Gordonia terrae: a difficult-to-diagnose emerging pathogen?
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Actinomycetales Infections↗
Biomedical subjects
V Blanc
Publications and source records attributed to V Blanc.
[Epidemiology of candidemia: results of a one month French hospitals-based surveillance study in 2004].
INTRODUCTION: The aim of our study was to determinate the frequency of candidemia, the species encountered and their susceptibility to antifungal agents in French hospitals in 2004. METHODS: A prospective survey of septicaemia, including candidaemia was conducted among 193 non teaching French hospitals during October 2004. All bloodstream infections were reported and the bloodstream isolates sent to two coordinating centers. Species identification and susceptibility were performed by biologists as usual, and further confirmed by use of alternative methods, including Minimal Inhibitory Concentration (MIC) determination. RESULTS: The survey was effective in 93 hospitals, where 2013 bloodstream infections were noticed, including 46 candidaemia (2.3%). Candida sp. Is the 7th pathogen responsible for bloodstream infection, without concern of the origin of the infection. Candidaemia was hospital acquired in 80.4% of cases (N=37/46). Candidaemia represents 0.7% (N=9/1211) of community-acquired bloodstream infections but 4.5% (N=37/802) of nosocomial cases, placing Candida sp. at the 5th row of frequency in this last group. No mistakes were noticed during identification, but two strains were not named. After confirmation of identification, species are distributed as follows: 23 C.albicans (50%), 13 C.glabrata (28.3%), 5 C.tropicalis (10.9%), 3 C.parapsilosis (6.5%), 1 C.krusei (2.2%), and 1 C.kefyr (2.2%). The overall susceptibility is 97.7% for amphotericin B, 93.2% for 5-fluorocytosin, 88.6% for fluconazole, 70.5% for itraconazole, 95.5% for voriconzole and 100% of strains had very low MIC for caspofungin. Concerning the two main species isolated, 95.5% of C.albicans strains remain susceptible to fluconazole, but only 69% of C.glabrata strains. Two strains, 1 C.albicans and 1 C.glabrata exhibit cross resistance to azoles. The majority of amphotericin, fluconazole, and itraconazole decreased susceptibility strains are found into C.glabrata strains, but susceptibility to 5-fluorocytosin and very low MCI to caspofungin remained in this species. CONCLUSION: Our survey allowed us to get data from French non-teaching hospitals in 2004 about frequency of candidaemia among septicaemia. Candida species distribution and in vitro susceptibility patterns of the strains isolated. Candidaemia is not a rare event, specially when septicaemia is nosocomially acquired. C.albicans is the main species isolated and remains highly in vitro susceptible to antifungals. One important feature is the frequency of C.glabrata in our survey. Considering its decreased susceptibility to azoles, this finding confirms the need for rapid identification of yeast isolated from bloodstream samples, and antifungal susceptibility testing each time it is available.
Antifungal Agents↗
Emergence of two populations of methicillin-resistant Staphylococcus aureus with distinct epidemiological, clinical and biological features, isolated from patients with community-acquired skin infections.
BACKGROUND: Community-acquired skin and soft-tissue infections due to methicillin-resistant Staphylococcus aureus (MRSA) are an emerging clinical and epidemiological problem. OBJECTIVES: To characterize community-acquired skin infections caused by S. aureus, and especially MRSA. METHODS: From November 1999 to December 2003, we conducted in a French hospital a prospective epidemiological, clinical and bacteriological study of skin infections acquired in the community, applying strict criteria for true community-acquired MRSA (CA-MRSA) and health-care-associated MRSA (HCA-MRSA). RESULTS: One hundred and ninety-seven patients had 207 skin infections (154 primary and 53 secondary infections). Twenty-two (11%) patients had skin infections caused by MRSA. The incidence of MRSA skin infections acquired in the community rose from 4% in 2000 to 17% in 2003, but the increase was not statistically significant. Six patients (3%) were infected by CA-MRSA and 15 (8%) by HCA-MRSA; one patient was lost to follow-up and could not be classified. CA-MRSA and HCA-MRSA had different epidemiological, clinical and biological characteristics. CA-MRSA infections were more severe than HCA-MRSA infections: all the CA-MRSA infections (six of six, 100%) required surgical treatment, compared with only two (15%) of 13 with HCA-MRSA infection (P < 0.001). CA-MRSA all belonged to the same clonal strain, harbouring an agr type 3 allele and the Panton-Valentine leucocidin genes (not detected in HCA-MRSA) and possessing a specific antibiotype. CONCLUSIONS: Two populations of MRSA causing skin infections are emerging in the French community, with distinct epidemiological, clinical and biological characteristics.
Adolescent↗
Isolation, characterization and developmental regulation of the human apobec-1 complementation factor (ACF) gene.
