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E Carniel

Publications and source records attributed to E Carniel.

At least 55 records · Page 3Linked to original sources
PubMed1995

Yersinia spp. HMWP2, a cytosolic protein with a cryptic internal signal sequence which can promote alkaline phosphatase export.

The iron starvation-induced, 2,042-amino-acid protein HMWP2 of Yersinia enterocolitica has two internal hydrophobic segments which might promote its export and association with the cytoplasmic membrane. To determine whether part of HMWP2 could be exported beyond the periplasmic face of the cytoplasmic membrane, we used TnphoA mutagenesis to construct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP1 (at amino acid positions 1751 and 1753 two independent isolates]) had high alkaline phosphate activity (close to that of the native enzyme), both in Escherichia coli and in Y. pseudotuberculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in the second hydrophobic segment of HMWP2. This result would be compatible with the topology of the protein in the cytoplasmic membrane predicted from the distribution of charged amino acids at either end of the two hydrophobic segments. However, two hybrids in which the junction was even further toward the C terminus of HMMWP2 (at positions 1793 and 1999) had only weak alkaline phosphatase activity, suggesting that the predicted topology is incorrect. The location of HMWP2 was therefore determined by subcellular fractionation. The results indicate that HMPW2 is mainly cytoplasmic, consistent with its presumed role in the ATP-dependent, nonribosomal synthesis of an unknown peptide. We propose that the high alkaline phosphatase activity associated with some of the HMWP-2-PhoA hybrids results from the unmasking of the cryptic export signal activity in the second hydrophobic segment of HMPW2.

Alkaline Phosphatase↗
PubMed1994

Assessment of a fluoroquinolone, three beta-lactams, two aminoglycosides, and a cycline in treatment of murine Yersinia pestis infection.

Amoxicillin, cefotaxime, ceftriaxone, gentamicin, doxycycline, and ofloxacin were active in vitro, like the reference drug streptomycin, against the virulent strain Yersinia pestis 6/69M. The comparative efficacies of these drugs in vivo were evaluated in a standardized and reproducible mouse model of systemic infection. Each antibiotic was injected intravenously once, at 24 h postinfection, and then repeatedly during 48 h. In vivo results were measured by counting the viable bacteria recovered from the whole spleens of mice sacrificed at selected times. All the drugs were manifestly successful; ceftriaxone, ofloxacine, and the reference drug were the most effective. Therefore, gentamicin and doxycycline could be used, depending on the clinical forms of the Y. pestis infection. Further investigations on beta-lactams, especially those used in the present study, could be carried out to confirm or not confirm their activities against Y. pestis. Ofloxacin appeared to be as active and to perform as rapidly as streptomycin in the treatment of murine Y. pestis infection, which is in agreement with the previous successes obtained with the use of fluoroquinolones in the treatment of murine infections caused by other pathogenic yersiniae.

Animals↗
PubMed1994

Efficient subtyping of pathogenic Yersinia enterocolitica strains by pulsed-field gel electrophoresis.

Yersinia enterocolitica is an enteropathogen that has recently and rapidly expanded over the world. There is a close correlation between the biotypes, serotypes, and phage types of the strains, making it virtually impossible to distinguish isolates of the same serotype with the classical phenotypic markers. In the present study, pulsed-field gel electrophoresis (PFGE) was used to compare the NotI genomic profile (i.e., pulsotype) of 20 strains each of serotypes O:3, O:9, and O:5. Eleven, 12, and 18 different pulsotypes were obtained, respectively, indicating that this technique is very efficient for subtyping pathogenic isolates of Y. enterocolitica. Within strains of serotype O:5, PFGE differentiated two subgroups that corresponded to two biotypes (biotypes 1A and 3). Comparison of the pulsotypes of three strains of biotype 3 and serotype O:3 (referred to as 3/O:3) with those of strains 4/O:3 and 3/O:5 suggested that the pulsotype is closer to the biotype than to the serotype. The pulsotypes of five pairs of strains isolated from the same patient or siblings were also analyzed. In four pairs, the two strains displayed identical pulsotypes, indicating that PFGE might be a powerful epidemiological tool. In the fifth pair, one restriction fragment differed, suggesting that genomic polymorphism may occur in vivo in Y. enterocolitica. Finally, the in vitro genomic stabilities of one strain each of Y. enterocolitica O:3, O:9, and O:5 were investigated. The pulsotypes of 10 isolated colonies were identical within each strain, indicating that in vitro, the genome of Y. enterocolitica is much more stable than that of Y. pestis.

Bacterial Typing Techniques↗
PubMed1994

Plague pandemics investigated by ribotyping of Yersinia pestis strains.

