blunt containing a spectinomycin resistance gene37
        pGZRS-39AHaemophilus-Actinobacillus pleuropneumoniae shuttle vector that contains the kanamycin resistance gene from Tn90332
        pSPEC1pGZRS-39A derivative in which the kanamycin resistance gene was replaced with the spectinomycin resistance gene from pSPECR28
        pLS88Broad-host-range plasmid isolated from Haemophilus ducreyi35
        pRSM2947Haemophilus-E. coli shuttle vector with a temperature-sensitive origin of replication from pLS88 and an Flp recombinase gene34
        pET-30bProtein expression vector with f1 origin of replicationEMD Millipore
        pSMART-LCKanLow-copy-number cloning vectorLucigen
        pTE. coli-Haemophilus shuttle vector which contains the tet repressor/promoter from pRSM2947This study
        pT-furpT containing the coding sequence for fur clones downstream of the tet repressor/promoterThis study
        pTS-furA derivative of pT-fur in which the kanamycin resistance gene was replaced with the spectinomycin resistance gene from pSPECRThis study
  • Table 2

    Fur-regulated genes in NTHi strain 86-028NP

    NTHI no.GeneFunctionFold changeaPresence of Fur boxb
    M/PNTHiType bRd
    NTHI0002Long-chain fatty acid coenzyme A ligase–2.22.421.42NSN
    NTHI0007fdxGFormate dehydrogenase major subunit–4.28.367.58NON
    NTHI0010fdxHFormate dehydrogenase, iron-sulfur subunit–3.97.496.003.40N
    NTHI0011fdxIFormate dehydrogenase, cytochrome b556 subunit–2.77.475.233.20N
    NTHI0012fdhEFormate dehydrogenase accessory protein–2.43.353.431.88N
    NTHI0088nrdDAnaerobic ribonucleoside triphosphate reductase9.9–2.95–2.54–4.03N
    NTHI0173Hypothetical protein3.1Y
    NTHI0175Hypothetical protein4.0–12.56–3.93–10.21Y
    NTHI0177hitAIron-utilization periplasmic protein hFbpA7.7–14.27–4.52–8.15Y
    NTHI0179hitBIron(III)-transport system permease protein hFbpB8.2NS–2.70–3.55Y
    NTHI0180hitCIron-utilization ATP-binding protein hFbpC6.9NS–2.16–3.56Y
    NTHI0202hemRHemin receptor3.6–4.72–2.28–2.36Y
    NTHI0209NTHI0209Hypothetical protein–2.2N
    NTHI0285fldAFlavodoxin FldA2.1N
    NTHI0358tonBTonB3.0–1.99–1.69–2.75Y
    NTHI0359exbDBiopolymer transport protein3.3–2.45–1.87–3.02Y
    NTHI0360exbBTransport protein ExbB3.0–3.07–2.26–2.69Y
    NTHI0364NTHI0364Hypothetical protein–2.2–2.27–1.52–1.61Y
    NTHI0369hxuCHeme/hemopexin utilization protein C6.0–24.69–4.03–10.30Y
    NTHI0370hxuBHeme/hemopexin-binding protein B5.5–29.31–3.29–9.20Y
    NTHI0390Metabolite transport protein2.7N
    NTHI0477hfeDPutative ABC-type chelated iron transport system, permease component3.3–2.921.21–4.35Y
    NTHI0478hfeCPutative ABC-type chelated iron transport system, permease component3.0–3.54NS–6.90Y
    NTHI0479hfeBPutative ABC-type chelated iron transport system, ATPase component2.5–7.89NS–8.91Y
    NTHI0782hgpBHemoglobin-haptoglobin binding protein B–8.0–6.52–4.14–1.20Y
    NTHI0783ABC transporter ATP-binding protein4.4NS–2.11–1.61N
    NTHI0785ABC transporter ATP-binding protein3.8NS–1.87–1.61N
    NTHI0805glpCsn-Glycerol-3-phosphate dehydrogenase subunit C–2.2–1.35–1.63NSY
