Proteomic investigation of the adaptation of Lactococcus lactis to the mouse digestive tract

Lactic acid bacteria are used on an industrial scale for the manufacturing of dairy products. It is now intended to develop novel applications of lactic acid bacteria that could be used as living vehicles for the targeting of antigens or therapeutics to the digestive mucosa. The aim of this study was to analyze the adaptations of Lactococcus lactis, a model lactic acid bacteria to the digestive tract and to identify functions required for colonization of the intestine. For this purpose, we combined gnotobiology with proteomics: axenic mice were colonized with a dairy L. lactis strain and the bacterial proteome was examined by 2-DE. As compared to cultures in broth, the proteome profile of bacteria grown in the intestine indicates the activation of metabolic pathways involved in various carbon sources assimilation and suggests the adoption of a mixed acids fermentative metabolism. We identified the product of the ywcC gene as essential for the colonization of the digestive tract and demonstrated that the corresponding gene product (YwcC) possesses a phosphogluconolactonase activity, suggesting an important role of the pentose phosphate pathway for the development of L. lactis in the digestive environment.     

Phylogenetic and Phyletic Studies of Informational Genes in Genomes Highlight Existence of a 4th Domain of Life Including Giant Viruses

The discovery of Mimivirus, with its very large genome content, made it possible to identify genes common to the three domains of life (Eukarya, Bacteria and Archaea) and to generate controversial phylogenomic trees congruent with that of ribosomal genes, branching Mimivirus at its root. Here we used sequences from metagenomic databases, Marseillevirus and three new viruses extending the Mimiviridae family to generate the phylogenetic trees of eight proteins involved in different steps of DNA processing. Compared to the three ribosomal defined domains, we report a single common origin for Nucleocytoplasmic Large DNA Viruses (NCLDV), DNA processing genes rooted between Archaea and Eukarya, with a topology congruent with that of the ribosomal tree. As for translation, we found in our new viruses, together with Mimivirus, five proteins rooted deeply in the eukaryotic clade. In addition, comparison of informational genes repertoire based on phyletic pattern analysis supports existence of a clade containing NCLDVs clearly distinct from that of Eukarya, Bacteria and Archaea. We hypothesize that the core genome of NCLDV is as ancient as the three currently accepted domains of life.     

Unfolding and refolding properties of S pili on extraintestinal pathogenic Escherichia coli

S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (S_II) and believed to play a role in ascending urinary tract infections (S_I). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of S_II and S_I pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for S_II pili and 21 pN for S_I pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX _II) indicated that absence of the sfaX _II gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of S_I are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.     

SAMPLEX: Automatic mapping of perturbed and unperturbed regions of proteins and complexes

Background  

The activity of proteins within the cell is characterized by their motions, flexibility, interactions or even the particularly intriguing case of partially unfolded states. In the last two cases, a part of the protein is affected either by binding or unfolding and the detection of the respective perturbed and unperturbed region(s) is a fundamental part of the structural characterization of these states. This can be achieved by comparing experimental data of the same protein in two different states (bound/unbound, folded/unfolded). For instance, measurements of chemical shift perturbations (CSPs) from NMR ¹H-¹⁵N HSQC experiments gives an excellent opportunity to discriminate both moieties.     

Sucrose deficiency delays lycopene accumulation in tomato fruit pericarp discs

Tomato (Solanum lycopersicum) fruit ripening is characterized by a massive accumulation of carotenoids (mainly lycopene) as chloroplasts change to chromoplasts. To address the question of the role of sugars in controlling carotenoid accumulation, fruit pericarp discs (mature green fruits) were cultured in vitro in the presence of various sucrose concentrations. A significant difference in soluble sugar content was achieved depending on external sucrose availability. Sucrose limitation delayed and reduced lycopene and phytoene accumulation, with no significant effect on other carotenoids. Chlorophyll degradation and starch catabolism were not affected by variations of sucrose availability. The reduction of lycopene synthesis observed in sucrose-limited conditions was mediated through metabolic changes illustrated by reduced hexose accumulation levels. In addition, variations of sucrose availability modulated PSY1 gene expression. Taken together our results suggest that the modulation of carotenoid accumulation by sucrose availability occurs at the metabolic level and involves the differential regulation of genes involved in carotenoid biosynthesis.     

Unravelling carbon metabolism in anaerobic cellulolytic bacteria

Carbon metabolism in anaerobic cellulolytic bacteria has been investigated essentially in Clostridium thermocellum, Clostridium cellulolyticum, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus. While cellulose depolymerization into soluble sugars by various cellulases is undoubtedly the first step in bacterial metabolisation of cellulose, it is not the only one to consider. Among anaerobic cellulolytic bacteria, C. cellulolyticum has been investigated metabolically the most in the past few years. Summarizing metabolic flux analyses in continuous culture using either cellobiose (a soluble cellodextrin resulting from cellulose hydrolysis) or cellulose (an insoluble biopolymer), this review aims to stress the importance of the insoluble nature of a carbon source on bacterial metabolism. Furthermore, some general and specific traits of anaerobic cellulolytic bacteria trends, namely, the importance and benefits of (i) cellodextrins with degree of polymerization higher than 2, (ii) intracellular phosphorolytic cleavage, (iii) glycogen cycling on cell bioenergetics, and (iv) carbon overflows in regulation of carbon metabolism, as well as detrimental effects of (i) soluble sugars and (ii) acidic environment on bacterial growth. Future directions for improving bacterial cellulose degradation are discussed.     

