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(A) Binding of the A. actinomycetemcomitans Fur protein to bacterial iron box. The DNA used in this assay was radiolabeled with ³²P and contained the iron box consensus sequence 5′-GATAATGATAATCATTATC-3′. The protein samples used were extracts of E. coli fur strain H1780 containing plasmid pUC18. Lane 1, Fur box DNA; lane 2, pUC18 plus H1780; lane 3, Aafur; lane 4, E. coli fur. (B) Binding of the A. actinomycetemcomitans Fur protein to the A. actinomycetemcomitans fur promoter region. Lane 1, E. coli fur; lane 2, Aafur; lane 3, empty; lane 4, pUC18 plus H1780; lane 5, A. actinomycetemcomitans fur box DNA (82 bp from the upstream region of Aa fur gene).
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In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants
through the fur gene product. Initial experiments showed that a cloned Escherichia coli fur gene probe hybridized to Southern blots of Actinobacillus actinomycetemcomitans strain JP2 (serotype b) chromosomal DNA. The A. actin...
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... Ketersediaan zat besi menjadi sinyal yang penting yang meregulasi ekspresi dari banyak faktor virulensi pada bakteri patogen. 7 Zat besi juga merupakan aktivator pembentukan biofilm, dan pada beberapa kasus, zat besi menghambat pembentukan biofilm. 5 Rhodes et al. 8 Prosedur untuk pembuatan antibodi primer terhadap PNAG meliputi: kultur bakteri, isolasi protein bakteri, elektroforesis, elektroelusi, imunisasi pada hewan coba dan isolasi serum, serta purifikasi antibodi. ...
... Isolasi protein bakteri dan elektroforesis pada prosedur ini dilakukan seperti isolasi protein bakteri dan elektroforesis pada prosedur sebelumnya. 7 Gel dari elektroforesis dilakukan running dengan Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE). Protein dari gel SDS PAGE ditransfer ke membran nitrocellulose dengan alat semi-dry (kertas saring, membran nitrocellulose, dan gel hasil running disusun seperti sandwich dengan komposisi: 9 lembar kertas saring bagian bawah, membran nitrocellulose, gel hasil running, dan 6 kertas saring). ...
... 9 Ketersediaan zat besi menjadi sinyal yang penting yang meregulasi ekspresi dari banyak faktor virulensi pada bakteri patogen. 7 Zat besi juga merupakan aktivator pembentukan biofilm, dan pada beberapa kasus, zat besi menghambat pembentukan biofilm. 5 Paparan zat besi pada A. actinomycetemcomitans menyebabkan protein Fur membentuk kompleks dengan Fe 2+ yang berikatan pada sekuen kosensus yang spesifik (yang disebut "Fur box") pada sRNA. ...
Background: The study of biofilms bacteria could be an alternative of preventive treatment in reducing prevalence of aggressive periodontitis in the community, because biofilm protects the bacteria from environmental conditions, including the attack of immune system and antimicrobial. Aggregatibacter actinomycetemcomitans is a major cause of bacterial aggressive periodontitis. Purpose: This study aims to examine the iron exposure to specific protein expression of extracellular polymeric substance (EPS) of Aggregatibacter actinomycetemcomitans biofilm. Methods: Protein containing EPS biofilm was isolated from cultures of A.actinomycetemcomitans. The protein was processed through several procedures: electrophoresis , electroelution , immunization of rabbits , serum isolation , and purification of antibodies. After the Western blotting procedure the antibody was used. Protein containing EPS biofilms exposed to iron, then once again isolated from cultures of A. actinomycetemcomitans. The electrophoresis and Western blotting were done on the isolated protein. Results: The result showed that the the expression of specific proteins in EPS biofilm decreased in response to iron exposure. Conclusions: Iron exposure could influenced the specific protein expression in EPS biofilm of Aggregatibacter actinomycetemcomitans.Latar belakang: Penelitian terhadap bakteri biofilm dapat menjadi alternatif perawatan preventif dalam menurunkan prevalensi periodontitis agresif di masyarakat, karena biofilm melindungi bakteri terhadap kondisi lingkungan, termasuk serangan sistem imun dan antimikroba. Aggregatibacter actinomycetemcomitans merupakan bakteri penyebab utama periodontitis agresif. Tujuan: Studi ini bertujuan meneliti paparan zat besi terhadap ekspresi protein spesifik extracellular polymeric substance (EPS) Aggregatibacter actinomycetemcomitans. Metode: Protein yang mengandung EPS biofilm diisolasi dari kultur A. actinomycetemcomitans. Protein yang diisolasi ini kemudian melalui beberapa prosedur: elektroforesis, elektroelusi, imunisasi pada kelinci, isolasi serum, dan purifikasi antibodi. Pada prosedur Western blotting di sesi penelitian berikutnya antibodi ini digunakan. Protein yang mengandung EPS biofilm dipapar dengan zat besi, kemudian diisolasi sekali lagi dari kultur A. actinomycetemcomitans. Protein yang diisolasi dilakukan elektroforesis dan Western blotting. Western blotting. Hasil: Penelitian ini menunjukkan hasil berupa penurunan ekspresi protein spesifik biofilm EPS sebagai respon terhadap paparan zat besi. Simpulan: Paparan zat besi memberi pengaruh ekspresi protein spesifik biofilm EPS Aggregatibacter actinomycetemcomitans.