Mammalian apolipoprotein B (apo B) mRNA undergoes site-specific C to U deamination which is mediated by a multicomponent enzyme complex containing a minimal core composed of apobec-1 and a complementation factor, ACF. We have isolated and characterized the human ACF gene and examined its tissue-specific and developmental expression. The ACF gene spans approximately 80 kb and contains 15 exons, three of which are non-coding. Multiple alternative splice acceptor sites were found, generating at least nine different transcripts. Of these, the majority (approximately 75-89%) encode functional protein. In order to examine the role of ACF mRNA expression in the regulation of apo B mRNA editing, we examined a panel of fetal intestinal and hepatic mRNAs as well as RNA from an intestinal cell line. A developmental increase in C to U RNA editing has been previously noted in the human intestine. In both instances, the pattern of alternative splicing and overall abundance of ACF mRNA was relatively constant during development in both liver and small intestine. Taken together, the data demonstrate a complex pattern of differential, tissue-specific splicing of ACF mRNA, but suggest that other mechanisms are responsible for the developmental increase noted in intestinal apo B mRNA editing in humans.
APOBEC-1 Deaminase↗
Mutagenesis of apobec-1 complementation factor reveals distinct domains that modulate RNA binding, protein-protein interaction with apobec-1, and complementation of C to U RNA-editing activity.
C to U editing of apolipoprotein B (apoB) RNA requires a multicomponent holoenzyme complex in which minimal constituents include apobec-1 and apobec-1 complementation factor (ACF). We have examined the predicted functional domains in ACF in binding apoB RNA, interaction with apobec-1, and complementation of RNA editing. We demonstrate that apoB RNA binding and apobec-1-interacting domains are defined by two partially overlapping regions containing the NH(2)-terminal RNA recognition motifs of ACF. Both apoB RNA binding and apobec-1 interaction are required for editing complementation activity. ACF is a nuclear protein that upon cotransfection with apobec-1 results in nuclear colocalization and redistribution of apobec-1 from the cytoplasm. ACF constructs with deletions or mutations in the putative nuclear localization signal (NLS) still localize in the nucleus of transfected cells but do not colocalize with apobec-1, the latter remaining predominantly cytoplasmic. These observations suggest that the putative NLS motif in ACF is not responsible for its nucleo-cytoplasmic trafficking. By contrast, protein-protein interaction is important for the nuclear import of apobec-1. Taken together, these data suggest that functional complementation of C to U RNA editing by apobec-1 involves the NH(2)-terminal 380 residues of ACF.
APOBEC-1 Deaminase↗
Identification of GRY-RBP as an apolipoprotein B RNA-binding protein that interacts with both apobec-1 and apobec-1 complementation factor to modulate C to U editing.
C to U editing of apolipoprotein B (apoB) mRNA involves the interaction of a multicomponent editing enzyme complex with a requisite RNA sequence embedded within an AU-rich context. This enzyme complex includes apobec-1, an RNA-specific cytidine deaminase, and apobec-1 complementation factor (ACF), a novel 65-kDa RNA-binding protein, that together represent the minimal core of the editing enzyme complex. The precise composition of the holo-enzyme, however, remains unknown. We have previously isolated an enriched fraction of S100 extracts, prepared from chicken intestinal cells, that displays apoB RNA binding and which, following supplementation with apobec-1, permits efficient C to U editing. Peptide sequencing of this most active fraction reveals the presence of ACF as well as GRY-RBP, an RNA-binding protein with approximately 50% homology to ACF. GRY-RBP was independently isolated from a two-hybrid screen of chicken intestinal cDNA. GRY-RBP binds to ACF, to apobec-1, and also binds apoB RNA. Experiments using recombinant proteins demonstrate that GRY-RBP binds to ACF and inhibits both the binding of ACF to apoB RNA and C to U RNA editing. This competitive inhibition is rescued by addition of ACF, suggesting that GRY-RBP binds to and sequesters ACF. As further evidence of the role of GRY-RBP, rat hepatoma cells treated with an antisense oligonucleotide to GRY-RBP demonstrated an increase in C to U editing of endogenous apoB RNA. ACF and GRY-RBP colocalize in the nucleus of transfected cells and, in cotransfection experiments with apobec-1, each appears to colocalize in a predominantly nuclear distribution. Taken together, the results indicate that GRY-RBP is a member of the ACF gene family that may function to modulate C to U RNA editing through binding either to ACF or to apobec-1 or, alternatively, to the target RNA itself.
APOBEC-1 Deaminase↗
C to U editing of the anticodon of imported mitochondrial tRNA(Trp) allows decoding of the UGA stop codon in Leishmania tarentolae.
All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNA(Trp)(CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNA(Trp) undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.
