LipL21 lipoprotein binding to peptidoglycan enables Leptospira interrogans to escape NOD1 and NOD2 recognition.

Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily digested than the parental or complemented strains. Muropeptides released from the PG of the lipl21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize PG of L. interrogans. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses.

Chemically-defined camelid antibody bioconjugate for the magnetic resonance imaging of Alzheimer's disease.

Today, molecular imaging of neurodegenerative diseases is mainly based on small molecule probes. Alternatively, antibodies are versatile tools that may be developed as new imaging agents. Indeed, they can be readily obtained to specifically target any antigen of interest and their scaffold can be functionalized. One of the critical issues involved in translating antibody-based probes to the clinic is the design and synthesis of perfectly-defined conjugates. Camelid single-domain antibody-fragments (VHHs) are very small and stable antibodies that are able to diffuse in tissues and potentially cross the blood brain barrier (BBB). Here, we selected a VHH (R3VQ) specifically targeting one of the main lesions of Alzheimer's disease (AD), namely the amyloid-beta (Aß) deposits. It was used as a scaffold for the design of imaging probes for magnetic resonance imaging (MRI) and labeled with the contrastophore gadolinium using either a random or site-specific approach. In contrast to the random strategy, the site-specific conjugation to a single reduced cysteine in the C-terminal part of the R3VQ generates a well-defined bioconjugate in a high yield process. This new imaging probe is able to cross the BBB and label Aß deposits after intravenous injection. Also, it displays improved r1 and r2 relaxivities, up to 30 times higher than a widely used clinical contrast agent, and it allows MRI detection of amyloid deposits in post mortem brain tissue of a mouse model of AD. The ability to produce chemically-defined VHH conjugates that cross the BBB opens the way for future development of tailored imaging probes targeting intracerebral antigens.

Camelid single-domain antibodies: A versatile tool for in vivo imaging of extracellular and intracellular brain targets.

Detection of intracerebral targets with imaging probes is challenging due to the non-permissive nature of blood-brain barrier (BBB). The present work describes two novel single-domain antibodies (VHHs or nanobodies) that specifically recognize extracellular amyloid deposits and intracellular tau neurofibrillary tangles, the two core lesions of Alzheimer's disease (AD). Following intravenous administration in transgenic mouse models of AD, in vivo real-time two-photon microscopy showed gradual extravasation of the VHHs across the BBB, diffusion in the parenchyma and labeling of amyloid deposits and neurofibrillary tangles. Our results demonstrate that VHHs can be used as specific BBB-permeable probes for both extracellular and intracellular brain targets and suggest new avenues for therapeutic and diagnostic applications in neurology.

Phosphoproteomic analysis of wild-type and antimony-resistant Leishmania braziliensis lines by 2D-DIGE technology.

Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.

Quality assessment and optimization of purified protein samples: why and how?

Purified protein quality control is the final and critical check-point of any protein production process. Unfortunately, it is too often overlooked and performed hastily, resulting in irreproducible and misleading observations in downstream applications. In this review, we aim at proposing a simple-to-follow workflow based on an ensemble of widely available physico-chemical technologies, to assess sequentially the essential properties of any protein sample: purity and integrity, homogeneity and activity. Approaches are then suggested to optimize the homogeneity, time-stability and storage conditions of purified protein preparations, as well as methods to rapidly evaluate their reproducibility and lot-to-lot consistency.

Characterization of the enzymatic activity of Clostridium perfringens TpeL.

TpeL is a toxin produced by Clostridium perfringens which belongs to the large clostridial glucosylating toxin family. It was shown that TpeL modifies Ras using UDP-glucose or UDP-N-acetylglucosamine as cosubstrates (Guttenberg et al., 2012; Nagahama et al., 2011). We confirmed that TpeL preferentially glucosaminates the three isoforms of Ras (cH-Ras, N-Ras, and K-Ras) from UDP-N-acetylglucosamine and to a lower extent Rap1a and R-Ras3, and very weakly Rac1. In contrast to previous report, we observed that Ral was not a substrate of TpeL. In addition, we confirmed by in vitro glucosylation and mass spectrometry that TpeL modifies cH-Ras at Thr35.