... These Fur box-like motifs were 63-74% (12-14 of the 19 bp) identical to the consensus Fur box of E. coli (supporting information, Table S3). Previously, a Fur box-like sequence with 68% identity to the consensus Fur box of E. coli was located downstream of a −35 and −10 sequence in the fur gene of A. actinomycetemcomitans (Haraszthy et al., 2002). The Fur box of the fur gene of A. actinomycetemcomitans is 52-65% identical to the putative sRNA Fur box motifs reported in this study. ...
Iron can regulate biofilm formation via non-coding small RNA (sRNA). To determine if iron-regulated sRNAs are involved in biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans, total RNA was isolated from bacteria cultured with iron supplementation or chelation. Transcriptional analysis demonstrated that the expression of four sRNA molecules (JA01-JA04) identified by bioinformatics was significantly upregulated in iron-limited medium compared with iron-rich medium. A DNA fragment encoding each sRNA promoter was able to titrate Escherichia coli ferric uptake regulator (Fur) from a Fur-repressible reporter fusion in an iron uptake regulator titration assay. Cell lysates containing recombinant AaFur shifted the mobility of sRNA-specific DNAs in a gel shift assay. Potential targets of these sRNAs, determined in silico, included genes involved in biofilm formation. The A. actinomycetemcomitans overexpressing JA03 sRNA maintained a rough phenotype on agar, but no longer adhered to uncoated polystyrene or glass, although biofilm determinant gene expression was only modestly decreased. In summary, these sRNAs have the ability to modulate biofilm formation, but their functional target genes remain to be confirmed.
... A. actinomycetemcomitans 67 was cultured in brain heart infusion broth supplemented with hemin and vitamin K at 37°C in an anaerobic chamber, and plated on brain heart infusion agar plates supplemented with hemin and vitamin K to assess purity every two weeks. E. coli was grown either in Luria broth or on Luria agar at 37°C in ambient air (Haraszthy et al., 2002). HepG cells were grown in RPMI 1640 media supplemented with 10% fetal calf serum, streptomycin and 10 mM HEPES in 5% CO 2 at 37°C. ...
Commercially available photodynamic therapy for periodontal diseases utilizes methylene blue as a photosensitizer. Here we propose a novel photosensitizer dye, indocyanine green (ICG), because it can be readily activated by commercially available dental 810 nm diode lasers and has an established safety record as an intravascular agent in cardiac imaging and ophthalmologic photodynamic therapy. Therefore, we aim to characterize ICG uptake and killing of key periodontal pathogens to explore its potential as a periodontal photodynamic therapy agent.
We tested ICG uptake by spectroscopy in Porphyromonas gingivalis 381 and Aggregatibacter actinomycetemcomitans, in addition to Escherichia coli DH5alpha and a human gingival epithelial cell line, HepG, in relation to ICG dose and exposure time. We then measured killing of bacteria by determining viable bacteria counts before and after exposure to ICG and 810 nm diode laser light (0-0.5 W output settings, 0-5 seconds). ICG was also applied to extracted, restored teeth, and the teeth inspected visually for staining after rinsing with saline.
