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Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
Tokano, Dorismey Vieira;Kawaichi, Marisa Emiko;Venancio, Emerson José;Vidotto, Marilda Carlos;
Brazilian Archives of Biology and Technology , 2008, DOI: 10.1590/S1516-89132008000300006
Abstract: the aim of this work was to isolate, clone and characterize the iron uptake gene iuta from avian pathogenic e. coli (apec). the iuta gene was isolated from the strain apec 9, serotype o2:h9, which was cloned in the expression vector pet101/d-topo. the gene of 2.2 kb was sequenced (ay602767, which showed high similarity to the iuta gene from three plasmids, two from apec, papec-02-colv (ay545598.4) and ptj100 (ay553855.1), and one from a human invasive e. coli strain, the pcolv k30. the recombinant protein iuta was over expressed in e. coli bl21(de-3) and was solubilized with urea and purified by ni-nta column. this method produced a relatively high yield of r-iuta of approximately 74kda, which was used to produce the antibody anti-iuta. this anti-iuta reacted with the protein r-iuta and native iuta of apec 9, as demonstrated by western blot, showing that the r-iuta conserved epitopes and its antigenicity was preserved. the anti-iuta igy was able to inhibit the iuta biological activity, inhibiting the sensitivity to cloacin df13 of apec9. however, it did not inhibit the growth of apec9 in m9 and did not protect the chickens inoculated with the apec, suggesting that the apec possessed another iron acquisition mechanism distinct of aerobactin.
Aerobactin Synthesis Genes iucA and iucC Contribute to the Pathogenicity of Avian Pathogenic Escherichia coli O2 Strain E058  [PDF]
Jielu Ling, Haizhu Pan, Qingqing Gao, Liping Xiong, Yefei Zhou, Debao Zhang, Song Gao, Xiufan Liu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057794
Abstract: Aerobactin genes are known to be present in virulent strains and absent from avirulent strains, but contributions of iucC and iucA, which are involved in aerobactin synthesis, to the pathogenicity of avian pathogenic Escherichia coli (APEC) have not been clarified. In this study, effects of double mutants (iucA/iutA or iucC/iutA) compared to those of single mutants (iucA, iucC or iutA) of aerobactin genes on the virulence of APEC strain E058 were examined both in vitro (aerobactin production, ingestion into HD-11 cells, survival in chicken serum) and in vivo (competitive growth against parental strain, colonization and persistence). In competitive co-infection assays, compared to the E058 parental strain, the E058ΔiucA mutant was significantly reduced in the liver, kidney, spleen (all P<0.01), heart and lung (both P<0.001). The E058ΔiutA mutant also was significantly reduced in the liver, lung, kidney (all P<0.01), heart and spleen (both P<0.001). The E058ΔiucC mutant was significantly attenuated in the heart and kidney (both P<0.05) and showed a remarkable reduction in the liver, spleen and lung (P<0.01); meanwhile, both E058ΔiucAΔiutA and E058ΔiucCΔiutA double mutants were sharply reduced as well (P<0.001). In colonization and persistence assays, compared with E058, recovered colonies of E058ΔiucA were significantly reduced from the lung, liver, spleen and kidney (P<0.01) and significantly reduced in the heart (P<0.001). E058ΔiutA was significantly reduced from the heart, lung, liver, spleen and kidney (P<0.01). E058ΔiucC, E058ΔiucAΔiutA and E058ΔiucCΔiutA were significantly decreased in all organs tested (P<0.001). These results suggest that iutA, iucA and iucC play important roles in the pathogenicity of APEC E058.
