Microdiversity of Enterococcus faecalis isolates in cases of infective endocarditis: selection of non-synonymous mutations and large deletions is associated with phenotypic modifications.

Context: Today, infective endocarditis (IE) caused by Enterococcus faecalis represents 10% of all IE and is marked by its difficult management and the frequency of relapses. Although the precise reasons for that remain to be elucidated, the evolution of the culprit strain under selective pressure through microdiversification could be, at least in part, involved. Material and methods: To further study the in situ genetic microdiversity and its possible phenotypic manifestations in E. faecalis IE, we sequenced and compared multiple isolates from the valves, blood culture and joint fluid of five patients who underwent valvular surgery. Growth rate and early biofilm production of selected isolates were also compared. Results: By sequencing a total of 58 E. faecalis genomes, we detected a considerable genomic microdiversity, not only among strains from different anatomical origins, but also between isolates from the same studied cardiac valves. Interestingly, deletions of thousands of bases including the well-known virulence factors ebpA/B/C, and srtC, as well as other large prophage sequences containing genes coding for proteins implicated in platelet binding (PlbA and PlbB) were evidenced. The study of mutations helped unveil common patterns in genes related to the cell cycle as well as central metabolism, suggesting an evolutionary convergence in these isolates. As expected, such modifications were associated with a significant impact on the in-vitro phenotypic heterogeneity, growth, and early biofilm production. Conclusion: Genome modifications associated with phenotypic variations may allow bacterial adaptation to both antibiotic and immune selective pressures, and thus promote relapses.

[The use of venous catheter : Which ones to choose, how to prevent their complications?] / Utilisation des cathéters veineux : lesquels choisir, comment prévenir leurs complications ?

Intravenous catheters are multiple and essential for daily practice. They are also responsible for high morbidity and mortality. Simple or echo-guided peripheral venous catheters, midlines, PICCline, tunneled or non-tunneled central venous catheters, and implantable venous access device are currently at our disposal. Thus, catheter selection, duration and indications for use, and prevention and treatment of complications vary according to the situation. The objective of this update is to provide the clinician with an overview of knowledge and rules of good practice on the use of catheters.

[Cerebral infarction and tuberculosis: case report and literature review]. / Infarctus cérébral et tuberculose : rapport d'un cas et analyse de la littérature.

INTRODUCTION: Although tuberculous meningitis is an uncommon presentation of tuberculosis, it still remains one of the deadliest forms of this disease. In this context, the occurrence of a cerebral infarct is an aggravating factor. OBSERVATION: A 48-year-old Asian man presented himself in the emergency room for dysarthria and dysphagia of progressive onset. Cerebral CT showed a recent ischemic defect of the right internal capsule. Lumbar puncture showed meningitis with low sugar levels. Pulmonary micronodules on the thoracic CT suggested tuberculosis, which was confirmed by a broncho-alveolar lavage. Anti-tuberculosis treatment and early corticosteroid resulted in an improvement of the patient's state. CONCLUSION: Cerebral infarctions in patients with tuberculous meningitis are events that cannot be underestimated in terms of frequency or severity. Their poor prognosis is partly the result of insufficiently defined management, which combines anti-tuberculosis treatment and early corticosteroid therapy.

[A tularemia mimicking lymphoma]. / Une tularémie mimant un lymphome.

INTRODUCTION: Adenopathies are a frequent cause of recourse in internal medicine. When histological analysis reveals the presence of granuloma, multiple infectious or non-infectious etiologies are considered. If diagnoses of lymphoma, sarcoidosis or tuberculosis are easily mentioned, tularemia should also be considered in the differential diagnosis. OBSERVATION: A 54-year-old patient had a fever at the evening with night sweats and a cough resistant to two lines of antibiotics. A thoraco-abdomino-pelvic CT scan revealed hilar and mediastinal adenopathies that appeared hypermetabolic with PET-TDM, as well as pulmonary nodules. A PCR performed on lymph node biopsy and serology allowed the diagnosis of tularemia. The evolution was favourable after antibiotic treatment. CONCLUSION: The association of fever, night sweats, altered general state and mediastinal adenopathies should be considered as a diagnosis of tularemia. Ganglionic biopsy, combined with molecular biology techniques and serology, can confirm the diagnosis.