Animals↗
The mitochondrial RNA ligase from Leishmania tarentolae can join RNA molecules bridged by a complementary RNA.
A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a K(m) of 25 +/- 0.75 nM and a V(max) of 1.0 x 10(-4) +/- 2.4 x 10(-4) nmol/min when ligating a nicked double-stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single-stranded substrate was 5-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion/deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.
Adenosine Triphosphate↗
[Infection after total hip replacement by Staphylococcus caprae. Case report and review of the literature].
We report a case of Staphylococcus caprae bone and joint infection, that illustrate difficulties to diagnose coagulase-negative staphylococci (CNS) orthopedic surgery infections, specially following implantation of prostheses. Four of 5 strains successivelly isolated from deep and/or peri-operative specimens during late infection after total hip replacement (THR) have been identified, using commercial systems and conventionnal tests, as S. caprae. Identity of biochemical profile, antibiotype and pulsotype of the 4 isolates confirmed the pathogenicity of this animal CNS, rarely described as a human pathogen. Analysis of the 24 S. caprae human cases previously described evidence a relation ship between this bacteria and bone and joint infections, with implantation of prosthetic material as supplementary risk factor. S. caprae, whose major identification criteria are resumed, may have previously been misidentified as some similar CNS; this bacteria is probably part of our normal flora but may be recognized as an opportunistic pathogen, responsible for both nosocomial and community acquired infections.
Aged↗
Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes.
A Streptomyces pristinaespiralis strain, which produces a streptogramin antibiotic pristinamycin II (PII) as a mixture of two biologically active molecules PIIB and PIIA, was genetically engineered to produce exclusively PIIA. The snaA,B genes, which encode a PIIA synthase that performs oxidation of the precursor (PIIB) to the final product (PIIA), were integrated in the chromosome of S. pristinaespiralis using an integrative derivative of the pSAM2 genetic element from Streptomyces ambofaciens. Integration was due to site-specific recombination at the attB site of S. pristinaespiralis, and no homologous recombination at the snaA,B locus was observed. The attB site of S. pristinaespiralis was sequenced and found to overlap the 3' end of a pro-tRNA gene. The integrants were stable in industrial conditions of pristinamycin production and showed no decrease in PII biosynthesis. Western blot analysis showed a constant production of the PIIA synthase in the overall fermentation process due to expression of the cloned snaA,B genes from the constitutive ermE promoter. This allows the complete conversion of the PIIB form into PIIA.
Attachment Sites, Microbiological↗
Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4-dimethylamino-L-phenylalanine precursor of pristinamycin I.
Four pap genes (papA, papB, papC, papM) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4-dimethylamino-L-phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI- phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N-methylation steps of 4-amino-L-phenylalanine leading to DMPAPA via 4-methylamino-L-phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.
Cloning, Molecular↗
Streptogramin B biosynthesis in Streptomyces pristinaespiralis and Streptomyces virginiae: molecular characterization of the last structural peptide synthetase gene.
Streptomyces pristinaespiralis and S. virginiae both produce closely related hexadepsipeptide antibiotics of the streptogramin B family. Pristinamycins I and virginiamycins S differ only in the fifth incorporated precursor, di(mono)methylated amine and phenylalanine, respectively. By using degenerate oligonucleotide probes derived from internal sequences of the purified S. pristinaespiralis SnbD and SnbE proteins, the genes from two streptogramin B producers, S. pristinaespiralis and S. virginiae, encoding the peptide synthetase involved in the activation and incorporation of the last four precursors (proline, 4-dimethylparaaminophenylalanine [for pristinamycin I(A)] or phenylalanine [for virginiamycin S], pipecolic acid, and phenylglycine) were cloned. Analysis of the sequence revealed that SnbD and SnbE are encoded by a unique snbDE gene. SnbDE (4,849 amino acids [aa]) contains four amino acid activation domains, four condensation domains, an N-methylation domain, and a C-terminal thioesterase domain. Comparison of the sequences of 55 amino acid-activating modules from different origins confirmed that these sequences contain enough information for the performance of legitimate predictions of their substrate specificity. Partial sequencing (1,993 aa) of the SnbDE protein of S. virginiae allowed comparison of the proline and aromatic acid activation domains of the two species and the identification of coupled frameshift mutations.
Amino Acid Sequence↗
Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes.
Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed.
Amino Acid Sequence↗
Two cases of epidemic mucormycosis infection in patients with acute lymphoblastic leukemia.
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Adult↗
Control of gene expression by base deamination: the case of RNA editing in wheat mitochondria.