Hyperthermophilic archaea produce membrane vesicles that can transfer DNA.

Thermococcales are hyperthermophilic archaea found in deep-sea hydrothermal vents. They have been recently reported to produce membrane vesicles (MVs) into their culture medium. Here, we have characterized the mode of production and determined the biochemical composition of MVs from two species of Thermococcales, Thermococcus gammatolerans and Thermococcus kodakaraensis. We observed that MVs are produced by a budding process from the cell membrane reminiscent of ectosome (microparticle) formation in eukaryotes. MVs and cell membranes from the same species have a similar protein and lipid composition, confirming that MVs are produced from cell membranes. The major protein present in cell membranes and MVs of both species is the oligopeptide binding protein OppA. This protein is also abundant in MVs from cells grown in minimal medium, suggesting that OppA could be involved in processes other than peptides scavenging. We have previously shown that MVs from Thermococcales harbour DNA and protect DNA against thermodegradation. Here, we show that T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harbouring this plasmid. Notably, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature.

Crosstalk between Helicobacter pylori and gastric epithelial cells is impaired by docosahexaenoic acid.

H. pylori colonizes half of the world's population leading to gastritis, ulcers and gastric cancer. H. pylori strains resistant to antibiotics are increasing which raises the need for alternative therapeutic approaches. Docosahexaenoic acid (DHA) has been shown to decrease H. pylori growth and its associated-inflammation through mechanisms poorly characterized. We aimed to explore DHA action on H. pylori-mediated inflammation and adhesion to gastric epithelial cells (AGS) and also to identify bacterial structures affected by DHA. H. pylori growth and metabolism was assessed in liquid cultures. Bacterial adhesion to AGS cells was visualized by transmission electron microscopy and quantified by an Enzyme Linked Immunosorbent Assay. Inflammatory proteins were assessed by immunoblotting in infected AGS cells, previously treated with DHA. Bacterial total and outer membrane protein composition was analyzed by 2-dimensional gel electrophoresis. Concentrations of 100 µM of DHA decreased H. pylori growth, whereas concentrations higher than 250 µM irreversibly inhibited bacteria survival. DHA reduced ATP production and adhesion to AGS cells. AGS cells infected with DHA pre-treated H. pylori showed a 3-fold reduction in Interleukin-8 (IL-8) production and a decrease of COX2 and iNOS. 2D electrophoresis analysis revealed that DHA changed the expression of H. pylori outer membrane proteins associated with stress response and metabolism and modified bacterial lipopolysaccharide phenotype. As conclusions our results show that DHA anti-H. pylori effects are associated with changes of bacteria morphology and metabolism, and with alteration of outer membrane proteins composition, that ultimately reduce the adhesion of bacteria and the burden of H. pylori-related inflammation.

A minimal bacterial RNase J-based degradosome is associated with translating ribosomes.

Protein complexes directing messenger RNA (mRNA) degradation are present in all kingdoms of life. In Escherichia coli, mRNA degradation is performed by an RNA degradosome organized by the major ribonuclease RNase E. In bacteria lacking RNase E, the existence of a functional RNA degradosome is still an open question. Here, we report that in the bacterial pathogen Helicobacter pylori, RNA degradation is directed by a minimal RNA degradosome consisting of Hp-RNase J and the only DExD-box RNA helicase of H. pylori, RhpA. We show that the protein complex promotes faster degradation of double-stranded RNA in vitro in comparison with Hp-RNase J alone. The ATPase activity of RhpA is stimulated in the presence of Hp-RNase J, demonstrating that the catalytic capacity of both partners is enhanced upon interaction. Remarkably, both proteins are associated with translating ribosomes and not with individual 30S and 50S subunits. Moreover, Hp-RNase J is not recruited to ribosomes to perform rRNA maturation. Together, our findings imply that in H. pylori, the mRNA-degrading machinery is associated with the translation apparatus, a situation till now thought to be restricted to eukaryotes and archaea.