Biomedical subjects

Alain Blanchard

Publications and source records attributed to Alain Blanchard.

12 recordsLinked to original sources

PubMed2007

Relating root structure and anatomy to whole-plant functioning in 14 herbaceous Mediterranean species.

This study investigated the relationships between root structure and anatomy and whole-plant functioning in herbaceous species. Fourteen annual and perennial species representative of a Mediterranean old-field succession were grown in monocultures in a common-garden experiment. Whole-plant functioning was assessed by inherent relative growth rate (RGR(max)), measured in standardized conditions, and maximum height (H(max)). Root tissue density (TMD(r)), considered as a major component of root structure, was measured on roots harvested within in-growth cores. Anatomical characteristics were analysed on cross-sectional areas (CSA). TMD(r) was correlated positively with H(max) and negatively with RGR(max). Root CSA explained interspecific variation in H(max) but not that in TMD(r) and RGR(max). Root xylem CSA and xylem proportion in root CSA were positively correlated with TMD(r) and H(max) and negatively with RGR(max). Mean xylem vessel CSA did not account for variations in TMD(r), H(max) and RGR(max). These results suggested that RGR(max) and H(max) are constrained by opposite root structural and anatomical traits, which have potential links with hydraulic conductance, support and longevity.

Magnoliopsida↗

PubMed2006

Activities of antimicrobial peptides and synergy with enrofloxacin against Mycoplasma pulmonis.

We showed in a previous study that associations of antimicrobial peptides (AMPs), which are key components of the innate immune systems of all living species, with the fluoroquinolone enrofloxacin can successfully cure HeLa cell cultures of Mycoplasma fermentans and M. hyorhinis contamination. In the present work, the in vitro susceptibility of M. pulmonis, a murine pathogen, to enrofloxacin and four AMPs (alamethicin, globomycin, gramicidin S, and surfactin) was investigated, with special reference to synergistic associations and the effect of the mycoplasma cell concentration. Enrofloxacin and globomycin displayed the lowest MICs (0.4 microM), followed by gramicidin S (3.12 microM), alamethicin (6.25 microM), and surfactin (25 microM). When the mycoplasma cell concentration was varied from 10(4) to 10(8) CFU/ml, the MICs of enrofloxacin and globomycin increased while those of the three other molecules remained essentially constant. The minimal bactericidal concentration of enrofloxacin (0.8 microM) was also lower than those of the peptides (6.25 to 100 microM), but the latter killed the mycoplasma cells much faster than enrofloxacin (2 h versus 1 day). The use of the AMPs in association with enrofloxacin revealed synergistic effects with alamethicin and surfactin. Interestingly, the mycoplasma-killing activities of the two combinations enrofloxacin (MIC/2) plus alamethicin (MIC/4) and enrofloxacin (MIC/2) plus surfactin (MIC/16) were about 2 orders of magnitude higher than those of the three molecules used separately. These results support the interest devoted to AMPs as a novel class of antimicrobial agents and pinpoint their ability to potentiate the activities of conventional antibiotics, such as fluoroquinolones.

Alamethicin↗

PubMed2006

Quantifying species composition in root mixtures using two methods: near-infrared reflectance spectroscopy and plant wax markers.

Understanding of plant interactions is greatly limited by our ability to identify and quantify roots belonging to different species. We proposed and compared two methods for estimating the root biomass proportion of each species in artificial mixtures: near-infrared reflectance spectroscopy (NIRS) and plant wax markers. Two sets of artificial root mixtures composed of two or three herbaceous species were prepared. The proportion of root material of each species in mixtures was estimated from NIRS spectral data (i) and the concentration patterns of n-alkanes (ii), n-alcohols (iii), and n-alkanes +n-alcohols combined (iv). For each data set, calibration equations were developed using multivariate statistical models. The botanical composition of root mixtures was predicted well for all the species considered. The accuracy varied slightly among methods: alkanes < alcohols = alkanes + alcohols < NIRS. Correlation coefficients between predicted and actual root proportions ranged from 0.89 to 0.99 for alkanes + alcohols predictions and from 0.97 to 0.99 for NIRS predictions. These two methods provide promising potential for understanding allocation patterns and competitive interactions.