The E3 ubiquitin ligase complex component COP1 regulates PEA3 group member stability and transcriptional activity.

In this study, we report that the PEA3 group members interact with the mammalian really interesting new gene (RING) E3 ubiquitin ligase constitutive photomorphogenetic 1 (COP1), which mediates ubiquitylation and subsequent proteasome degradation of the p53 and c-Jun transcription factors. This interaction is mediated by the central region of COP1 including the coiled-coil domain and two COP1-interacting consensus motifs localized in the well-conserved N-terminal transactivation domain of the PEA3 group members. At the transcriptional level, COP1 reduces the transcriptional activity of ERM and the two other PEA3 group proteins on Ets-responsive reporter genes; this effect being dependent on the RING domain of COP1 and the two COP1-interacting motifs of ERM. Reduced transcriptional activity was, however, not related to COP1-induced changes in ERM stability. In fact, increased ubiquitylation and subsequent proteasome-mediated degradation of ERM is achieved only when COP1 is expressed with DET1, a key COP1 partner within the ubiquitylation complex. Conversely, we show that the depletion of COP1 or DET1 by small interference RNA (siRNA) in U2OS cells stabilizes endogenous ERM whereas only COP1 knockdown enhances expression of ICAM-1, a gene regulated by this transcription factor. These results indicate that COP1 is a complex regulator of ERM and the two other PEA3 group members.

Expression patterns of the Ets transcription factors from the PEA3 group during early stages of mouse development.

erm, er81 and pea3 are three related genes that define a novel Ets-related subfamily of transcription factors. The expression patterns of these genes has been previously characterized in the mouse from embryonic day (E) 9.5 to birth (Oncogene 15 (1997) 937). In this study, we report differential expression patterns of the PEA3 group genes during early mouse post-implantation development. erm and pea3 expression patterns were partly overlapping. erm was activated prior to pea3 in the distal tip of the embryonic epiblast but, at primitive streak-stages, both genes were coexpressed in the posterior region of the embryo in epiblast, primitive streak and adjacent mesoderm. Similar erm and pea3 expression patterns were seen later in posterior neural plate, presomitic and lateral mesoderm, mesonephros, and tail bud. Only erm, however, was expressed in specific brain regions corresponding to prospective midbrain and ventral forebrain. erm was also strongly expressed as early as E8 in the developing branchial region, especially at the level of branchial pouches, whereas pea3 transcripts appeared later in frontonasal and first arch mesenchyme. er81 transcripts were not detected prior to E9.0-9.5, suggesting that this gene may not be involved in early developmental events.

Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway.

Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its approximately 130 kDa C-terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria. This is reminiscent of pro-protein convertases of eukaryotic cells.

Cloning and expression analysis of a cDNA that encodes a leech hemerythrin.

We report the cDNA sequence of a leech hemerythrin. A cDNA was isolated from a Theromyzon tessulatum cDNA library and encodes a 120 amino acid protein of about 14 kDa. The predicted protein contains the hemerythrin signature sequence and the iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin. The protein displayed the highest identity to myohemerythrin, a non-heme iron-binding protein described in sipunculids. Expression analysis indicated that the mRNA is widely expressed in leech and is stage specific in appearance, being absent after the two first blood meals, appearing after the last blood meal during the period preceding oogenesis and disappearing after egg laying.

Rapid PCR-based procedure to identify lactic acid bacteria: application to six common Lactobacillus species.