    NTHI0806glpBAnaerobic glycerol-3-phosphate dehydrogenase subunit B–2.8NS–1.72–1.24Y
    NTHI0808glpAsn-Glycerol-3-phosphate dehydrogenase subunit A–2.8Y
    NTHI0813glpKGlycerol kinase–2.1–5.71–3.40–1.61Y
    NTHI0831tnaATryptophanase2.1NHN
    NTHI0998frdDFumarate reductase subunit D–2.4NS–1.95NSN
    NTHI0999frdCFumarate reductase subunit C–2.4NS–1.33NSN
    NTHI1000frdBFumarate reductase iron-sulfur subunit–2.1N
    NTHI1021hbpAHeme-binding protein A–2.2Y
    NTHI1164iga1IgA-specific serine endopeptidase2.1–1.82–1.45–1.52Y
    NTHI1168tbp1Transferrin-binding protein 117.4–9.56–8.16–12.40Y
    NTHI1169tbp2Transferrin-binding protein 2 precursor35.2–19.45–16.53–13.94Y
    NTHI1171Hypothetical protein16.6–15.68–14.61NOY
    NTHI1203Ferredoxin–2.8N
    NTHI1205dmsCAnaerobic dimethyl sulfoxide reductase chain C–4.3NS–1.98–1.06Y
    NTHI1206dmsBAnaerobic dimethyl sulfoxide reductase chain B–4.4NS–2.43NSY
    NTHI1207dmsAAnaerobic dimethyl sulfoxide reductase chain A precursor–5.0NS–4.99–1.10Y
    NTHI1226nrfDNrfD, formate-dependent nitrite reductase, membrane component–3.46.342.241.88Y
    NTHI1227nrfCNrfC, Fe-S-cluster-containing hydrogenase component 1–3.16.991.701.52Y
    NTHI1229nrfBCytochrome c nitrite reductase pentaheme subunit–3.89.49NS2.00Y
    NTHI1230nrfACytochrome c552–3.88.332.532.13Y
    NTHI1321hptHypoxanthine-guanine phosphoribosyltransferase3.3–1.901.15–1.20N
    NTHI1344NTHI1344Hypothetical protein–2.7N
    NTHI1345artMArginine transporter permease subunit ArtM–4.7–4.54–2.24NSN
    NTHI1346artQArginine transporter permease subunit ArtQ–4.8–7.73–2.77NSN
    NTHI1347artIArginine-binding periplasmic protein–4.5–5.87–2.83–1.40N
    NTHI1348artPArginine transporter ATP-binding subunit–2.7–5.68–2.47–1.20N
    NTHI1449hmw2BHMW2B, OMP-85-like protein required for HMW1A and HMW2A secretion–2.3NHN
    NTHI1649fabA3-Hydroxydecanoyl-(acyl carrier protein) dehydratase–2.31.661.77NSN
    NTHI1707ABC transporter periplasmic protein2.3–6.60–2.32–6.12Y
    NTHI1712Hypothetical protein3.4N
    NTHI1716Hypothetical protein2.7NHN
    NTHI1717Hypothetical protein2.2NHY
    NTHI1721Hypothetical protein2.9NHN
    NTHI1722Hypothetical protein3.2NHN
    NTHI1723Hypothetical protein3.4NHN
    NTHI1724Hypothetical protein3.0NHN
    NTHI1726Hypothetical protein2.8NHN
    NTHI1727ninBPutative recombination protein NinB2.4NHN
    NTHI1772ftnBFerritin–2.44.132.242.94Y
    NTHI1773ftnAFerritin-like protein 1–2.73.212.012.60Y
    NTHI1796pqqLPutative zinc protease2.3NSNS–1.86N
    NTHI1961nrdARibonucleotide-diphosphate reductase subunit alpha2.6N
    NTHI1962nrdBRibonucleotide-diphosphate reductase subunit beta2.9NS–1.56–1.54N
    NTHI1979Regulator of cell morphogenesis and NO signaling–2.6Y
    NTHI1984hmw1BHMW1B, OMP-85-like protein required for secretion of HMW1A and HMW2A–2.2NHN
    • ↵a M, 86-028NPΔfur::Tn903; P, 86-028NP. Data for NTHi, type b, and Rd were from Whitby et al. (53) following transition from iron-heme restricted to iron-heme replete medium. NS, not significant; NH, no homolog of 86-028NP gene in strain Rd; NO, not on, i.e., there was no homologue of NTHi or type b gene in strain Rd.

