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1.
Eur J Clin Microbiol Infect Dis ; 37(4): 723-728, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29270862

RESUMO

Campylobacter jejuni-related diarrheal diseases is one of the major health issues among young children (0-59 months old) in low-income countries. Monitoring of the capsular (capsule polysaccharide, CPS) types of virulent C. jejuni strains in regions where the disease is endemic is of great importance for the development of a customized capsule-based multivalent vaccine. Therefore, we aimed to determine the prevalence of CPS genotypes among C. jejuni strains isolated from young children with enteritis (n = 152) and asymptomatic carriers matched by age, sex, and residence defined as the control group (n = 215) in Bangladesh. CPS genotyping was performed using a newly established multiplex polymerase chain reaction (PCR) method and lipooligosaccharide (LOS) locus classes (A-E) were characterized using PCR as well. We identified 24 different CPS genotypes among the 367 isolates. Four prevalent capsular types, HS5/31 complex (n = 27, 18%), HS3 (n = 26, 17%), HS4A (n = 10, 7%), and HS8/17 (n = 10, 7%) covered almost 50% of the strains from enteritis patients and 43% of the isolates from controls. In combination, the CPS genotype and LOS class was not discriminative between cases and controls. Dominant capsular types previously identified in C. jejuni strains isolated from patients with Guillain-Barré syndrome in Bangladesh were rarely detected in strains isolated from the young children. A similar distribution was evident among enteritis- and control-related strains when comparison was done between CPS types and LOS classes. This is the first systematic study presenting the distribution of CPS genotypes of C. jejuni strains isolated in Bangladesh from children with diarrhea and controls, with capsular genotypes HS5/31 complex, HS3, HS4A, and HS8/17 being prevalent in both. In conclusion, systematic studies are required to develop a multivalent capsule-based vaccine for children in low-income countries.


Assuntos
Cápsulas Bacterianas/genética , Infecções por Campylobacter , Campylobacter jejuni , Portador Sadio , Diarreia , Lipopolissacarídeos/genética , Bangladesh/epidemiologia , Infecções por Campylobacter/epidemiologia , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/classificação , Campylobacter jejuni/genética , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Criança , Pré-Escolar , DNA Bacteriano/análise , DNA Bacteriano/genética , Diarreia/epidemiologia , Diarreia/microbiologia , Feminino , Genótipo , Humanos , Lactente , Masculino
2.
Clin Microbiol Infect ; 21(9): 852.e1-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26070960

RESUMO

In about one in a thousand cases, a Campylobacter jejuni infection results in the severe polyneuropathy Guillain-Barré syndrome (GBS). It is established that sialylated lipo-oligosaccharides (LOS) of C. jejuni are a crucial virulence factor in GBS development. Frequent detection of C. jejuni with sialylated LOS in stools derived from patients with uncomplicated enteritis implies that additional bacterial factors should be involved. To assess whether the polysaccharide capsule is a marker for GBS, the capsular genotypes of two geographically distinct GBS-associated C. jejuni strain collections and an uncomplicated enteritis control collection were determined. Capsular genotyping of C. jejuni strains from the Netherlands revealed that three capsular genotypes, HS1/44c, HS2 and HS4c, were dominant in GBS-associated strains and capsular types HS1/44c and HS4c were significantly associated with GBS (p 0.05 and p 0.01, respectively) when compared with uncomplicated enteritis. In a GBS-associated strain collection from Bangladesh, capsular types HS23/36c, HS19 and HS41 were most prevalent and the capsular types HS19 and HS41 were associated with GBS (p 0.008 and p 0.02, respectively). Next, specific combinations of the LOS class and capsular genotypes were identified that were related to the occurrence of GBS. Multilocus sequence typing revealed restricted genetic diversity for strain populations with the capsular types HS2, HS19 and HS41. We conclude that capsular types HS1/44c, HS2, HS4c, HS19, HS23/36c and HS41 are markers for GBS. Besides a crucial role for sialylated LOS of C. jejuni in GBS pathogenesis, the identified capsules may contribute to GBS susceptibility.