The goal of this study was to develop a method allowing rapid identification of the lactic acid bacteria strains in use in the laboratory (Lactobacillus plantarum NCIMB8826; L. fermentum KLD; L. reuteri 100-23; L. salivarius UCC43321; L. paracasei LbTGS1.4; L. casei ATCC393), based on PCR amplification of 16S RNA coding sequences. First, specific forward oligonucleotides were designed in the variable regions of 16S RNA coding sequences of six Lactobacillus strains. The reverse oligonucleotide was designed in the region where the sequences were homologous for the six strains. The expected size of the amplification product was +/-1000 bp. The specificity of the method was tested on total chromosomal DNA. For five out of the six strains, the amplification of the fragment was strain-specific and the method was directly applicable to colonies. For the strain L. casei ATCC393, an additional argument to the classification of this bacteria in the paracasei group could be proposed. Validation of the developed method was performed by applying it to six Lactobacillus reference strains and to various species of bacteria.

New virulence-activated and virulence-repressed genes identified by systematic gene inactivation and generation of transcriptional fusions in Bordetella pertussis.

An in silico scan of the partially completed genome sequence of Bordetella pertussis and analyses of transcriptional fusions generated with a new integrational vector were used to identify new potential virulence genes. The genes encoding a putative siderophore receptor, adhesins, and an autotransporter protein appeared to be regulated in a manner similar to Bordetella virulence genes by the global virulence regulator BvgAS. In contrast, the gene encoding a putative intimin-like protein appeared to be repressed under conditions of virulence.

The PEA3 group of ETS-related transcription factors. Role in breast cancer metastasis.

The ets genes encode eukaryotic transcription factors that are involved in tumorigenesis and developmental processes. The signature of the Ets family is the ETS-domain, which binds to sites containing a central 5'-GGAA/T-3' motif. They can be sub-classified primarily because of the high amino acid conservation in their ETS-domains and, in addition, in the conservation of other domains generally characterized as transactivating. This is the case for the PEA3 group, which is currently made up of three members, PEA3/E1AF, ER81/ETV1 and ERM, which are more than 95% identical in the ETS-domain and more than 85% in the transactivation acidic domain. The members of the PEA3 group are activated through both the Ras-dependent and other kinase pathways, a function which emphasizes their involvement in several oncogenic mechanisms. The expression pattern of the three PEA3 group genes during mouse embryogenesis suggests that they are differentially regulated, probably to serve important functions such as tissue interaction. Although the target genes of these transcription factors are multiple, their most frequently studied role concerns their involvement in the metastatic process. In fact, PEA3 group members are over-expressed in metastatic human breast cancer cells and mouse mammary tumors, a feature which suggests a function of these transcription factors in mammary oncogenesis. Moreover, when they are ectopically over-expressed in non-metastatic breast cancer cells, these latter become metastatic with the activation of transcription of matrix metalloproteinases or adhesion molecules, such as ICAM-1.

Characterization of the human and mouse ETV1/ER81 transcription factor genes: role of the two alternatively spliced isoforms in the human.

The Ets transcription factors of the PEA3 group--E1AF/PEA3, ETV1/ER81 and ERM--are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1 alpha) or the absence (ETV1 beta) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1 beta) and the full-length isoform (human ETV1 alpha) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1 alpha and human ETV1 beta expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

Genomic organization of the human e1af gene,a member of Ets transcription factors.

The E1AF protein belongs to the family of Ets transcription factors and is involved in the regulation of metastasis gene expression. It has recently been reported in an undifferentiated child sarcoma that part of this gene could be fused by translocation to the ews gene. We show here that the human e1af gene, which is located in the q21 region of chromosome 17, is organized in 13 exons distributed along 19kb of genomic DNA. Its two main functional domains, the acidic domain and the DNA-binding ETS domain, are each encoded by three different exons. The 3'-untranslated region of e1af is 0.7kb. The 5'-untranslated region is about 0.3kb and is composed of a first exon upstream from the exon containing the first methionine. These data could possibly accelerate an understanding of the molecular basis of putative inherited diseases linked to E1AF.

Structure-function relationships of the PEA3 group of Ets-related transcription factors.