Alkanes↗

PubMed2005

Resistance to antimicrobial peptides and stress response in Mycoplasma pulmonis.

Antimicrobial peptides are widely distributed in nature, and in vertebrates, they play a key function in the innate immune defense system. It is generally agreed that these molecules may provide new antibiotics with therapeutic value. However, there are still many unsolved questions regarding the mechanisms underlying their antimicrobial activity as well as the mechanisms of resistance evolved by microorganisms against these molecules. The second point was addressed in this study. After determining the activity of 10 antimicrobial peptides against Mycoplasma pulmonis, a murine respiratory pathogen, the development of resistance was investigated. Following in vitro selection using subinhibitory concentrations of peptides, clones of this bacterium showing increased resistance to melittin or gramicidin D were obtained. For some of the clones, a cross-resistance was observed between these two peptides, in spite of their deep structural differences, and also with tetracycline. A proteomic analysis suggested that the stress response in these clones was constitutively activated, and this was confirmed by finding mutations in the hrcA gene; in mycoplasmas, bacteria which lack alternative sigma factors, the HrcA protein is supposed to play a key role as a negative regulator of heat shock proteins. By complementation of the hrcA mutants with the wild-type gene, the initial MICs of melittin and gramicidin D decreased to values close to the initial ones. This indicates that the resistance of M. pulmonis to these two antimicrobial peptides could result from a stress response involving HrcA-regulated genes.

Anti-Infective Agents↗

PubMed2005

Versatile use of oriC plasmids for functional genomics of Mycoplasma capricolum subsp. capricolum.

Replicative oriC plasmids were recently developed for several mollicutes, including three Mycoplasma species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: Mycoplasma mycoides subsp. mycoides small-colony type, Mycoplasma mycoides subsp. mycoides large-colony type, and Mycoplasma capricolum subsp. capricolum. In this study, oriC plasmids were evaluated in M. capricolum subsp. capricolum as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene lacZ, encoding beta-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri, were successfully expressed. Functional Escherichia coli beta-galactosidase was detected in transformed Mycoplasma capricolum subsp. capricolum cells despite noticeable codon usage differences. The expression of spiralin in M. capricolum subsp. capricolum was assessed by colony and Western blotting. Accessibility of this protein at the cell surface and its partition into the Triton X-114 detergent phase suggest a correct maturation of the spiralin precursor. The expression of a heterologous lipoprotein in a mycoplasma raises potentially interesting applications, e.g., the use of these bacteria as live vaccines. Targeted inactivation of gene lppA encoding lipoprotein A was achieved in M. capricolum subsp. capricolum with plasmids harboring a replication origin derived from S. citri. Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of oriC plasmids derived from related mollicute species. Mycoplasma gene inactivation opens the way to functional genomics in a group of bacteria for which a large wealth of genome data are already available and steadily growing.

Bacterial Outer Membrane Proteins↗

PubMed2004

MolliGen, a database dedicated to the comparative genomics of Mollicutes.

Bacteria belonging to the class Mollicutes were among the first ones to be selected for complete genome sequencing because of the minimal size of their genomes and their pathogenicity for humans and a broad range of animals and plants. At this time six genome sequences have been publicly released (Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum-parvum, Mycoplasma pulmonis, Mycoplasma penetrans and Mycoplasma gallisepticum) and as the number of available mollicute genomes increases, comparative genomics analysis within this model group of organisms becomes more and more instructive. However, such an analysis is difficult to carry out without a suitable platform gathering not only the original annotations but also relevant information available in public databases or obtained by applying common bioinformatics methods. With the aim of solving these difficulties, we have developed a web-accessible database named MolliGen (http://cbi.labri.fr/outils/molligen/). After selecting a set of genomes the user can launch various types of search based on annotation, position on the chromosomes or sequence similarity. In addition, relationships of putative orthology have been precomputed to allow differential genome queries. The results are presented in table format with multiple links to public databases and to bioinformatic analyses such as multiple alignments or BLAST search. Specific tools were also developed for the graphical visualization of the results, including a multi- genome browser for displaying dynamic pictures with clickable objects and for viewing relationships of precomputed similarity. MolliGen is designed to integrate all the complete genomes of mollicutes as they become available.

Computational Biology↗

PubMed2003

Host specificity of mollicutes oriC plasmids: functional analysis of replication origin.

Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis. In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp. mycoides LC (MmmLC), M.mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capricolum. Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids. All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes. However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species. For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC. In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum. Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.

Amino Acid Sequence↗

PubMed2002

Genomic repeats, genome plasticity and the dynamics of Mycoplasma evolution.

Mycoplasmas evolved by a drastic reduction in genome size, but their genomes contain numerous repeated sequences with important roles in their evolution. We have established a bioinformatic strategy to detect the major recombination hot-spots in the genomes of Mycoplasma pneumoniae, Mycoplasma genitalium, Ureaplasma urealyticum and Mycoplasma pulmonis. This allowed the identification of large numbers of potentially variable regions, as well as a comparison of the relative recombination potentials of different genomic regions. Different trends are perceptible among mycoplasmas, probably due to different functional and structural constraints. The largest potential for illegitimate recombination in M.pulmonis is found at the vsa locus and its comparison in two different strains reveals numerous changes since divergence. On the other hand, the main M.pneumoniae and M.genitalium adhesins rely on large distant repeats and, hence, homologous recombination for variation. However, the relation between the existence of repeats and antigenic variation is not necessarily straightforward, since repeats of P1 adhesin were found to be anti-correlated with epitopes recognized by patient antibodies. These different strategies have important consequences for the structures of genomes, since large distant repeats correlate well with the major chromosomal rearrangements. Probably to avoid such events, mycoplasmas strongly avoid inverse repeats, in comparison to co-oriented repeats.

Adhesins, Bacterial↗

PubMed2002

Transgenic B lymphocytes expressing a human cold agglutinin escape tolerance following experimental infection of mice by Mycoplasma pulmonis.

Several microbial infections, including Mycoplasma pneumoniae respiratory infection, are capable, in man, of transiently inducing the expression of anti-red blood cell autoantibody called cold agglutinins (CA). To analyze the mechanisms by which immune tolerance is broken following a mycoplasma infection, we used transgenic mice expressing a pathogenic human CA, designated CA-GAS, specific for sialylated carbohydrates. In these mice peripheral deletion of autoreactive B lymphocytes and receptor editing, prevent the development of autoimmune hemolytic anemia. Experimental infections of transgenic mice with Mycoplasma pulmonis resulted in a high anti-mycoplasma antibody response (despite a severe B cell depletion at the onset of infection), and an important induction of serum CA concentrations, reaching in some mice pathological titers. Whereas in naïve mice, only a small percentage of CA-expressing cells could be detected, in infected mice, a majority of circulating B lymphocytes were large B220(-) cells, which expressed the transgenic immunoglobulin. Immunization of the transgenic mice with keyhole limpet hemocyanin and Freund's adjuvant, to nonspecifically stimulate the expression of the passenger transgenes, only moderately increased the CA titers. These results indicate that M. pulmonis infection is capable of breaking immune tolerance in the CA-transgenic mice, in part through specific activation of CA-expressing B lymphocytes. This experimental infection mimics the induction of CA in humans and provide an animal model for studying the genesis of the autoimmune hemolytic anemia.

Agglutinins↗

PubMed2002

Susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to membrane-active peptides and enrofloxacin in human tissue cell cultures.

Mycoplasmas, which are bacteria that are devoid of a cell wall and which belong to the class Mollicutes, are pathogenic for humans and animals and are frequent contaminants of tissue cell cultures. Although contamination of cultures with mycoplasma can easily be monitored with fluorescent dyes that stain DNA and/or with molecular probes, protection and decontamination of cultures remain serious challenges. In the present work, we investigated the susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to the membrane-active peptides alamethicin, dermaseptin B2, gramicidin S, and surfactin by growth inhibition and lethality assays. In the absence of serum, the four peptides killed mycoplasmas at minimal bactericidal concentrations that ranged from 12.5 to 100 microM, but in all cases the activities were decreased by the presence of serum. As a result, under standard culture conditions (10% serum) only alamethicin and gramicidin S were able to inhibit mycoplasma growth (MICs, 50 microM), while dermaseptin B2 and surfactin were ineffective. Furthermore, 8 days of treatment of HeLa cell cultures experimentally contaminated with either mycoplasma species with 70 microM enrofloxacin cured the cultures of infection, whereas treatment with alamethicin and gramicidin S alone was not reliable because the concentrations and treatment times required were toxic to the cells. However, combination of alamethicin or gramicidin S with 70 microM enrofloxacin allowed mycoplasma eradication after 30 min or 24 h of treatment, depending on the mycoplasma and peptide considered. HeLa cell cultures experimentally infected with mycoplasmas should prove to be a useful model for study of the antimycoplasma activities of antibiotics and membrane-active peptides under conditions close to those found in vivo.

