Biomedical subjects

Bernard Martin

Publications and source records attributed to Bernard Martin.

16 recordsLinked to original sources

PubMed2006

Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae.

Natural transformation is a widespread mechanism for genetic exchange in bacteria. Aminoglycoside and fluoroquinolone antibiotics, as well as mitomycin C, a DNA-damaging agent, induced transformation in Streptococcus pneumoniae. This induction required an intact competence regulatory cascade. Furthermore, mitomycin C induction of recA was strictly dependent on the development of competence. In response to antibiotic stress, S. pneumoniae, which lacks an SOS-like system, exhibited genetic transformation. The design of antibiotherapy should take into consideration this potential of a major human pathogen to increase its rate of genetic exchange in response to antibiotics.

Aminoglycosides↗

PubMed2006

Independent evolution of competence regulatory cascades in streptococci?

Natural genetic transformation is a mechanism of horizontal gene transfer that is widely distributed in bacteria and requires assembly of a DNA uptake machinery. Transformable bacteria use fundamentally the same machine, which in most species is assembled only in cells that are developing competence. Competence regulation usually differs between unrelated species. Here, we examine whether related streptococci use the same competence regulatory cascade. Phylogenetic analyses of streptococcal genome sequences reveal the existence of two paralogous two-component regulatory systems, either of which might control competence. This suggests the distribution of streptococci into two groups that use competence regulatory cascades that have at least partly evolved independently. Comparison of data obtained with two transformable streptococci, Streptococcus pneumoniae and Streptococcus mutans, provides support to this suggestion.

DNA, Bacterial↗

PubMed2006

Inhibition of competence development in Streptococcus pneumoniae by increased basal-level expression of the ComDE two-component regulatory system.

Natural competence for genetic transformation in Streptococcus pneumoniae is controlled by the ComCDE signal-transduction pathway. Together, ComD, a membrane histidine kinase, and ComE, its cognate response regulator, constitute a typical two-component regulatory system involved in sensing the comC-encoded competence-stimulating peptide (CSP). The comCDE operon is strongly upregulated when CSP reaches a critical threshold, probably to coordinate competence induction throughout the population. During a study of the early regulation of the comCDE operon, a mutation which resulted in increased beta-galactosidase production from a comC : : lacZ fusion was isolated. This mutation, which was characterized as a G-->T change in the transcription terminator of the tRNA(Arg) located immediately upstream of comCDE, is suggested to destabilize the terminator and to allow transcriptional readthrough of comCDE. Here, it is shown that, quite unexpectedly, the mutation confers reduced transformability. A series of experiments undertaken with the aim of understanding this surprising phenotype is described. Evidence is presented that increased basal-level expression of comDE impedes both spontaneous and CSP-induced competence in S. pneumoniae. There is a discussion of how an increased concentration of ComD and/or ComE could affect competence development.

Bacterial Proteins↗

PubMed2006

Construction and evaluation of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Streptococcus pneumoniae.

In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome of Streptococcus pneumoniae, is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication in Escherichia coli, but not in S. pneumoniae, was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, P(M), separated from a kanamycin-resistance gene by NcoI and BamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of P(M) can be obtained through direct transformation of an S. pneumoniae recipient with ligation products between that gene and NcoI/BamHI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.

Bacteriological Techniques↗

PubMed2006

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor.

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.

Adhesins, Bacterial↗

PubMed2006

Induction of competence regulons as a general response to stress in gram-positive bacteria.

Bacterial transformation, a programmed mechanism for genetic exchange originally discovered in Streptococcus pneumoniae, is widespread in bacteria. It is based on the uptake and integration of exogenous DNA into the recipient genome. This review examines whether induction of competence for genetic transformation is a general response to stress in gram-positive bacteria. It compares data obtained with bacteria chosen for their different lifestyles, the soil-dweller Bacillus subtilis and the major human pathogen S. pneumoniae. The review focuses on the relationship between competence and other global responses in B. subtilis, as well as on recent evidence for competence induction in response to DNA damage or antibiotics and for the ability of S. pneumoniae to use competence as a substitute for SOS. This comparison reveals that the two species use different fitness-enhancing strategies in response to stress conditions. Whereas B. subtilis combines competence and SOS induction, S. pneumoniae relies only on competence to generate genetic diversity through transformation.