The PEA3 group of transcription factors belongs to the Ets family and is composed of PEA3, ERM, and ER81, which are more than 95% identical within the DNA-binding domain--the ETS domain--and which demonstrate 50% aa identity overall. We present here a review of the current knowledge of these transcription factors, which possess functional domains responsible for DNA-binding, DNA-binding inhibition, and transactivation. Recent data suggest that these factors are targets for signaling cascades, such as the Ras-dependent ones, and thus may contribute first to the nuclear response to cell stimulation and second to Ras-induced cell transformation. The expression of the PEA3 group members in numerous developing murine organs, and, especially, in epithelial-mesenchymal interaction events, suggests a key role in murine organogenesis. Moreover, their expression in certain breast cancer cells suggests a possible involvement of these genes in the appearance, progression, and invasion of malignant cells.

Genomic organization of the human ERM (ETV5) gene, a PEA3 group member of ETS transcription factors.

The ERM protein belongs to the family of Ets transcription factors. We show here that the human ERM gene is organized into 14 exons distributed along 65 kb of genomic DNA on chromosome 3. The two main functional domains of ERM, the acidic domain and the DNA-binding ETS domain, are overlapped by three different exons each. The 3'-untranslated region of ERM is 2.1 kb, whereas the 5'-untranslated region is about 0.3 kb; this allows the transcription of ERM transcripts of approximately 4 kb. The human ERM gene is localized to the q27-q29 region of chromosome 3.

Molecular characterization of the ets-related human transcription factor ER81.

The PEA3 group is a homogeneous group of the ets transcription factor family and is composed of three known members, PEA3, ERM and ER81, which, on the amino acid (AA) level, are more than 95% identical within the DNA-binding domain (the Ets domain), more than 85% within a 32 AA domain (the acidic domain) localized in the amino-terminus and almost 50% identical overall. By screening a human kidney cDNA library with a specific probe obtained from mouse ER81, we isolated two clones of 1.6 and 1.5 kb in length encoding a 458 AA open reading frame. When compared to mouse ER81, the present human ER81 lacks the last 13 AA of the acidic domain and the 5 AA contiguous to the carboxy-terminal part of the acidic domain. Of the 458 AA of the human ER81 protein, 97% are identical to mouse ER81. Gel shift analysis indicates that the full-length human ER81 protein is able to bind specifically to an oligonucleotide containing the binding sites recognized by most of the Ets proteins. By transient expression in RK13 mammalian cells, human ER81 protein transactivated a reporter plasmid containing Ets binding sites, indicating that this molecule is a bonafide transcriptional activator, while by expression in Cos-1 transfected cells, we detected the presence of human ER81 protein in the nucleus using immunocytochemistry. In human tissues, ER81 mRNA is very highly expressed in brain, highly expressed in testis, lung and heart, moderately in spleen, small intestine, pancreas and colon, weakly in liver, prostate and thymus, very weakly in skeletal muscle, kidney and ovary and not in placenta and peripheral blood leukocytes. Analysis of human solid or haematopoietic tumour cell lines showed that most of them did not express ER81 detectably. Database searches revealed that ETV1 mRNA is highly similar to human ER81 described here, although it contains the full-length acidic domain present in mouse ER81. By screening a genomic DNA library, we characterized the intron-exon junction within the acidic domain of human ER81 and we showed that this junction corresponds to the site where the remaining AA of the acidic domain of ETV1 or mouse ER81 are inserted.

[Transcription factors of the PEA3 group in mammary cancer]. / Les facteurs de transcription du groupe PEA3 dans le cancer mammaire.

Prognosis factors such as mutated or amplified oncogenes are used in the treatment of breast cancer. We have recently shown that the members of the PEA3 group (ER81, ERM and PEA3) from the transcription factor family of the ets genes are overexpressed in breast cancer tumors arising from MMTV-neu transgenic animals. Moreover, we have shown that ER81, and in a lesser extent ERM and PEA3, are not expressed in the estrogen and/or progesterone receptor-positive mammary cancer cell lines, whereas they are expressed in the receptor negative ones. Our research interest in now focused on the role(s) of these oncogenes in the development and the regulation of breast tumors.