Plasmacytoid dendritic cell dynamics and alpha interferon production during Simian immunodeficiency virus infection with a nonpathogenic outcome

We addressed the role of plasmacytoid dendritic cells (PDC) in protection against AIDS in nonpathogenic simian immunodeficiency virus (SIVagm) infection in African green monkeys (AGMs). PDC were monitored in blood and lymph nodes (LNs) starting from day 1 postinfection. We observed significant declines in blood during acute infection. However, PDC then returned to normal levels, and chronically infected AGMs showed no decrease of PDC in blood. There was a significant increase of PDC in LNs during acute infection. Blood PDC displayed only weak alpha interferon (IFN-alpha) responses to TLR9 agonist stimulation before infection. However, during acute infection, both blood and LN PDC showed a transiently increased propensity for IFN-alpha production. Bioactive IFN-alpha was detected in plasma concomitant with the peak of viremia, though levels were only low to moderate in some animals. Plasma interleukin 6 (IL-6) and IL-12 were not increased. In conclusion, PDC were recruited to the LNs and displayed increased IFN-alpha production during acute infection. However, increases in IFN-alpha were transient. Together with the lack of inflammatory cytokine responses, these events might play an important role in the low level of T-cell activation which is associated with protection against AIDS in nonpathogenic SIVagm infection.     

Impact of strong selection for the PrP major gene on genetic variability of four French sheep breeds (Open Access publication)

Effective selection on the PrP gene has been implemented since October 2001 in all French sheep breeds. After four years, the ARR "resistant" allele frequency increased by about 35% in young males. The aim of this study was to evaluate the impact of this strong selection on genetic variability. It is focussed on four French sheep breeds and based on the comparison of two groups of 94 animals within each breed: the first group of animals was born before the selection began, and the second, 3–4 years later. Genetic variability was assessed using genealogical and molecular data (29 microsatellite markers). The expected loss of genetic variability on the PrP gene was confirmed. Moreover, among the five markers located in the PrP region, only the three closest ones were affected. The evolution of the number of alleles, heterozygote deficiency within population, expected heterozygosity and the Reynolds distances agreed with the criteria from pedigree and pointed out that neutral genetic variability was not much affected. This trend depended on breed, i.e. on their initial states (population size, PrP frequencies) and on the selection strategies for improving scrapie resistance while carrying out selection for production traits.     

A solid phase parallel synthesis of diverse amides as dopamine D3 receptor ligands

A solid phase parallel synthesis using SynPhase? technology was used to couple a series of 21 carboxylic with three different 4-(4-arylpiperazinyl)butanamines. The resulting library was evaluated as dopamine D₃ receptor ligands giving rise to several compounds with affinities in the low nanomolar concentration range (9e and 9n with binding affinities at D₃ receptors of 0.10 and 0.35 nM respectively).     

Illumination of Murine Gammaherpesvirus-68 Cycle Reveals a Sexual Transmission Route from Females to Males in Laboratory Mice

Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which have evolved to be transmitted in presence of specific immune response. Identifying their mode of transmission and their mechanisms of immune evasion is therefore essential to develop prophylactic and therapeutic strategies against these infections. As the known human gammaherpesviruses, Epstein-Barr virus and Kaposi''s Sarcoma-associated Herpesvirus are host-specific and lack a convenient in vivo infection model; related animal gammaherpesviruses, such as murine gammaherpesvirus-68 (MHV-68), are commonly used as general models of gammaherpesvirus infections in vivo. To date, it has however never been possible to monitor viral excretion or virus transmission of MHV-68 in laboratory mice population. In this study, we have used MHV-68 associated with global luciferase imaging to investigate potential excretion sites of this virus in laboratory mice. This allowed us to identify a genital excretion site of MHV-68 following intranasal infection and latency establishment in female mice. This excretion occurred at the external border of the vagina and was dependent on the presence of estrogens. However, MHV-68 vaginal excretion was not associated with vertical transmission to the litter or with horizontal transmission to female mice. In contrast, we observed efficient virus transmission to naïve males after sexual contact. In vivo imaging allowed us to show that MHV-68 firstly replicated in penis epithelium and corpus cavernosum before spreading to draining lymph nodes and spleen. All together, those results revealed the first experimental transmission model for MHV-68 in laboratory mice. In the future, this model could help us to better understand the biology of gammaherpesviruses and could also allow the development of strategies that could prevent the spread of these viruses in natural populations.