The term 'RNA editing' was used for the first time in 1986 to describe the process of uridylate insertion into trypanosomal mitochondrial transcripts. Since then, the term has been used more generally to describe a large variety of processes involving base insertions, deletions and conversions that generate RNAs with a primary sequence different to those encoded by the gene. RNA editing has been observed in the mitochondrial fraction of trypanosomes, plants and other organisms, in the animal nuclear fraction in the case of the apolipoprotein B and glutamate brain receptors mRNAs as well as in viruses like paramyxovirus, hepatitis delta and probably HIV. The role of cytidine and adenine deamination leading to C to U and A to I transitions has became pivotal to explain this process by base conversion. In this review we will focus mainly on the work performed in our group on plant mitochondria and more specifically on the mechanism and the functional significance of RNA editing in wheat organelles. The original contributions of our laboratory in this field are: i) showing that RNA editing is reflected at the protein level; ii) settling three in vitro systems to assay C to U conversion using a wheat mitochondrial lysate as source of enzymes and factors, and unedited mRNA from the same source, as substrate; iii) determination by double labelling of the unedited substrate that RNA editing in wheat mitochondria occurs via a deamination step; and iv) that introducing unedited proteins in the mitochondria of transgenic plants leads to the emergence of cytoplasmic male sterility supporting the idea that the role of this process is to produce functional proteins. Using the antisense approach in transgenic plants we were able to obtain a significant male fertility restoration.
Cytosine Deaminase↗
RNA editing in wheat mitochondria proceeds by a deamination mechanism.
Most if not all mitochondrial messenger RNAs from seed plants undergo a post-transcriptional modification (RNA editing) involving the conversion of some cytidine residues to uridine. Using a molecular hybridization approach, an in vitro RNA editing system, able to faithfully reproduce the in vivo observed C to U changes of subunit 9 (atp9) of wheat mitochondrial ATP synthase mRNA, has been described [Araya et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1040-1044]. In this work we extend these studies to better understand the biochemical mechanism of this process. RNA editing was analysed by P1 nuclease digestion of the reaction product followed by thin layer chromatography. Experiments performed with unedited [3H]RNA labelled on the base and with unedited [32P]RNA labelled at the alpha-phosphate of cytidine residues, indicate that plant mitochondrial RNA editing operates through a deamination mechanism.
Base Sequence↗
RNA editing in wheat mitochondria.
C to U transitions in plant mitochondrial mRNA (RNA editing) lead to amino acid changes as well as to the creation of new initiation or termination codons. We established an in vitro system to assay and to dissect the process of wheat mitochondrial mRNA editing. A deamination mechanism explains most easily the observed C to U transitions. Several fractions of organellar protein participate in the editing machinery. Some of these proteins presumably carry the catalytic activity while others are typical RNA binding proteins and may confer specificity to the 'editosome' complex. To investigate the functional properties of protein products synthesized from unedited mRNAs, we constructed transgenic tobacco plants carrying an unedited gene coding for subunit 9 (ATP9) of the ATP synthase complex. The nuclear encoded 'unedited' protein product is targeted to the mitochondria with a heterologous presequence. A significant number of male sterile tobacco plants were obtained suggesting that at least the functional ATP9 protein requires RNA editing. This result suggests a novel approach to obtain artificial male sterile plants by using a physiological effect resulting in CMS which mimics the situation found in many natural populations.
Codon, Initiator↗
Molecular characterization and transcriptional analysis of a multidrug resistance gene cloned from the pristinamycin-producing organism, Streptomyces pristinaespiralis.
A multidrug resistance gene (mdr) has been cloned from Streptomyces pristinaespiralis, a producer of two antibiotics having synergistic activities together known as pristinamycin. This gene, ptr, provides resistance not only to two structurally dissimilar compounds (pristinamycin I, PI; pristinamycin II, PII) and the natural pristinamycin mixture but also to rifampicin. Mutagenesis and subcloning of ptr localized it to a 2 kb region which was sequenced and analyzed. It contained an open reading frame of 1506 bp which encoded a putative membrane protein with 14 hydrophobic domains, and showed sequence similarity to a superfamily of bacterial proteins that employ transmembrane electrochemical gradients to catalyse active efflux of various antibiotics and toxic compounds. Ptr was most similar to a subfamily which included other mdr genes and antibiotic transport genes associated with antibiotic biosynthetic gene clusters in actinomycetes. In vitro coupled transcription-translation experiments were used to identify the ptr gene product. Analysis of the upstream region did not reveal a divergently transcribed repressor gene, as is the case for several related resistance determinants involved in antibiotic transport, suggesting that ptr is regulated by a different mechanism. Transcriptional analyses of this gene, carried out in both S. pristinaespiralis and Streptomyces lividans, indicated the same transcriptional start point and predicted -10 and -35 hexamers which were somewhat similar to Streptomyces vegetative-type promoters.
Amino Acid Sequence↗