    • ↵b Y, yes; N, no.

  • Table 3

    Fur-regulated genes that encode proteins with hypothetical functions in NTHi strain 86-028NP

    NTHI no.Predicted functionaPredicted motif(s)b
    NTHI0173Nucleoside-diphosphate-sugar epimerasePermease involved in gluconate uptake
    NTHI0175MethylaseSAM-dependent methyltransferase
    NTHI0209
    NTHI0364Ribosome-binding proteinSigma 54 modulation protein
    NTHI1171Type III secretion chaperoneTetratricopeptide repeats
    NTHI1344YadA-like C-terminalYadA-like C-terminal
    NTHI1712DNA binding excisionase
    NTHI1716DNA bindingWinged-helix-like domain
    NTHI1717Hydrolase
    NTHI1721Transcription factorDNA-binding motif
    NTHI1722Transcription factor
    NTHI1723Transcription factorDNA-binding motif
    NTHI1724Primosomal proteinDNA-binding motif
    NTHI1726Metal-binding protein
    • ↵a According to PHYRE2.

    • ↵b According to EMBL InterProScan.

  • Table 4

    Confirmation of Fur-regulated genes in NTHi strain 86-028NPa

    NTHI no.GeneArray data (M/P)qRT-PCR
    M/PC/PFe(–)/Fe(+)
    NTHI0007fdxG–4.2–5.3–1.4–6.0
    NTHI0177hitA5.711.4–6.694.6
    NTHI0360exbB3.05.82.05.5
    NTHI0369hxuC6.09.4–1.57.4
    NTHI0371hxuA1.52.4–2.12.3
    NTHI0479hfeB2.53.7–14.19.5
    NTHI0481hfeA1.34.3–3.89.7
    NTHI0998frdD–2.4–1.41.4–2.1
    NTHI1169tbp235.218.0–2.979.0
    NTHI1207dmsA–5.0–1.11.82.4
    NTHI1230nrfA–3.8–7.2–3.1–17.1
    NTHI1347artI–4.5–2.7b–1.2–5.4
    NTHI1348artP–2.71.61.4–5.0
    NTHI1721Hypothetical2.91.3–1.41.4
    NTHI1773ftnA–2.7–4.8–4.3–1.7
    NTHI1961nrdA2.62.1–3.4–3.5
    • ↵a M, 86-028NPΔfur::Tn903; P, 86-028NP; C, 86-028NPΔfur::Tn903(pTS-fur); Fe(–), 86-028NP (iron chelated); Fe(+), 86-028NP (no iron chelation).

    • ↵b P = 0.2.

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Ferric Uptake Regulator and Its Role in the Pathogenesis of Nontypeable Haemophilus influenzae
Alistair Harrison, Estevan A. Santana, Blake R. Szelestey, David E. Newsom, Peter White, Kevin M. Mason
Infection and Immunity Mar 2013, 81 (4) 1221-1233; DOI: 10.1128/IAI.01227-12

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Ferric Uptake Regulator and Its Role in the Pathogenesis of Nontypeable Haemophilus influenzae
Alistair Harrison, Estevan A. Santana, Blake R. Szelestey, David E. Newsom, Peter White, Kevin M. Mason
Infection and Immunity Mar 2013, 81 (4) 1221-1233; DOI: 10.1128/IAI.01227-12
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