Assuntos
Cápsulas Bacterianas/genética , Infecções por Campylobacter/complicações , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/genética , Genótipo , Síndrome de Guillain-Barré/epidemiologia , Síndrome de Guillain-Barré/etiologia , Bangladesh/epidemiologia , Campylobacter jejuni/classificação , Humanos , Países Baixos/epidemiologia
4.
J Phys Condens Matter ; 19(41): 415116, 2007 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-28192328

RESUMO

Neutron diffraction and 31P nuclear magnetic resonance spectroscopy were used to probe the structure of phosphate glass biomaterials of general composition (CaO)0.5-x(Na2O)x(P2O5)0.5 (x = 0, 0.1 and 0.5). The results suggest that all three glasses have structures based on chains of Q2 phosphate groups. Clear structural differences are observed between the glasses containing Na2O and CaO. The P-O bonds to bridging and non-bridging oxygens are less well resolved in the neutron data from the samples containing CaO, suggesting a change in the nature of the bonding as the field strength of the cation increases [Formula: see text]. In the (CaO)0.5(P2O5)0.5 glass most of the Ca2+ ions are present in isolated CaOx polyhedra whereas in the (Na2O)0.5(P2O5)0.5 glass the NaOx polyhedra share edges leading to a Na-Na correlation. The results of the structural study are related to the properties of the (CaO)0.4(Na2O)0.1(P2O5)0.5 biomaterial.

5.
J Biol Chem ; 276(37): 34862-70, 2001 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-11461915

RESUMO

Flagellins from three strains of Campylobacter jejuni and one strain of Campylobacter coli were shown to be extensively modified by glycosyl residues, imparting an approximate 6000-Da shift from the molecular mass of the protein predicted from the DNA sequence. Tryptic peptides from C. jejuni 81-176 flagellin were subjected to capillary liquid chromatography-electrospray mass spectrometry with a high/low orifice stepping to identify peptide segments of aberrant masses together with their corresponding glycosyl appendages. These modified peptides were further characterized by tandem mass spectrometry and preparative high performance liquid chromatography followed by nano-NMR spectroscopy to identify the nature and precise site of glycosylation. These analyses have shown that there are 19 modified Ser/Thr residues in C. jejuni 81-176 flagellin. The predominant modification found on C. jejuni flagellin was O-linked 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) with additional heterogeneity conferred by substitution of the acetamido groups with acetamidino and hydroxyproprionyl groups. In C. jejuni 81-176, the gene Cj1316c, encoding a protein of unknown function, was shown to be involved in the biosynthesis and/or the addition of the acetamidino group on Pse5Ac7Ac. Glycosylation is not random, since 19 of the total 107 Ser/Thr residues are modified, and all but one of these are restricted to the central, surface-exposed domain of flagellin when folded in the filament. The mechanism of attachment appears unrelated to a consensus peptide sequence but is rather based on surface accessibility of Ser/Thr residues in the folded protein.


Assuntos
Campylobacter jejuni/química , Flagelina/química , Glicopeptídeos/análise , Sequência de Aminoácidos , Glicosilação , Espectrometria de Massas , Dados de Sequência Molecular
6.
Mol Microbiol ; 40(3): 769-77, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11359581

RESUMO

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Cápsulas Bacterianas/fisiologia , Campylobacter jejuni/patogenicidade , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Clonagem Molecular , Escherichia coli/genética , Teste de Complementação Genética , Metabolismo dos Lipídeos , Mutagênese , Polissacarídeos/metabolismo , Análise de Sequência de DNA , Virulência
7.
Infect Immun ; 68(12): 6535-41, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11083762

RESUMO

Live cells of Campylobacter jejuni and Campylobacter coli can induce release of interleukin-8 (IL-8) from INT407 cells. Additionally, membrane fractions of C. jejuni 81-176, but not membrane fractions of C. coli strains, can also induce release of IL-8. Membrane preparations from 81-176 mutants defective in any of the three membrane-associated protein subunits of cytolethal distending toxin (CDT) were unable to induce IL-8. The presence of the three cdt genes on a shuttle plasmid in trans restored both CDT activity and the ability to release IL-8 to membrane fractions. However, CDT mutations did not affect the ability of 81-176 to induce IL-8 during adherence to or invasion of INT407 cells. When C. jejuni cdt genes were transferred on a shuttle plasmid into a C. coli strain lacking CDT, membrane preparations became positive in both CDT and IL-8 assays. Growth of C. jejuni in physiological levels of sodium deoxycholate released all three CDT proteins, as well as CDT activity and IL-8 activity, from membranes into supernatants. Antibodies against recombinant forms of each of the three CDT subunit proteins neutralized both CDT activity and the activity responsible for IL-8 release. The data suggest that C. jejuni can induce IL-8 release from INT407 cells by two independent mechanisms, one of which requires adherence and/or invasion and the second of which requires CDT.