Anti-Bacterial Agents↗

PubMed1999

Antigenic characterization and cytolocalization of P35, the major Mycoplasma penetrans antigen.

Mycoplasma penetrans is a mycoplasma with unique morphology, recently identified in urine samples collected from HIV-infected patients. This mycoplasma has been found to be statistically associated with HIV infection, and to be cytopathic in vitro. The dominant antigen recognized during natural and experimental infections is an abundant lipoprotein, P35, which, upon extraction, segregates in the Triton X-114 detergent phase. It is used as the basis of M. penetrans-specific serological assays. Although mycoplasma lipoproteins, including M. penetrans P35, are the main antigens recognized by the host humoral immune response, very little is known about the nature of the epitopes involved. Immunoelectron microscopy revealed that all P35 is exposed at the cell surface and is distributed all over the membrane. P35 linear B-epitopes were mapped by an ELISA approach based on a set of overlapping peptides covering the entire mature polypeptide. The immunoreactivity of the peptides was first tested with sera from immunized animals. The dominant B-epitopes were found at the C- and N-terminal regions, in partial agreement with algorithmic predictions. Patient sera were evaluated with the same assay. Only some reacted with linear epitopes whereas others did not, indicating the importance of P35 nonsequential epitopes. Statistical analysis of the results allowed the definition of a set of peptides which were clearly immunodominant. Finally, the P35-encoding gene was modified by in vitro mutagenesis to allow the production and purification of a recombinant protein (rP35delta0) in Escherichia coil. The antigenicity of rP35delta0 was tested by Western blotting and compared to that of another recombinant product, rP35delta3, a truncated P35 polypeptide. Although rP35delta0 reacted with the M. penetrans-seropositive patient sera tested, rP35delta3 was only immunoreactive with one of six sera. This result confirmed that P35-nonsequential epitopes dominate during M. penetrans infection. Our results have important implications for the understanding of lipoprotein antigenicity during mycoplasma infections. In addition, the P35-derived immunodominant synthetic peptides defined in this study, as well as the purified rP35delta0, provide the antigenic material for the necessary improvement of M. penetrans serological assays.

Amino Acid Sequence↗

PubMed1998

Identification of two glycosylated components of Mycoplasma penetrans: a surface-exposed capsular polysaccharide and a glycolipid fraction.

Among the wall-less mycoplasmas only a few species have been identified with a capsule at their cell surface. Mycoplasma penetrans is a recently identified mycoplasma with unique morphology, isolated from HIV-infected patients. Using transmission electron microscopy, it was found that M. penetrans is surrounded by capsular material 11 nm (strain GTU-54-6A1) to 30 nm (strain HF-2) thick, which can be stained with ruthenium red and labelled with cationized ferritin. The polysaccharide composition of this capsule was indicated by its staining with periodic acid-thiocarbohydrazide silver proteinate and the abolition of ruthenium red staining of the cell surface by neuraminidase treatment. In addition, proteinase K treatment of the M. penetrans cells resulted in removal of the capsule, suggesting that polypeptides may contribute in anchoring it to the membrane or in its stability. Two different types of glycosylated material were detected in mycoplasma extracts by SDS-PAGE and periodic acid-Schiff staining. The first component was a high-molecular-mass material, which was heat- and proteinase-K-labile and which probably constitutes the capsular polymer. The other component was a low-molecular-mass glycolipid fraction, which was proteinase-K-, heat- and EDTA-resistant. The identification of a capsule at the M. penetrans cell surface is of particular interest for a mycoplasma which has been shown to adhere to various host cells and to penetrate into their intracellular compartments. The capsule may have significance in the pathogenesis of disease associated with infection by this organism.

Bacterial Capsules↗