We found rapid and significant uptake of indocyanine green into P. gingivalis 381 and A. actinomycetemcomitans 67, compared to E. coli DH5alpha and HepG gingival cell line. This correlated with significant killing of strains 381 and 67 compared to E.coli, with less than 10% survival. ICG does not appear to stain tooth surfaces and materials except calculus.
ICG combined with an 810 nm diode laser may be useful as a photodynamic adjunct for reduction of bacterial load in periodontal pockets.
... To probe its possible roles, the gene was aimed to be inactivated by in-frame deletion mutagenesis. On the other hand, findings were made in several bacteria that mutants of iron-responsive Fur genes show a strong deregulation of siderophore production, as in Legionella pneumophila (Hickey et al., 1997) or Actinobacillus actinomycetemcomitans (Haraszthy et al., 2002;Haraszthy et al., 2006). Anyway, the discrepancy between the low myxochelin production (ca. ...
The myxobacterium Myxococcus xanthus DK1622 is a reliable producer of different secondary metabolites with partially unknown bioactivities. In the present work the response of iron availibility were evaluated, concerning effects on growth, proteome profile and secondary metabolite production. The production of the siderophore myxochelin A was increased by the factor 81, myxochelin B by the factor 678. Unexpectedly, several other secondary metabolite production rates were found influenced, as e.g. myxochromids und cittilins. In proteome analysis, 1979 protein spots were detected in average, whereof 172 exhibited an iron-induced change in expression. A subsequent analysis by tandem mass spectrometry identified 169 of these spots as 131 individual proteins, some with up to 3 protein-phosphorylations. Furthermore, the functions of some, interesting proteins were investigated by knockout of the respective coding gene. At all, 12 single crossover mutants were generated and compared in iron-rich environment concerning effects on growth and rates of iron uptake or secondary metabolite production to the wild type strain. Typically, mutant strains show variations in all three parameters. An in-frame deletion mutant in one of the two fur genes (MXAN_6967) exhibited reduced growth and a decrease in iron uptake (ca. 49 % of the wild type). Additionally, production of all seven monitored secondary metabolites cannot be explained with the traditional Fur model, which suggests a new, unexpected regulation in M. xanthus.
... Putative Fur boxes or fur genes have been described in different Pasteurellaceae members, including P. multocida, H. ducreyi, A. suis, Agg. actinomycetemcomitans and A. pleuropneumoniae (Haraszthy et al., 2002;Cox et al., 2003;Bahrami et al., 2003;Hsu et al., 2003). ...
When Avibacterium paragallinarum reference strain 0083 (serovar A) was grown in an iron-restricted culture medium, the expression of the 60, 68 and 93 kDa outer membrane proteins increased as compared with normal media. Sera of chickens experimentally infected with Av. paragallinarum recognized these iron-restriction induced proteins, suggesting their expression in vivo. The three outer membrane proteins were identified as transferrin receptor and iron transport proteins by mass spectroscopy and a search in sequence databases. As these proteins have been reported to be regulated by the Fur protein in many bacteria, we investigated, through molecular methods, the presence of the fur gene in Av. paragallinarum. A candidate fur gene of Av. paragallinarum was amplified by polymerase chain reaction using complementary primers to conserved regions of fur gene sequences from members of the Pasteurellaceae family. The nucleotide sequence of the cloned gene, from ATG to TAA stop codon, was 453 base pairs in length and the deduced amino acid sequence showed 94% identity with Fur sequences of Actinobacillus pleuropneumoniae and Haemophilus ducreyi. The Av. paragallinarum deduced Fur protein (17.8 kDa) amino acid sequence contains the N-terminal helix-turn-helix DNA-binding domain and the two iron-binding sites in the C-terminal end, typical of other described Fur proteins. The study of iron-restriction-induced proteins and the mechanism regulating their expression could lead to an understanding of the responses of Av. paragallinarum to survive in an iron-restricted environment on host mucosal surfaces.