In vivo bioluminescence imaging of Escherichia coli O104:H4 and role of aerobactin during colonization of a mouse model of infection
Alfredo G Torres, Roberto J Cieza, Maricarmen Rojas-Lopez, Carla A Blumentritt, Cristiane S Souza, R. Katie Johnston, Nancy Strockbine, James B Kaper, Elena Sbrana, Vsevolod L Popov
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-112
Abstract: A bioluminescent reporter construct was used to perform real-time monitoring of E. coli O104:H4 in a mouse model of infection. We verified that our reporter strain maintained characteristics and growth kinetics that were similar to those of the wild-type E. coli strain. We found that the intestinal cecum of ICR (CD-1) mice was colonized by O104:H4, with bacteria persisting for up to 7?days after intragastric inoculation. MALDI-TOF analysis of heat-extracted proteins was performed to identify putative surface-exposed virulence determinants. A protein with a high similarity to the aerobactin iron receptor was identified and further demonstrated to be up-regulated in E. coli O104:H4 when grown on MacConkey agar or during iron-depleted conditions. Because the aerobactin iron acquisition system is a key virulence factor in Enterobacteriaceae, an isogenic aerobactin receptor (iutA) mutant was created and its intestinal fitness assessed in the murine model. We demonstrated that the aerobactin mutant was out-competed by the wild-type E. coli O104:H4 during in vivo competition experiments, and the mutant was unable to persist in the cecum.Our findings demonstrate that bioluminescent imaging is a useful tool to monitor E. coli O104:H4 colonization properties, and the murine model can become a rapid way to evaluate bacterial factors associated with fitness and/or colonization during E. coli O104:H4 infections.
Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coli: salmochelin and aerobactin contribute more to virulence than heme in a chicken infection model  [cached]
Gao Qingqing,Wang Xiaobo,Xu Huiqing,Xu Yaya
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-143
Abstract: Background Avian pathogenic Escherichia coli (APEC) and uropathogenic E. coli (UPEC) are the two main subsets of extraintestinal pathogenic E. coli (ExPEC). Both types have multiple iron acquisition systems, including heme and siderophores. Although iron transport systems involved in the pathogenesis of APEC or UPEC have been documented individually in corresponding animal models, the contribution of these systems during simultaneous APEC and UPEC infection is not well described. To determine the contribution of each individual iron acquisition system to the virulence of APEC and UPEC, isogenic mutants affecting iron uptake in APEC E058 and UPEC U17 were constructed and compared in a chicken challenge model. Results Salmochelin-defective mutants E058ΔiroD and U17ΔiroD showed significantly decreased pathogenicity compared to the wild-type strains. Aerobactin defective mutants E058ΔiucD and U17ΔiucD demonstrated reduced colonization in several internal organs, whereas the heme defective mutants E058ΔchuT and U17ΔchuT colonized internal organs to the same extent as their wild-type strains. The triple mutant ΔchuTΔiroDΔiucD in both E058 and U17 showed decreased pathogenicity compared to each of the single mutants. The histopathological lesions in visceral organs of birds challenged with the wild-type strains were more severe than those from birds challenged with ΔiroD, ΔiucD or the triple mutants. Conversely, chickens inoculated with the ΔchuT mutants had lesions comparable to those in chickens inoculated with the wild-type strains. However, no significant differences were observed between the mutants and the wild-type strains in resistance to serum, cellular invasion and intracellular survival in HD-11, and growth in iron-rich or iron-restricted medium. Conclusions Results indicated that APEC and UPEC utilize similar iron acquisition mechanisms in chickens. Both salmochelin and aerobactin systems appeared to be important in APEC and UPEC virulence, while salmochelin contributed more to the virulence. Heme bounded by ChuT in the periplasm appeared to be redundant in this model, indicating that other periplasmic binding proteins likely contributed to the observed no phenotype for the heme uptake mutant. No differences were observed between the mutants and their wild-type parents in other phenotypic traits, suggesting that other virulence mechanisms compensate for the effect of the mutations.
Examination of uropathogenic Escherichia coli strains conferring large plasmids
SUHARTONO
Biodiversitas , 2010,
Abstract: Suhartono (2010) Examination of uropathogenic Escherichia coli strains conferring large plasmids. Biodiversitas 11: 59-64. Of major uropathogens, Escherichia coli has been widely known as a main pathogen of UTIs globally and has considerable medical and financial consequences. A strain of UPEC, namely E. coli ST131, confers a large plasmid encoding cephalosporinases (class C β-lactamase) or AmpC that may be disseminated through horizontal transfer among bacterial populations. Therefore, it is worth examining such large plasmids by isolating, purifying, and digesting the plasmid with restriction enzymes. The examination of the large plasmids was conducted by isolating plasmid DNA visualized by agarose gel electrophoresis as well as by PFGE. The relationship of plasmids among isolates was carried out by HpaI restriction enzyme digestion. Of 36 isolates of E. coli ST 131, eight isolates possessed large plasmids, namely isolates 3, 9, 10, 12, 17, 18, 26 and 30 with the largest molecular size confirmed by agarose gel electrophoresis and PFGE was ~42kb and ~118kb respectively. Restriction enzyme analysis revealed that isolates 9, 10, 12, 17 and 18 have the common restriction patterns and those isolates might be closely related.