Assuntos
Toxinas Bacterianas/toxicidade , Campylobacter jejuni/patogenicidade , Interleucina-8/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Toxinas Bacterianas/genética , Células Cultivadas , Escherichia coli/patogenicidade , Teste de Complementação Genética , Humanos , Mucosa Intestinal/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Infect Immun ; 68(12): 6656-62, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11083778

RESUMO

Three genes involved in biosynthesis of the lipooligosaccharide (LOS) core of Campylobacter jejuni MSC57360, the type strain of the HS:1 serotype, whose structure mimics GM(2) ganglioside, have been cloned and characterized. Mutation of genes encoding proteins with homology to a sialyl transferase (cstII) and a putative N-acetylmannosamine synthetase (neuC1), part of the biosynthetic pathway of N-acetylneuraminic acid (NeuNAc), have identical phenotypes. The LOS cores of these mutants display identical changes in electrophoretic mobility, loss of reactivity with cholera toxin (CT), and enhanced immunoreactivity with a hyperimmune polyclonal antiserum generated against whole cells of C. jejuni MSC57360. Loss of sialic acid in the core of the neuC1 mutant was confirmed by fast atom bombardment mass spectrometry. Mutation of a gene encoding a putative beta-1,4-N-acetylgalactosaminyltransferase (Cgt) resulted in LOS cores intermediate in electrophoretic mobility between that of wild type and the mutants lacking NeuNAc, loss of reactivity with CT, and a reduced immunoreactivity with hyperimmune antiserum. Chemical analyses confirmed the loss of N-acetylgalactosamine (GalNAc) and the presence of NeuNAc in the cgt mutant. These data suggest that the Cgt enzyme is capable of transferring GalNAc to an acceptor with or without NeuNAc and that the Cst enzyme is capable of transferring NeuNAc to an acceptor with or without GalNAc. A mutant with a nonsialylated LOS core is more sensitive to the bactericidal effects of human sera than the wild type or the mutant lacking GalNAc.


Assuntos
Atividade Bactericida do Sangue , Campylobacter jejuni/imunologia , Lipopolissacarídeos/química , Ácido N-Acetilneuramínico/metabolismo , Animais , Campylobacter jejuni/patogenicidade , Flagelina/genética , Mutagênese Insercional , Fases de Leitura Aberta , Coelhos
9.
Infect Immun ; 68(8): 4384-90, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10899834

RESUMO

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.


Assuntos
Campylobacter jejuni/patogenicidade , Plasmídeos/genética , Fatores de Virulência , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Conjugação Genética , Diarreia , Feminino , Furões , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Fatores R/genética , Análise de Sequência de DNA , Especificidade da Espécie , Resistência a Tetraciclina/genética , Transformação Bacteriana
10.
Infect Immun ; 67(11): 5799-805, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10531231

RESUMO

A recombinant protein comprising the maltose-binding protein (MBP) of Escherichia coli fused to amino acids 5 to 337 of the FlaA flagellin of Campylobacter coli VC167 was evaluated for immunogenicity and protective efficacy against challenge by a heterologous strain of campylobacter, Campylobacter jejuni 81-176, in two murine models. The sequence of the flaA gene of strain 81-176 revealed a predicted protein which was 98.1% similar to that of VC167 FlaA over the region expressed in the fusion protein. Mice were immunized intranasally with two doses of 3 to 50 microgram of MBP-FlaA, given 8 days apart, with or without 5 microgram of the mutant E. coli heat-labile enterotoxin (LT(R192G)) as a mucosal adjuvant. The full range of MBP-FlaA doses were effective in eliciting antigen-specific serum immunoglobulin G (IgG) responses, and these responses were enhanced by adjuvant use, except in the highest dosing group. Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA (>/=25 microgram) or coadministration of lower doses with the adjuvant. When vaccinated mice were challenged intranasally 26 days after immunization, the best protection was seen in animals given 50 microgram of MBP-FlaA plus LT(R192G). The protective efficacies of this dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized with 50 microgram of MBP-FlaA plus LT(R192G) intranasally were challenged orally with 8 x 10(10), 8 x 10(9), or 8 x 10(8) cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively.