... Iron is an important environmental signal that controls gene expression. Although there are some reports that describe the production of the Fur iron-dependent transcriptional regulator and the effect of iron on protein synthesis (Fong et al., 2003; Graber et al., 1998; Haraszthy et al., 2002; Rhodes et al., 2005; Spitznagel et al., 1995; Willemsen et al., 1997; Winston et al., 1993), the extent and the components of the A. actinomycetemcomitans iron modulon and Fur regulon remain virtually unexplored. Therefore, we decided to test the effects of iron-rich and iron-chelated conditions on the formation of biofilms on abiotic surfaces. ...
Actinobacillus actinomycetemcomitans, a pathogen associated with oral and extra-oral infections, requires iron to grow under limiting conditions. Although incapable of producing siderophores, this pathogen could acquire iron by direct interaction with compounds such as haemin, haemoglobin, lactoferrin and transferrin. In this work the ability of different A. actinomycetemcomitans strains to bind and use different iron sources was tested. None of the strains tested used haemoglobin, lactoferrin or transferrin as sole sources of iron. However, all of them used FeCl(3) and haemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, haemoglobin and haemin, but not transferrin. Insertion inactivation of hmsF, which encodes a predicted cell-envelope protein related to haemin-storage proteins produced by other pathogens, reduced haemin and Congo red binding drastically without affecting haemin utilization as an iron source under chelated conditions. Biofilm assays showed that all strains tested attached to and formed biofilms on plastic under iron-rich and iron-chelated conditions. However, scanning electron microscopy showed that smooth strains formed simpler biofilms than rough isolates. Furthermore, the incubation of rough cells in the presence of FeCl(3) or haemin resulted in the formation of more aggregates and microcolonies compared with the fewer cell aggregates formed when cells were grown in the presence of the synthetic iron chelator dipyridyl. These cell responses to changes in extracellular iron concentrations may reflect those that this pathogen expresses under the conditions it encounters in the human oral cavity.
... The analysis of the huvA promoter region revealed a potential Fur-box downstream of its transcriptional start site. This location is unusual in the Fur-binding sequences, but a similar situation has been described in the fur gene of Actinobacillus actinomycetemcomitans (Haraszthy et al., 2002). Transcriptional fusion results obtained in low-iron conditions and in the fur mutant confirmed a Fur-mediated regulation of huvA. ...
Vibrio anguillarum can utilize heme and hemoglobin as iron sources. Nine genes, huvA, huvZ, huvX, tonB1, exbB1, exbD1, huvB, huvC, huvD, encoding the proteins involved in heme transport and utilization, are clustered in a 10-kb region of chromosomal DNA. Reverse Transcriptase-PCR analysis demonstrated that the gene cluster is arranged into three transcriptional units: (1) huvA, (2) huvXZ, and (3) tonB1exbB1D1-huvBCD. Transcriptional start sites for each huvA, huvX, and tonB1 promoters were identified by primer extension analysis, and their respective -10 and -35 regions were shown to exhibit similarity to those of sigma70-recognized promoters. Expression from the three promoters, as analyzed by transcriptional fusions to a promoter less lacZ gene, was regulated by the iron concentration. Furthermore, analysis of the beta-galactosidase activities of these fusions in a V. anguillarum fur mutant demonstrated that the ferric uptake regulator repressor protein (Fur) is directly involved in the negative iron-mediated regulation of the heme uptake cluster.
... We have cloned and characterized the fur gene in A. actinomycetemcomitans (Haraszthy et al., 2002) – one of the first global regulatory genes identified in an oral pathogen. ...
... Furta is a powerful tool for identifying Fur-regulated genes, but it is extremely sensitive to both iron and Fur concentrations. A. actinomycetemcomitans fur has 62 % homology to E. coli fur gene and the cloned A. actinomycetemcomitans fur gene was able to complement the fur mutation in E. coli (Haraszthy et al., 2002). However, the assay probably targets clones containing Fur boxes with a high degree of similarity to the E. coli Fur box since the E. coli Fur protein will bind those sites preferentially. ...
... Total cellular proteins were extracted and equal amounts were loaded to each lane. The Western blots were probed with antiserum to the P. aeruginosa Fur protein (a gift from Dr M. L. Vasil, Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO, USA) that was previously shown to react with both the E. coli and the A. actinomycetemcomitans Fur protein (Haraszthy et al., 2002). As expected, the wild-type A. actinomycetemcomitans strain responded to available iron by expressing significantly more Fur protein under iron-rich conditions than under ironchelated conditions (Fig. 2). ...