Bacillus subtilis↗

PubMed2005

A complementation method for functional analysis of mammalian genes.

Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies.

Adenoviridae↗

PubMed2005

Competence-programmed predation of noncompetent cells in the human pathogen Streptococcus pneumoniae: genetic requirements.

Natural competence for genetic transformation is the best-characterized feature of the major human pathogen Streptococcus pneumoniae. Recent studies have shown the virulence of competence-deficient mutants to be attenuated, but the nature of the connection between competence and virulence remained unknown. Here we document the release, triggered by competent cells, of virulence factors (e.g., the cytolytic toxin pneumolysin) from noncompetent cells. This phenomenon, which we name allolysis, involves a previously undescribed bacteriocin system consisting of a two-peptide bacteriocin, CibAB, and its immunity factor, CibC; the major autolysin, LytA, and lysozyme, LytC; and a proposed new amidase, CbpD. We show that CibAB are absolutely required for allolysis, whereas LytA and LytC can be supplied either by the competent cells or by the targeted cells. We propose that allolysis constitutes a competence-programmed mechanism of predation of noncompetent cells, which benefits to the competent cells and contributes to virulence by coordinating the release of virulence factors.

Amidohydrolases↗

PubMed2005

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference.

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-alpha, IL-1beta or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-kappaB, or on the protein levels of IkappaB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases.

Adenoviridae↗

PubMed2004

Modelling of shoulder and torso perception of effort in manual transfer tasks.

The aim of the present study was to develop statistical models of perceived effort at the shoulder and torso levels associated with manual load transfer tasks. The motions were directed from a home location toward one of twenty-two target shelves distributed in the right hemisphere. A total of 2149 ratings were obtained from 31 subjects for effort perception at the selected joints, using a ten-point modified Borg scale. Regression models, developed for the perception associated with each body part, included target locations (azimuth, height and distance), posture constraints (standing or sitting), task types (one or two handed transfer conditions), and demographic and anthropometric measures (stature, body weight, gender, and age) as parameters. The models provide a prediction of effort perception with adjusted r-square coefficients of 0.41 and 0.50 for the shoulder and torso, respectively. The results indicate that space and posture interact in a complex way to affect the rating of perceived effort, and are in agreement with the hypothesis that the 'sense of effort' is primarily associated with the efference copy of the descending motor command. Since a level of effort is not associated with a unique pattern of motor command, it is proposed that effort perception is likely to result from a summation of the components of the motor command. The models can be applied to optimize the spatial organization of the work environment in an attempt to reduce the risk of musculoskeletal injury.

Adult↗

PubMed2004

Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells.

Of the 13 two-component signal transduction systems (TCS) identified in Streptococcus pneumoniae, two, ComDE and CiaRH, are known to affect competence for natural genetic transformation. ComD and ComE act together with the comC-encoded competence-stimulating peptide (CSP) and with ComAB, the CSP-dedicated exporter, to co-ordinate activation of genes required for differentiation to competence. Several lines of evidence suggest that the CiaRH TCS and competence regulation are interconnected, including the observation that inactivation of the CiaR response regulator derepresses competence. However, the nature of the interconnection remains poorly understood. Interpretation of previous transcriptome analyses of ciaR mutants was complicated by competence derepression in the mutants. To circumvent this problem, we have used microarray analysis to investigate the transition from non-competence to competence in a comC-null wild-type strain and its ciaR derivative after the addition of CSP. This study increased the number of known CSP-induced genes from approximately 47 to 105 and revealed approximately 42 genes with reduced expression in competent cells. Induction of the CiaR regulon, as well as the entire HrcA and part of the CtsR stress response regulons, was observed in wild-type competent cells. Enhanced induction of stress response genes was detected in ciaR competent cells. In line with these observations, CSP was demonstrated to trigger growth arrest and stationary phase autolysis in ciaR cells. Taken together, these data strongly suggest that differentiation to competence imposes a temporary stress on cells, and that the CiaRH TCS is required for the cells to exit normally from the competent state.