Assuntos
Vacinas Bacterianas/imunologia , Campylobacter jejuni/imunologia , Flagelina/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Flagelina/química , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular
11.
Mol Microbiol ; 32(5): 1022-30, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10361304

RESUMO

A genetic locus from Campylobacter jejuni 81-176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5alpha containing some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site-specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site-specific mutation of each of the seven genes in 81-176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild-type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chemical deglycosylation of protein fractions from the 81-176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Proteínas de Membrana/metabolismo , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Campylobacter jejuni/imunologia , DNA Bacteriano/química , DNA Bacteriano/genética , Ensaio de Imunoadsorção Enzimática , Flagelina/genética , Flagelina/metabolismo , Genes Bacterianos , Glicosilação , Immunoblotting , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Mutação , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA , Aglutininas do Germe de Trigo/metabolismo
12.
Infect Immun ; 67(1): 88-93, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9864200

RESUMO

Incubation of INT407 cells with various clinical isolates of Campylobacter jejuni resulted in secretion of interleukin-8 (IL-8) at levels ranging from 96 to 554 pg/ml at 24 h. The strains which produced the highest levels of IL-8 secretion were 81-176 and BT44. Induction of IL-8 secretion required live cells of 81-176 and was dependent on de novo protein synthesis. Site-specific mutants of 81-176, which were previously shown to be defective in adherence and invasion, resulted in reduced levels of secretion of IL-8, and cheY mutants of strains 81-176 and 749, which are hyperadherent and hyperinvasive, resulted in higher levels of IL-8 secretion. Another mutant of 81-176, which adheres at about 43% of the wild-type levels but is noninvasive, also showed marked reduction in IL-8 levels, suggesting that invasion is necessary for high levels of IL-8 secretion. When gentamicin was added to INT407 cells at 2 h after infection with 81-176, IL-8 secretion 22 h later was equivalent to that of controls without gentamicin, suggesting that the events which trigger induction and release of IL-8 occur early in the interactions of bacteria and eukaryotic cells.


Assuntos
Campylobacter jejuni/imunologia , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Aderência Bacteriana/imunologia , Campylobacter jejuni/crescimento & desenvolvimento , Campylobacter jejuni/patogenicidade , Linhagem Celular , Quimiotaxia/imunologia , Contagem de Colônia Microbiana , Embrião de Mamíferos , Gentamicinas/farmacologia , Humanos , Interleucina-8/biossíntese , Mucosa Intestinal/citologia , Cinética , Testes de Sensibilidade Microbiana
13.
J Infect Dis ; 176 Suppl 2: S122-4, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9396694

RESUMO

Among the protein antigens of Campylobacter species, flagellin, the subunit of the flagellar filament, is the best characterized. The motility imparted by this locomotory organelle is absolutely essential for Campylobacter organisms to colonize the gastrointestinal tract and to cause diarrheal disease. Flagellin is the immunodominant protein recognized during infection and has been suggested to be involved in the protective immune response. Campylobacter flagellins are glycosylated, which is an unusual posttranslational modification for prokaryotic proteins. Although the chemical structure of the glycosylated moiety is undetermined, the posttranslational modification includes sialic acid. The association of glycosylated flagellin with development of Guillain-Barré syndrome remains speculative, but the possibility of molecular mimicry between glycosylated flagellin and eukaryotic glycoproteins exists.


Assuntos
Antígenos de Bactérias/imunologia , Campylobacter/imunologia , Campylobacter/metabolismo , Flagelina/imunologia , Flagelina/metabolismo , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Campylobacter/química , Proteínas de Fímbrias , Flagelos/química , Flagelos/imunologia , Flagelos/fisiologia , Glicosilação , Glicoproteínas de Membrana/imunologia , Mimetismo Molecular , Ácido N-Acetilneuramínico/metabolismo , Polirradiculoneuropatia/metabolismo , Polirradiculoneuropatia/microbiologia , Porinas/imunologia , Processamento de Proteína Pós-Traducional
14.
J Bacteriol ; 179(15): 4647-53, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9244248