Actinobacillus actinomycetemcomitans is an oral pathogen that causes aggressive periodontitis as well as sometimes life-threatening, extra-oral infections. Iron regulation is thought to be important in the pathogenesis of A. actinomycetemcomitans infections and, consistent with this hypothesis, the fur gene has recently been identified and characterized in A. actinomycetemcomitans. In this study, 14 putatively Fur-regulated genes were identified by Fur titration assay (Furta) in A. actinomycetemcomitans, including afuA, dgt, eno, hemA, tbpA, recO and yfe - some of which are known to be Fur regulated in other species. A fur mutant A. actinomycetemcomitans strain was created by selecting for manganese resistance in order to study the Fur regulon. Comparisons between the fur gene sequences revealed that nucleotide 66 changed from C in the wild-type to T in the mutant strain, changing leucine to isoleucine. The fur mutant strain expressed a nonfunctional Fur protein as determined by Escherichia coli-based ferric uptake assays and Western blotting. It was also more sensitive to acid stress and expressed higher levels of minC than the wild-type strain. minC, which inhibits cell division in other bacterial species and whose regulation by iron has not been previously described, was found to be Fur regulated in A. actinomycetemcomitans by Furta, by gel shift assays, and by RT-qPCR assays for gene expression.
... The sequence TAAAAT, separated from the start codon by 20 nt, may constitute a k10 promoter region, whereas a k35 region (Hawley & McClure, 1983) was not evident. Fifty-nine nucleotides upstream of the start codon the sequence AGAAATGAATTTC- ATTATC has 13 of 19 positions identical to the consensus Fur box (Escolar et al., 1999), suggesting that expression of hgpA is iron-responsively regulated by the Fur protein that has been shown to be functional in A. actinomycetemcomitans (Haraszthy et al., 2002). An inverted repeat structure of 46 nt located 44 nt downstream of the TAA stop codon presumably constitutes a transcriptional terminator. ...
To get a better insight into the physiology of the high-toxic JP2 clone of Actinobacillus actinomycetemcomitans serotype b, which is strongly associated with juvenile periodontitis in adolescents of African descent, the modes of iron acquisition in this clone were examined and compared to those of other strains of the species. None of the strains examined could utilize human transferrin as a source of iron. This was in accordance with the presence of a non-functional tbpA gene, which normally encodes the A subunit of the transferrin-binding-protein complex. Southern blot analysis indicated that functional duplications of tbpA were not present in the genome. Thus, A. actinomycetemcomitans seems to be in a process of evolution, in which iron acquisition from host transferrin is not essential as in many other members of the pasteurellaceae. All strains could utilize haem as a source of iron. All 11 A. actinomycetemcomitans strains examined harboured a single genomic sequence with homology to the hgpA gene encoding haemoglobin-binding protein A in Haemophilus influenzae. However, in all three strains belonging to the JP2 clone and in one serotype e strain hgpA was a pseudogene. Seven other strains possessed a functional hgpA gene which, according to insertion mutagenesis experiments, was responsible for the ability of these strains to utilize haemoglobin as a source of iron. Thus, the presence of an hgpA pseudogene and the inability to use human haemoglobin as an iron source discriminate the high-toxic JP2 clone from low-toxic serotype b strains and most other strains of A. actinomycetemcomitans.
... Actinobacillus actinomycetemcomitans, E. coli, Klebsiella pneumoniae, Haemophilus influenzae, Pasteurella multocida, and Yersinia pestis, are preceded by fldA which encodes a flavodoxin homologue (Achenbach and Genova, 1997;Haraszthy et al., 2002;Mayet al., 2001;Zheng et al., 1999). It is speculated that flavodoxin may play a role in the response to oxidative stress by maintaining the reduced state of enzymatic [4Fe-4S] clusters, thereby protecting them from superoxide attack (Zheng et al., 1999). ...
Written for the Dept. of Natural Resource Sciences, Macdonald College of McGill University. Thesis (Ph.D.). Includes bibliographical references.