Bacterial Proteins↗

PubMed2003

Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia.

We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.

Analysis of Variance↗

PubMed2003

Bacterial "competence" genes: signatures of active transformation, or only remnants?

An exhaustive review published ten years ago reported natural genetic transformation, a potential mechanism for intra- and interspecies gene transfer, in approximately 40 species belonging to different taxonomic and trophic groups. Since then, considerable progress has been made in characterizing DNA-uptake machineries and regulatory circuits controlling their expression in cells competent for genetic transformation. In this article, in light of the recent description of a Group A streptococcal isolate capable of DNA transfer in mixed cultures, we discuss whether the detection in completely sequenced microbial genomes of intact homologues of key competence-regulatory and/or DNA-uptake proteins enables the prediction of new transformable species.

Bacteria↗

PubMed2003

Transformation of Streptococcus pneumoniae relies on DprA- and RecA-dependent protection of incoming DNA single strands.

Seventy-five years after the discovery of transformation with Streptococcus pneumoniae, it is remarkable how little we know of the proteins that interact with incoming single strands in the early processing of transforming DNA. In this work, we used as donor DNA in transformation a radioactively labelled homologous fragment to examine the fate of the single-stranded (ssDNA) products of uptake in cells mutant for DprA or RecA, two proteins essential for transformation. Fifteen minutes after uptake, the labelling of specific chromosomal restriction fragments that demonstrated homologous integration in the wild type was not detected in dprA or recA cells, indicating that in the mutants incoming ssDNA could not be processed into recombinants. Investigation of the fate of donor label 1 min after uptake revealed that incoming ssDNA was immediately degraded in the absence of DprA or RecA. Our results demonstrate that incoming ssDNA requires active protection prior to the RecA-driven search for homology and that both DprA and RecA are needed for this protection.

Bacterial Proteins↗

PubMed2002

Uptake of transforming DNA in Gram-positive bacteria: a view from Streptococcus pneumoniae.

In a working model for the uptake of transforming DNA based on evidence taken from both Bacillus subtilis and Streptococcus pneumoniae, the ComG proteins are proposed to form a structure that provides access for DNA to the ComEA receptor through the peptidoglycan. DNA would then be delivered to the ComEC-ComFA transport complex. A DNA strand would be degraded by a nuclease, while its complement is pulled into the cell by ComFA through an aqueous pore formed by ComEC. The nuclease is known in S. pneumoniae only as EndA. We have examined the processing (i.e. binding, degradation and internalization) of DNA in S. pneumoniae strains lacking candidate uptake proteins. Mutants were generated by transposon insertion in endA, comEA/C, comFA/C, comGA and dprA. Processing of DNA was abolished only in a comGA mutant. As significant binding was measured in comEA mutants, we suggest the existence of two stages in binding: surface attachment (abolished in a comGA mutant) required for and preceding deep binding (by ComEA). Abolition of degradation in comGA and comEA mutants indicated that, despite its membrane location, EndA cannot access donor DNA by itself. We propose that ComEA is required to deliver DNA to EndA. DNA was still bound and degraded in comEC and comFA mutants. We conclude that recruitment of EndA can occur in the absence of ComEC or ComFA and that EndA is active even when the single strands it produces are not pulled into the cell. Finally, inactivation of dprA had no effect on the internalization of DNA, indicating that DprA is required at a later stage in transformation.

Bacterial Proteins↗

PubMed2002

Identification of a protein that inactivates the competence-stimulating peptide of Streptococcus pneumoniae.

Competence for genetic transformation of Streptococcus pneumoniae is a transient physiological property inducible by a competence-stimulating peptide (CSP). A 68-kDa CSP-inactivating protein was previously obtained following lithium chloride (LiCl) extraction. By the same protocol, a CSP-inactivating protein was purified and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as an endopeptidase, PepO. Analysis of a pepO mutant provided no support for the hypothesis that PepO participates in competence regulation. To reconcile in vitro and in vivo data, we suggest that LiCl treatment results in the release of intracellular molecules, including PepO.

Bacterial Proteins↗