RESUMO

The flgE gene encoding the flagellar hook protein of Campylobacter coli VC167-T1 was cloned by immunoscreening of a genomic library constructed in lambdaZAP Express. The flgE DNA sequence was 2,553 bp in length and encoded a protein with a deduced molecular mass of 90,639 Da. The sequence had significant homology to the 5' and 3' sequences of the flgE genes of Helicobacter pylori, Treponema phagedenis, and Salmonella typhimurium. Primer extension analysis indicated that the VC167 flgE gene is controlled by a sigma54 promoter. PCR analysis showed that the flgE gene size and the 5' and 3' DNA sequences were conserved among C. coli and C. jejuni strains. Southern hybridization analyses confirmed that there is considerable sequence identity among the hook genes of C. coli and C. jejuni but that there are also regions within the genes which differ. Mutants of C. coli defective in hook production were generated by allele replacement. These mutants were nonmotile and lacked flagellar filaments. Analyses of flgE mutants indicated that the carboxy terminus of FlgE is necessary for assembly of the hook structure but not for secretion of FlgE and that, unlike salmonellae, the lack of flgE expression does not result in repression of flagellin expression.


Assuntos
Proteínas de Bactérias/genética , Campylobacter coli/genética , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA/fisiologia , Flagelos , Regulação Bacteriana da Expressão Gênica , Fator sigma/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Flagelina , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54 , Coelhos , Homologia de Sequência de Aminoácidos
15.
J Bacteriol ; 179(12): 3997-4002, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9190817

RESUMO

Two genes involved in iron utilization in Campylobacter coli VC167 T1 have been characterized. The cfrA gene encodes a protein with a predicted Mr of 77,653 which, after processing of the leader sequence, has a predicted Mr of 75,635. This protein has significant sequence identity to siderophore receptors of several bacteria, and site-specific mutants defective in cfrA do not synthesize one of two major iron-repressible outer membrane proteins. An adjacent gene encodes a TonB-like protein; a mutant in this gene lost the ability to utilize hemin, ferrichrome, and enterochelin as iron sources. The cfrA and tonB genes of VC167 T1 hybridized to all strains of C. coli and most strains of C. jejuni examined but did not hybridize to several other strains of C. jejuni, suggesting that the thermophilic campylobacters can be separated into two categories based on the presence of these two iron utilization genes.


Assuntos
Campylobacter/genética , Genes Bacterianos , Ferro/metabolismo , Sequência de Aminoácidos , Campylobacter/metabolismo , Mapeamento Cromossômico , DNA Bacteriano/análise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Hibridização de Ácido Nucleico , Fases de Leitura Aberta
16.
Mol Microbiol ; 23(5): 1021-31, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9076738

RESUMO

Four motile, non-adherent and non-invasive mutants of Campylobacter jejuni 81-176 generated by a site-specific insertional mutagenesis scheme were characterized at the molecular level and all contained a duplication of the same region of the chromosome. When this region was cloned from wild-type 81-176 and transferred into 81-176 on a shuttle plasmid, the same non-invasive phenotype as the original mutants was observed, suggesting that the region contained a repressor of adherence and invasion. The smallest piece of DNA identified which was capable of repressing adherence and invasion was a 0.8 kb fragment encoding the cheY gene of C.jejuni. To confirm further that CheY was responsible for the observed non-adherent and non-invasive phenotypes, the cheY gene was inserted into the arylsulfatase gene of 81-176 to generate a strain with two chromosomal copies of cheY. This diploid strain displayed the same non-adherent and non-invasive phenotype as the original mutants. Insertional inactivation of the cheY gene in 81-176 resulted in an approx. threefold increase in adherence and invasion in vitro, but this strain was unable to colonize or cause disease in animals. The diploid cheY strain, although able to colonize mice, was attenuated in a ferret disease model.


Assuntos
Proteínas de Bactérias , Infecções por Campylobacter/genética , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidade , Proteínas de Membrana/genética , Virulência/genética , Sequência de Aminoácidos , Animais , Aderência Bacteriana/genética , Southern Blotting , Western Blotting , Quimiotaxia , Mapeamento Cromossômico , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/fisiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Furões , Regulação Bacteriana da Expressão Gênica , Proteínas Quimiotáticas Aceptoras de Metil , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Recombinação Genética , Alinhamento de Sequência , Análise de Sequência de DNA , Sulfatases/genética , Transcrição Gênica , Transformação Bacteriana
17.
J Bacteriol ; 178(11): 3335-8, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8655516

RESUMO

The arylsulfatase gene from Campylobacter jejuni 81-176 encodes a predicted protein of 69,293 Da which shows no sequence similarity with other known arylsulfatases. The gene hybridizes to other Ast+ strains of C. jejuni and Campylobacter sputorum subsp. bubulus, as well as to many Ast- strains of C. jejuni.


