HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources.

Despite the high prevalence of both cervico-vaginal human papillomavirus (HPV) infection and bacterial vaginosis (BV) worldwide, their causal relationship remains unclear. While BV has been presumed to be a risk factor for HPV acquisition and related carcinogenesis for a long time, here, supported by both a large retrospective follow-up study (n = 6,085) and extensive in vivo data using the K14-HPV16 transgenic mouse model, we report a novel blueprint in which the opposite association also exists. Mechanistically, by interacting with several core members (NEMO, CK1 and ß-TrCP) of both NF-κB and Wnt/ß-catenin signaling pathways, we show that HPV E7 oncoprotein greatly inhibits host defense peptide expression. Physiologically secreted by the squamous mucosa lining the lower female genital tract, we demonstrate that some of these latter are fundamental factors governing host-microbial interactions. More specifically, several innate molecules down-regulated in case of HPV infection are hydrolyzed, internalized and used by the predominant Lactobacillus species as amino acid source sustaining their growth/survival. Collectively, this study reveals a new viral immune evasion strategy which, by its persistent/negative impact on lactic acid bacteria, ultimately causes the dysbiosis of vaginal microbiota.

Discovery of biomarker candidates associated with the risk of short-term and mid/long-term relapse after infliximab withdrawal in Crohn's patients: a proteomics-based study.

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New biomarkers of relapse were searched in the baseline serum of CD patients stopping infliximab when they were under combined therapy (antimetabolite and infliximab) and stable clinical remission (diSconTinuation in CrOhn's disease patients in stable Remission on combined therapy with Immunosuppressors cohort, n=102). From shotgun proteomics experiment (discovery step), biomarker candidates were identified and further targeted by selected reaction monitoring (verification step). The dataset was stratified to search for markers of short-term (<6 months) or mid/long-term relapse (>6 months). The risk of relapse and the predicting capacity associated with biomarker candidates were evaluated using univariate Cox model and log-rank statistic, respectively. To test their complementary predicting capacity, biomarker candidates were systematically combined in pairs. RESULTS: Distinct biomarker candidates were associated with the risk (HR) of short-term (15 proteins, 2.9

Characterization of New Fengycin Cyclic Lipopeptide Variants Produced by Bacillus amyloliquefaciens (ET) Originating from a Salt Lake of Eastern Algeria.

Fengycin antibiotic displays a strong antifungal activity and inhibits the growth of a wide range of plant pathogens especially filamentous fungi. The main objective of the present study is to characterize fengycin variants produced by B. amyloliquefaciens strain (ET). LC-MS analysis of fengycin extracts has shown several molecular ion peaks corresponding to conventional fengycin homologues (MH + : m/z 1463.9; 1491.9; 1506) and some new ones (MH + : m/z 1433; 1447; 1461; and 1477). Further characterization of these precursor ions was carried out by LC-MS.MS analysis. Reporter fragment ions were observed (named A and B), they correspond to the cleavage of Orn2-Tyr3 (A), Glu1-Orn2 (B), and used for identifying fengycin variants. The reporter fragment couple ions [A/B] at [m/z 966.5/1080.5] and [m/z 994.4 /1108.5] represent fengycin A and B, respectively. The diagnostic ions at ([m/z 980/1094]) may correspond to fengycin C3, D, S or B2. Interestingly, unknown diagnostic product ions at [m/z 951/1065] and [m/z 979/1093] were detected for the first time in this study which prove that they correspond to new fengycin variants, named fengycin X and fengycin Y, respectively. The fengycin X results from a substitution of the glutamine amino acid (Q), at position 8 of the fengycin A peptide part, by an isoleucine (I) or a leucine (L) residue. This mutation should be the same in fengycin Y but compared to fengycin B.

Proteomics Highlights Common and Distinct Pathophysiological Processes Associated with Ileal and Colonic Ulcers in Crohn's Disease.

BACKGROUND AND AIMS: Based on genetics and natural history, Crohn's disease can be separated into two entities, an ileal and a colonic disease. Protein-based approaches are needed to elucidate whether such subphenotypes are related to distinct pathophysiological processes. METHODS: The proteome of ulcer edges was compared with that of paired control tissue samples [n = 32 biopsies] by differential proteomics in the ileum and the colon of Crohn's disease patients [n = 16]. The results were analysed using a hypothesis-driven approach [based on the literature] and a hypothesis-free approach [pathway enrichment analyses] to determine common and segment-specific pathophysiological processes associated with ileal and colonic CD ulcer edges. To confirm the involvement of a key pathway highlighted by proteomics, two proteins were also studied by immunochemistry. RESULTS: In the ileum and the colon, 4428 and 5204 proteins, respectively, were identified and quantified. Ileal and colonic ulcer edges differed in having a distinct distribution of proteins associated with epithelial-mesenchymal transition, neutrophil degranulation, and ribosomes. Ileal and colonic ulcer edges were similarly characterized by an increase in the proteins implicated in the endoplasmic reticulum protein-processing pathway and a decrease in mitochondrial proteins. Immunochemistry confirmed the presence of endoplasmic reticulum stress in the mucosa of ileal and colonic ulcer edges. CONCLUSION: This study provides protein-based evidence for partially distinct pathophysiological processes being associated with ileal and colonic ulcer edges in Crohn's disease patients. This could constitute a first step toward the development of gut segment-specific diagnostic markers and therapeutics.