Dual agarose magnetic (DAM) ChIP
Lata Balakrishnan, Barry Milavetz
BMC Research Notes , 2009, DOI: 10.1186/1756-0500-2-250
Abstract: Protein A agarose and protein G magnetic beads bound with different IgGs have been combined in a single Chromatin Immunoprecipitation (ChIP) assay to analyze for the presence of two epitopes on the same chromatin at the same time. This procedure has been used with non-immune rabbit IgG bound to either the agarose or beads in order to include an internal negative control for non-specific binding of chromatin. The procedure has also been used with various antibodies including those targeting RNA Polymerase II and replication protein A 70 to determine whether specific forms of modified histones are present in either transcribing or replicating forms of SV40 minichromosomes respectively.The DAM ChIP procedure is a rapid, simple, and sensitive technique to characterize two epitopes located in the same chromatin. It should be particularly useful for the rapid screening of epigenetic modifications present in biologically active chromatin.Chromatin Immunoprecipitation (ChIP) has become an extremely powerful tool for the characterization of biologically functional chromatin. Specific antibodies bound to either protein A agarose or protein G magnetic beads are used to immune-select fragments of chromatin containing the target epitope of the antibody followed by PCR amplification to identify and quantitate the DNA present in the chromatin [1-4]. A carrier like agarose or magnetic beads is required in both of these procedures because it is necessary to separate the chromatin bound to antibody from contaminating chromatin during purifications. In the standard procedure this is done by sedimenting the agarose by low speed centrifugation, while in the modification with magnetic beads a magnet is used to bind the beads.We have taken advantage of the distinct properties of agarose and magnetic beads and developed a procedure which allows chromatin immunoprecipitation with two antibodies present in the same tube. A schematic representation of this procedure is shown in Figure 1. One
Anisotropic elasticity of magnetically ordered agarose gel
Isao Yamamoto et al
Science and Technology of Advanced Materials , 2008,
Abstract: The physical properties of agarose gel prepared under strong magnetic fields were investigated. The storage modulus was measured by the reflection method with an ultrasonic pulse. The measurement results of the gel's elasticity indicate that agarose gel has anisotropic properties. The elasticity and its anisotropy depend on the concentration of the gel and the magnetic field to which it is exposed. The experimental results indicate that the anisotropic network structure of the gel is induced by the exposure to the magnetic field during gelation. The gelation mechanism under a magnetic field is discussed
从agarose胶回收dna的方法  [PDF]
刘佑国
中国生物工程杂志 , 1984,
Abstract: 到目前为止,已经建立了很多从agarose胶回收dna的方法,但所有这些方法中没有一种令人完全满意。两个主要的问题是:第一,大部分agarose均被硫粘多糖所污染,当抽提dna时,它们被一起抽提出来。
Biological and genetic characteristics of uropathogenic Escherichia coli strains
SILVEIRA, Wanderley Dias da;BENETTI, Fabiane;LANCELLOTTI, Marcelo;FERREIRA, Alessandra;SOLFERINI, Vera Nisaka;BROCCHI, Marcelo;
Revista do Instituto de Medicina Tropical de S?o Paulo , 2001, DOI: 10.1590/S0036-46652001000600001
Abstract: the aim of the present study was to determine biological characteristics such as expression of fimbriae, congo red binding, production of hemolysin and aerobactin, adhesion to hela and uroepithelial cells and invasion of hela cells by escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (uti). also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (cnf1, cnf2) was assayed using specific primers in pcr. the data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (mlee), restriction fragment length polymorphism (rflp) of the rdna (ribotyping) and enterobacterial repetitive intergenic consensus-pcr (eric-pcr). all isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (pais). diffuse adherence type to hela cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. although four strains seemed to be able to invade hela cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. mlee, rflp and eric-pcr were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.
Biological and genetic characteristics of uropathogenic Escherichia coli strains  [cached]
SILVEIRA Wanderley Dias da,BENETTI Fabiane,LANCELLOTTI Marcelo,FERREIRA Alessandra
Revista do Instituto de Medicina Tropical de S?o Paulo , 2001,
Abstract: The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.
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