Yersinia pestis is the causative agent of plague, a disease which has caused the deaths of millions of people and which persists now in endemic foci. The rRNA gene restriction patterns (i.e., ribotypes) of 70 strains of Y. pestis, isolated on the five continents over a period of 72 years, were determined by hybridization with a 16S-23S rRNA probe from Escherichia coli. The combination of the EcoRI and EcoRV patterns resulted in the elucidation of 16 ribotypes. Two of them (B and O) characterized 65.7% of the strains studied, while the 14 other ribotypes were found in no more than three strains each. A relationship was established between biovars and ribotypes: strains of biovar Orientalis were of ribotypes A to G, those of biovar Antiqua were of ribotypes F to O, and those of biovar Medievalis were of ribotypes O and P. Great heterogeneity in rRNA restriction patterns was found among strains isolated in Africa; this heterogeneity was less pronounced among Asian isolates and was completely absent from the American strains. Pulsed-field gel electrophoresis was performed on the DNAs of some strains, but it appeared that different colonies from the same strain displayed different pulsed-field gel electrophoresis patterns and therefore that this technique was not suitable for comparison of Y. pestis isolates. In contrast, the ribotypes of individual colonies within a given strain were stable and were not modified after five passages in vivo. A clear correlation between the history of the three plague pandemics and the ribotypes of the strains could be established.

Bacterial Typing Techniques↗
PubMed1993

Chromosomal irp2 gene in Yersinia: distribution, expression, deletion and impact on virulence.

Iron starvation induces the synthesis of two high molecular weight proteins (HMWP1 and 2) in Yersinia. The presence of the irp2 gene coding for the HMWP2 was investigated in 170 Yersinia strains. This gene was absent from all avirulent or weakly pathogenic species and was restricted to highly pathogenic strains. One hundred percent of the potentially highly pathogenic Yersinia enterocolitica biotype 1B harbored irp2 but surprisingly, 70.4% of the Yersinia pseudotuberculosis tested lacked the gene. Only serotypes I and III of Y. pseudotuberculosis harbored the locus, however, synthesis of HMWPs was detected uniquely in the former. In Yersinia pestis, overall 55.3% of the strains tested had the gene, with an uneven distribution among Orientalis (65.2%), Antiqua (66.6%) and Medievalis (0%) geographic variants. Except for one Y. pestis strain, the irp2 restriction profiles were identical for all strains of Y. pestis and Y. pseudotuberculosis tested. All Y. enterocolitica 1B displayed the same irp2 pattern, different from that of the other two species. In vitro spontaneous deletion of irp2 was not obtained in Y. enterocolitica 1B but was observed in both Y. pseudotuberculosis and Y. pestis. Repeated subcultures of Y. pestis increased progressively the proportion of irp2-deleted colonies, yielding an almost pure irp2-deleted strain after 16 subcultures. A clear correlation was established between the presence of irp2 and the level of virulence of Y. pseudotuberculosis and Y. pestis.

Animals↗
PubMed1993

Relationship between loss of pigmentation and deletion of the chromosomal iron-regulated irp2 gene in Yersinia pestis: evidence for separate but related events.

The irp2 gene, coding for a 190-kDa iron-regulated protein (HMWP2), and the hemin storage locus (hms), which determines Yersinia pestis pigmentation, are each located on a large chromosomal fragment which carries virulence genes and deletes spontaneously. To determine whether the two loci are located on one unstable fragment or on two different excisable DNA segments, the pigmentation status and the presence of irp2 in 43 strains of Y. pestis isolated in various parts of the world were examined. Three different types were observed: Pgm+ Irp2+ (39.5%), Pgm- Irp2- (44.2%), and Pgm- Irp2+ (16.3%). No Pgm+ Irp2- strain was found. These three types were also recovered in vitro from the parental strain Saigon 55-12-39 (Pgm+ Irp2+), but again, no Pgm+ Irp2- colony was observed. Pgm- Irp2- derivatives were obtained from a single Pgm- Irp2+ colony, indicating sequential loss of the two traits. The fact that the genomic SpeI restriction patterns obtained by pulsed-field gel electrophoresis were specific for each of the three variants suggested that distinct large-scale chromosomal rearrangements had occurred in the Pgm- Irp2+ and Pgm- Irp2- derivatives. The virulence of Pgm- Irp2+ bacteria in mice was ca. 10(7)-fold lower than that of the Pgm+ Irp2+ strains injected subcutaneously but was not significantly decreased when injected intravenously. In contrast, the Pgm- Irp2- microorganisms were markedly less pathogenic (10(6)-fold) than the Pgm+ Irp2+ strains injected intravenously and were 100 times less virulent than the Pgm- Irp2+ strains injected subcutaneously.

Animals↗
PubMed1993

High-molecular-weight protein 2 of Yersinia enterocolitica is homologous to AngR of Vibrio anguillarum and belongs to a family of proteins involved in nonribosomal peptide synthesis.

