Biomedical subjects

Ludovic Pelosi

Publications and source records attributed to Ludovic Pelosi.

6 recordsLinked to original sources

PubMed2006

Identification of the first Oomycete annexin as a (1-->3)-beta-D-glucan synthase activator.

(1-->3)-beta-D-Glucans are major components of the cell walls of Oomycetes and as such they play an essential role in the morphogenesis and growth of these microorganisms. Despite the biological importance of (1-->3)-beta-D-glucans, their mechanisms of biosynthesis are poorly understood. Previous studies on (1-->3)-beta-D-glucan synthases from Saprolegnia monoica have shown that three protein bands of an apparent molecular weight of 34, 48 and 50 kDa co-purify with enzyme activity. However, none of the corresponding proteins have been identified. Here we have identified, purified, sequenced and characterized a protein from the 34 kDa band and clearly shown that it has all the biochemical properties of proteins from the annexin family. In addition, we have unequivocally demonstrated that the purified protein is an activator of (1-->3)-beta-D-glucan synthase. This represents a new type of function for proteins belonging to the annexin family. Two other proteins from the 48 and 50 kDa bands were identified as ATP synthase subunits, which most likely arise from contaminations by mitochondria during membrane preparation. The results, which are discussed in relation with the possible regulation mechanisms of (1-->3)-beta-D-glucan synthases, represent a first step towards a better understanding of cell wall polysaccharide biosynthesis in Oomycetes.

Amino Acid Sequence↗

PubMed2006

Parallel changes in global protein profiles during long-term experimental evolution in Escherichia coli.

Twelve populations of Escherichia coli evolved in and adapted to a glucose-limited environment from a common ancestor. We used two-dimensional protein electrophoresis to compare two evolved clones, isolated from independently derived populations after 20,000 generations. Exceptional parallelism was detected. We compared the observed changes in protein expression profiles with previously characterized global transcription profiles of the same clones; this is the first time such a comparison has been made in an evolutionary context where these changes are often quite subtle. The two methodologies exhibited some remarkable similarities that highlighted two different levels of parallel regulatory changes that were beneficial during the evolution experiment. First, at the higher level, both methods revealed extensive parallel changes in the same global regulatory network, reflecting the involvement of beneficial mutations in genes that control the ppGpp regulon. Second, both methods detected expression changes of identical gene sets that reflected parallel changes at a lower level of gene regulation. The protein profiles led to the discovery of beneficial mutations affecting the malT gene, with strong genetic parallelism across independently evolved populations. Functional and evolutionary analyses of these mutations revealed parallel phenotypic decreases in the maltose regulon expression and a high level of polymorphism at this locus in the evolved populations.

Adaptation, Physiological↗

PubMed2005

The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H).

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one alpha-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->4)-beta-D-Glcp-(1-->]n). Expression of WciS-His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/DeltagalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [beta-D-GlcAp-(1-->4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by alpha-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS-His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

Bacterial Capsules↗

PubMed2005

The glucosyl-1-phosphate transferase WchA (Cap8E) primes the capsular polysaccharide repeat unit biosynthesis of Streptococcus pneumoniae serotype 8.

The gene wchA (cap8E) belongs to the cps8 locus that is involved in biosynthesis of the capsular polysaccharide (CPS) repeat unit (RU) of the virulent Streptococcus pneumoniae serotype 8. We report here the biochemical characterization of the membrane-associated protein WchA (Cap8E), overexpressed in Escherichia coli BL21(DE3)/pLysS. Our results demonstrate that the recombinant enzyme transfers in vitro a glucosyl-1-phosphate from UDP-glucose to an endogenous phosphoryl-polyprenol, thereby priming the RU biosynthetic pathway of S. pneumoniae CPS 8. We also show that the C-terminal half of WchA is the glycosyltransferase domain as observed for the galactosyl-1-phosphate transferase WbaP from Salmonella enterica, previously described to prime the first step of O-antigen biosynthesis. These results demonstrate that WchA plays a prominent function in the capsule biosynthesis and explain the key role it occupies in the pneumococcal capsule variation.

Bacterial Capsules↗

PubMed2003

Structural and morphological diversity of (1-->3)-beta-D-glucans synthesized in vitro by enzymes from Saprolegnia monoïca. Comparison with a corresponding in vitro product from blackberry (Rubus fruticosus).

Detergent extracts of microsomal fractions from Saprolegnia monoïca and blackberry (Rubus fruticosus) cells were incubated with UDP-glucose to yield in vitro (1-->3)-beta-d-glucans. The insoluble products were analyzed by conventional and cryo transmission electron microscopy, X-ray diffraction, and (13)C CP/MAS NMR, and their molecular weights were determined by light scattering experiments. All the products were microfibrillar, but for the detergent extracts from S. monoïca, important morphological differences were observed when the pH of the synthesizing medium was modified. At pH 6, the product had a weight average degree of polymerization () exceeding 20 000 and consisted of endless ribbon-like microfibrils. The microfibrils obtained at pH 9 had a length of only 200-300 nm, and their was approximately 5000. Of all the in vitro (1-->3)-beta-d-glucans, the one from R. fruticosus had the shortest length and the smallest. Crystallographic and spectroscopic data showed that the three in vitro samples consisted of triple helices of (1-->3)-beta-d-glucans and contained substantial amounts of water molecules in their structure, the shortest microfibrils being more hydrated. In addition, the long microfibrils from S. monoïca synthesized at pH 6 were more resistant toward the action of an endo-(1-->3)-beta-d-glucanase than the shorter ones obtained at pH 9. These results are discussed in terms of molecular biosynthetic mechanisms of fungal and plant (1-->3)-beta-d-glucans, and in relation with the possible existence of several (1-->3)-beta-d-glucan synthases in a given organism. The interpretation and discussion of these observations integrate the current knowledge of the structure and function of (1-->3)-beta-d-glucans.

Crystallography, X-Ray↗

PubMed2002

Mutated barley (1,3)-beta-D-glucan endohydrolases synthesize crystalline (1,3)-beta-D-glucans.

Barley (1,3)-beta-D-glucan endohydrolases (EC ), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with alpha-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with alpha-laminaribiosyl fluoride and 4'-nitrophenyl beta-D-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k(cat) K(m)(-1)) of mutants E231G, E231S, and E231A as glycosynthases are 28.9, 0.9, and 0.5 x 10(-4) m(-1) s(-1), respectively. Glycosynthase reactions appear to be processive and proceed with pH optima of 6-8 and yields of up to 75%. Insoluble products formed during the glycosynthase reaction appear as lamellar, hexagonal crystals when observed by electron microscopy. Methylation, NMR, and matrix-assisted laser desorption ionization time-of-flight analyses show that the reaction products are linear (1,3)-beta-D-glucans with a degree of polymerization of 30-34, whereas electron and x-ray diffraction patterns indicate that these (1,3)-beta-D-glucan chains adopt a parallel, triple helical conformation. The (1,3)-beta-D-glucan triple helices are orientated perpendicularly to the plane of the lamellar crystals. The barley (1,3)-beta-D-glucan glycosynthases have considerable potential for tailored and high efficiency synthesis of (1,3)-beta-D-linked oligo- and polysaccharides, some of which could have immunomodulating activity, or for the coupling of (1,3)-beta-D-linked glucosyl residues onto other oligosaccharides or glycoproteins.

Base Sequence↗