Role of Gp120 Glycosylation in Sexual Transmission of HIV

Background: In chronic HIV patients, the viral populations are genetically diverse due to mutations introduced by the viral reverse transcriptase during HIV replication. However, more than 80% new infections result from single transmission founder (TF) viruses; therefore, targeting the TFs is key to control AIDS worldwide. Gp120 is a glycosylated envelope protein required for HIV infection, propagation, and transmission. Glycans on gp120 influence HIV infectivity through their interactions with lectins, the carbohydrate-binding immune proteins in the host mucosa. To transmit sexually, viruses must overcome the lectin traps to access more target T cells. Hypothesis: TF viruses are less likely to be trapped by host lectins due to their reduced gp120 glycosylation, thus more infectious. Methods: We aim to characterize and compare the gp120 glycosylation signatures in TF and chronic HIV strains, B4 and Q0 respectively, using mass spectrometry (MS), surface plasmon resonance (SPR), and capillary electrophoresis (CE). To date, we have established a work flow to purify gp120 glycoproteins, perform MS using ETHcD methods, and analyze raw MS data using the GlycoPAT software. We are currently analyzing MS data for three replicates of B4 and the first replicate of Q0. Then we will compare the glycosylation patterns between the two strains. CE and SPR will be performed to test the glycan enrichment and functional interactions between gp120 and lectins, respectively. Discussion: Our results will provide qualitative and quantitative details about gp120 glycosylation underlying the strong infectivity of TF viruses, shedding light on new strategies to develop HIV vaccines

Effect of environmental stress factors on the uptake and survival of Campylobacter jejuni in Acanthamoeba castellanii

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni</it> is a major cause of bacterial food-borne illness in Europe and North America. The mechanisms allowing survival in the environment and transmission to new hosts are not well understood. Environmental free-living protozoa may facilitate both processes. Pre-exposure to heat, starvation, oxidative or osmotic stresses encountered in the environment may affect the subsequent interaction of <it>C. jejuni</it> with free-living protozoa. To test this hypothesis, we examined the impact of environmental stress on expression of virulence-associated genes (<it>ciaB, dnaJ,</it> and <it>htrA</it>) of <it>C. jejuni</it> and on its uptake by and intracellular survival within <it>Acanthamoeba castellanii</it>.</p> <p>Results</p> <p>Heat, starvation and osmotic stress reduced the survival of <it>C. jejuni</it> significantly, whereas oxidative stress had no effect. Quantitative RT-PCR experiments showed that the transcription of virulence genes was slightly up-regulated under heat and oxidative stresses but down-regulated under starvation and osmotic stresses, the <it>htrA</it> gene showing the largest down-regulation in response to osmotic stress. Pre-exposure of bacteria to low nutrient or osmotic stress reduced bacterial uptake by amoeba, but no effect of heat or oxidative stress was observed. Finally, <it>C. jejuni</it> rapidly lost viability within amoeba cells and pre-exposure to oxidative stress had no significant effect on intracellular survival. However, the numbers of intracellular bacteria recovered 5 h post-gentamicin treatment were lower with starved, heat treated or osmotically stressed bacteria than with control bacteria. Also, while ~1.5 × 10³ colony forming unit/ml internalized bacteria could typically be recovered 24 h post-gentamicin treatment with control bacteria, no starved, heat treated or osmotically stressed bacteria could be recovered at this time point. Overall, pre-exposure of <it>C. jejuni</it> to environmental stresses did not promote intracellular survival in <it>A. castellanii</it>.</p> <p>Conclusions</p> <p>Together, these findings suggest that the stress response in <it>C. jejuni</it> and its interaction with <it>A. castellanii</it> are complex and multifactorial, but that pre-exposure to various stresses does not prime <it>C. jejuni</it> for survival within <it>A. castellanii</it>.</p

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

WbpO is associated with B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O6. This protein is thought to catalyze the enzymatic conversion of UDP-N-acetyl-d-galactosamine (UDP-GalNAc) to UDP-N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA). WbpO was overexpressed with a C-terminal hexahistidine tag. The soluble form of expressed WbpO (WbpO(Sol)) exhibited a secondary structure with 29.2% alpha-helix and 20.1% beta-strand. However, no enzymatic activity could be detected using either high performance anion exchange chromatography or capillary electrophoresis-mass spectrometry analysis. An insoluble form of expressed WbpO was purified in the presence of guanidine hydrochloride by immobilized metal ion affinity chromatography. After refolding, this preparation of WbpO (designated as WbpO(Rf)) exhibited stable secondary structure at pH 7.5 to 8.2, and it was enzymatically active. Capillary electrophoresis-mass spectrometry and tandem mass spectrometry analysis showed that WbpO(Rf) catalyzed the conversion of UDP-GalNAc to UDP-GalNAcA. 26 and 22% of the substrate could be converted to UDP-GalNAcA in the presence of NAD(+) and NADP(+) as the cofactors, respectively. The K(m) values of WbpO(Rf) for UDP-GalNAc, NAD(+), and NADP(+) were 7.79, 0.65, and 0.44 mm, respectively. WbpO(Rf) can also catalyze the conversion of UDP-GlcNAc to UDP-GlcNAcA. In conclusion, this is the first report of the overexpression, purification, and biochemical characterization of an NAD(+)/NADP(+)-dependent UDP-GalNAc dehydrogenase. Our results also complete the biosynthetic pathway for GalNAcA that is part of the O-antigen of P. aeruginosa serotype O6 lipopolysaccharide

FlaA1, a New Bifunctional UDP-GlcNAc C6Dehydratase/ C4 Reductase from Helicobacter pylori

FlaA1 is a small soluble protein of unknown function in Helicobacter pylori. It has homologues that are essential for the virulence of numerous medically relevant bacteria. FlaA1 was overexpressed as a histidine-tagged protein and purified to homogeneity by nickel chelation and cation exchange chromatography. Spectrophotometric assays, capillary electrophoresis, and mass spectrometry analyses showed that FlaA1 is a novel bifunctional C(6) dehydratase/C(4) reductase specific for UDP-GlcNAc. It converts UDP-GlcNAc into a UDP-4-keto-6-methyl-GlcNAc intermediate, which is stereospecifically reduced into UDP-QuiNAc. Substrate conversions as high as 80% were obtained at equilibrium. The K(m) and V(max) for UDP-GlcNAc were 159 microm and 65 pmol/min, respectively. No exogenous cofactor was required to obtain full activity of FlaA1. Additional NADH was only used with poor efficiency for the reduction step. The biochemical characterization of FlaA1 is important for the elucidation of biosynthetic pathways that lead to the formation of 2,6-deoxysugars in medically relevant bacteria. It establishes unambiguously the first step of the pathway and provides the means of preparing the substrate UDP-QuiNAc, which is necessary for the study of downstream enzymes

Protein Glycosylation in Helicobacter pylori: Beyond the Flagellins?

Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac) previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac) which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori

Synthese des peptides signaux de caseines. Etude de leurs proprietes et de leurs interactions avec quelques phospholipides

* CR de Nantes. Laboratoire d'Etude des Interactions des Molecules Alimentaires. BP 527, 44026 Nantes Cedex 03 Diffusion du document : CR de Nantes. Laboratoire d'Etude des Interactions des Molecules Alimentaires. BP 527, 44026 Nantes Cedex 03 Diplôme : Dr. d'Universit

Interaction of alphaS2- and beta-casein signal peptides with DMPC and DMPG liposomes

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Alkylation de la beta-lactoglobuline et de la beta-caseine en milieu aqueux ou organique

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Synthesis and purification of casein signal peptides

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