The iron-regulated irp2 gene is specific for the highly pathogenic Yersinia species and encodes high-molecular-weight protein 2 (HMWP2). Despite the established correlation between the presence of HMWP2 and virulence, the role of this protein is still unknown. To gain insight into the function of HMWP2, the entire coding sequence and the promoter of irp2 were sequenced. Two putative -35 and -10 promoter sequences were identified upstream of a large open reading frame, and two potential Fur-binding sites were found overlapping the second -35 box. The large open reading frame is composed of 6,126 nucleotides and may encode a protein of 2,035 amino acids (ca. 228 kDa) with a pI of 5.81. A signal sequence was not present at the N terminus of the protein. Despite the existence of 30 cysteine residues, carboxymethylation prevented the formation of most if not all disulfide bonds that otherwise occurred when the cells were sonicated. The protein was composed of three main domains: a central region of ca. 850 residues, bordered on each side by a repeat of 550 residues. A high degree of identity (44.5%) was found between HMWP2 and the protein AngR of Vibrio anguillarum. The central part of HMWP2 (after removal of a loop of 337 residues) also displayed significant homology with proteins belonging to the superfamily of adenylate-forming enzymes and, like them, possessed a putative ATP-binding motif that is also present in AngR. In addition, HMWP2 shared with the group of antibiotic and enterochelin synthetases a potential amino acid-binding site. Six consensus sequences defining the superfamily and four defining the family of synthetases were derived from the multiple alignment of the 30 sequences of proteins or repeated domains. A phylogenetic tree that was constructed showed that HMWP2 and AngR are in a family composed of Lys2, EntF, and the tyrocidine, gramicidin, and Beta-lactam synthetases. This finding suggests that HMWP2 may participate in the nonribosomal synthesis of small biologically active peptides.

Amino Acid Sequence↗
PubMed1992

Molecular cloning, iron-regulation and mutagenesis of the irp2 gene encoding HMWP2, a protein specific for the highly pathogenic Yersinia.

Under iron-starvation, the highly pathogenic Yersinia synthesize several iron-regulated proteins including two high-molecular-weight polypeptides (HMWP1 and HMWP2). From the chromosome of Yersinia enterocolitica serovar O:8 (strain Ye 8081), the genes coding for the HMWP2 (irp2) and its promoter were cloned into plasmid pUC18 (pIR2) and used as a probe. We show here that the irp2 gene is present only in the highly pathogenic strains and that its promoter is iron-regulated in Escherichia coli. After introduction of the pIR2 plasmid into a fur mutant of E. coli, both the iron-starved and the iron-replete bacteria expressed the HMWP2. Repressibility of irp2 by iron was restored by introduction of a plasmid carrying the fur gene. These results demonstrate that the irp2 promoter is controlled by the Fur repressor in E. coli. Mutagenesis of the chromosomal irp2 gene of Yersinia pseudotuberculosis was obtained by homologous recombination with a 1 kb fragment of this gene cloned on the suicide plasmid pJM703.1. Inactivation of irp2 resulted in the non-expression of both HMWPs, while introduction of plasmid pIR2 into the mutant strain led to the synthesis of the HMWP2 only. Therefore, it is probable that the genes coding for the HMWPs constitute an operon where irp2 is upstream of irp1. When comparing the virulence of the wild-type strain and of its irp2 mutant derivative, we found that the 50% lethality (LD50) for mice of the mutant strain was increased, whatever the route of infection, but more markedly when injected parenterally. Accordingly, these data demonstrate that a mutation in the irp2 gene alters the pathogenicity of Y. pseudotuberculosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Use of pulsed field gel electrophoresis to size the chromosome of the bacterial fish pathogen Yersinia ruckeri.

Field inversion gel electrophoresis (FIGE) and contour-clamped homogeneous field (CHEF) electrophoresis were used to analyse the chromosome of Yersinia ruckeri. The 8 base-pair recognition endonucleases, NotI and SfiI, generated less than 47 DNA fragments whose size and distribution were appropriate for pulsed field separation. Each isolate displayed a characteristic restriction pattern, with about 20% of bands in common. Depending on the strain used, the estimated genome size for this bacterial fish pathogen ranged from 4460 to 4770 kilobase pairs.

Animals↗
PubMed1990

A highly efficient electroporation system for transformation of Yersinia.

The various pathogenic Yersinia species are not readily and efficiently transformed by classical methods. For this reason, the electroporation technique was applied for genetic transformation of these species. Using optimal conditions, we were able to transform the six Yersinia strains studied with the two most widely used groups of plasmids: pSU2718 (a pACYC184 derivative) and pK19 (a pUC19 derivative). Only Yersinia enterocolitica (Y. e.) serotype 0:8 gave poor results (less than 5 x 10(2) transformants/microgram) DNA). Electrical transformation of the other species resulted in high efficiencies, up to 10(5) transformants/microgram DNA for Y. e. serotypes 0:3 and 0:9, 10(6) for Y. pseudotuberculosis and 10(7) for Y. pestis. The results varied for each strain with the type of plasmid used. Neither the introduced foreign plasmid nor the resident 72-kb virulence plasmid underwent detectable deletions. Transformation was most efficient with supercoiled DNA, decreasing by one and four orders of magnitude for relaxed circular and linearized plasmids, respectively. The ability to easily and efficiently transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA technology for direct cloning and analysis of significant genes into Yersinia.

Electric Stimulation↗
PubMed1990

Yersiniosis.

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Animals↗