Assuntos
Arilsulfatases/genética , Campylobacter jejuni/genética , Genes Bacterianos , Sequência de Aminoácidos , Arilsulfatases/biossíntese , Sequência de Bases , Campylobacter jejuni/enzimologia , Clonagem Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Plasmídeos
18.
Mol Microbiol ; 20(4): 885-94, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8793884

RESUMO

Examination of strains of Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus by electron microscopy revealed that they produced peritrichous pilus-like appendages when the bacteria were grown in the presence of bile salts. Various bile-salt supplements were used and it was found that deoxycholate and chenodeoxycholic acid caused a significant enhancement of pilus production and resulted in a highly aggregative phenotype. Morphologically, the pili were between 4 and 7 nm in width and were greater than 1 micron in length. A gene, termed pspA, which encodes a predicted protein resembling protease IV of Escherichia coli, was identified in C. jejuni strain 81-176. A site-specific insertional mutation within this gene resulted in the loss of pilus synthesis as determined by electron microscopy. Insertions upstream and downstream of the gene had no effect on pilus production. The non-piliated mutant of strain 81-176 showed no reduction in adherence to or invasion of INT 407 cells in vitro. However, this mutant, while still possessing the ability to colonize ferrets, caused significantly reduced disease symptoms in this animal model.


Assuntos
Campylobacter/patogenicidade , Proteínas de Escherichia coli , Fímbrias Bacterianas/genética , Serina Endopeptidases , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ácidos e Sais Biliares/farmacologia , Campylobacter/ultraestrutura , Cromossomos Bacterianos/química , Clonagem Molecular , Sondas de DNA/metabolismo , Modelos Animais de Doenças , Feminino , Furões , Proteínas de Fímbrias , Fímbrias Bacterianas/ultraestrutura , Proteínas de Choque Térmico/química , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Peptídeo Hidrolases/química , Alinhamento de Sequência
19.
J Clin Microbiol ; 34(5): 1216-9, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8727905

RESUMO

The conservation of flagellin genes from thermophilic Campylobacter spp. strains isolated in Egypt was evaluated by a restriction fragment length polymorphism (RFLP) assay. The flaA and flaB genes were amplified from 59 independent clinical isolates and digested with EcoRI and PstI, and the resulting patterns were compared with each other and with previously described RFLP groups. The results indicate that the isolates fell into 14 groups for flaA and 11 groups for flaB, 9 of which have been described, and that considerable genetic variability exists among isolates belonging to the same LIO serogroup. In most cases, the flaB gene displayed the same RFLP pattern as that of the flaA gene of the same strain, although some variability was observed. The data suggest that more variability of flagellin genes exists within the LIO serogroups common to Campylobacter field isolates from Egypt than has previously been reported for North American isolates.


Assuntos
Campylobacter coli/genética , Campylobacter jejuni/genética , Flagelina/genética , Genes Bacterianos , Polimorfismo de Fragmento de Restrição , Sequência de Bases , Infecções por Campylobacter/microbiologia , Campylobacter coli/classificação , Campylobacter coli/isolamento & purificação , Campylobacter jejuni/classificação , Campylobacter jejuni/isolamento & purificação , Estudos de Casos e Controles , Criança , Sequência Conservada , Diarreia/microbiologia , Egito , Humanos , Sorotipagem , Especificidade da Espécie
20.
J Med Chem ; 39(9): 1864-71, 1996 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-8627610

RESUMO

The synthesis and structure-activity relationships of a new class of vinylcephalosporins substituted with a lactamyl residue are described. These compounds show excellent activity against enterococci and retain the broad spectrum activity of third-generation cephalosporins such as ceftriaxone.


Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Cefalosporinas/síntese química , Cefalosporinas/farmacologia , Enterococcus/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Antibacterianos/química , Cefalosporinas/química , Testes de Sensibilidade Microbiana , Análise Espectral , Relação Estrutura-Atividade
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