Kendrick Mass Defect Approach Combined to NORINE Database for Molecular Formula Assignment of Nonribosomal Peptides.

The identification of known (dereplication) or unknown nonribosomal peptides (NRPs) produced by microorganisms is a time consuming, expensive, and challenging task where mass spectrometry and nuclear magnetic resonance play a key role. The first step of the identification process always involves the establishment of a molecular formula. Unfortunately, the number of potential molecular formulae increases significantly with higher molecular masses and the lower precision of their measurements. In the present article, we demonstrate that molecular formula assignment can be achieved by a combined approach using the regular Kendrick mass defect (RKMD) and NORINE, the reference curated database of NRPs. We observed that irrespective of the molecular formula, the addition and subtraction of a given atom or atom group always leads to the same RKMD variation and nominal Kendrick mass (NKM). Graphically, these variations translated into a vector mesh can be used to connect an unknown molecule to a known NRP of the NORINE database and establish its molecular formula. We explain and illustrate this concept through the high-resolution mass spectrometry analysis of a commercially available mixture composed of four surfactins. The Kendrick approach enriched with the NORINE database content is a fast, useful, and easy-to-use tool for molecular mass assignment of known and unknown NRP structures.

A Single Biosynthetic Gene Cluster Is Responsible for the Production of Bagremycin Antibiotics and Ferroverdin Iron Chelators.

Biosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, representing amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with p-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on p-vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus, our work shows a unique exception to the concept that BGCs should only produce a single family of molecules with one type of bioactivity and that in fact different bioactive molecules may be produced depending on the environmental conditions.IMPORTANCE Access to whole-genome sequences has exposed the general incidence of the so-called cryptic biosynthetic gene clusters (BGCs), thereby renewing their interest for natural product discovery. As a consequence, genome mining is the often first approach implemented to assess the potential of a microorganism for producing novel bioactive metabolites. By revealing a new level of complexity of natural product biosynthesis, we further illustrate the difficulty of estimation of the panel of molecules associated with a BGC based on genomic information alone. Indeed, we found that the same gene cluster is responsible for the production of compounds which differ in terms of structure and bioactivity. The production of these different compounds responds to different environmental triggers, which suggests that multiplication of culture conditions is essential for revealing the entire panel of molecules made by a single BGC.

Exploring the N-Glycosylation Profile of Glycoprotein B from Human Cytomegalovirus Expressed in CHO and Nicotiana tabacum BY-2 Cells.

The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.

Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.

The N-glycan profile of the peritrophic membrane in the Colorado potato beetle larva (Leptinotarsa decemlineata).

The insect peritrophic membrane (PM) is a non-cellular structure composed of secreted proteins imbedded in a proteoglycan matrix together with chitin. It separates the midgut epithelium from the intestinal contents, and functions in the digestion of food. Furthermore it acts as a protective barrier against abrasive particles and microbial infections. Here we studied for the first time the N-glycome of the PM. We identified the N-glycan structures present in the PM of the Colorado potato beetle (CPB) at the fourth larval stage using MALDI-TOF mass spectrometry. In parallel, we correlated the N-glycan data to the presence of the N-glycosylation related genes (NGRGs) in the transcriptome of epithelial midgut cells. The presumed activities of the identified genes support the N-glycan profile resolved for the proteins in the PM. To our knowledge these data are the first report on the N-glycome of the PM of a pest insect. These results will contribute to the study of the importance of N-glycosylation in the function and structure of the PM. In addition, the data can help to find novel targets and design better biopesticides for pest control.

The N-glycome of the hemipteran pest insect Nilaparvata lugens reveals unexpected sex differences.

The brown planthopper, Nilaparvata lugens, is a model species for hemimetabolous development and the most important pest insect in rice, which is the major staple crop for about half of the world population. Despite its importance, little is known of the N-glycosylation process in this insect. Here we report on the N-glycome for the post-embryonic stages of N. lugens, revealing unique features that are different from the holometabolous insect models, as the fruit fly Drosophila melanogaster and the beetle Tribolium castaneum. Analysis of the N-glycan fingerprint for male and female adults showed sex-specific N-glycosylation in insects. Specifically, the female adults progress towards a unique glycan profile with a striking increase in high mannose N-glycans. The N-glycome of N. lugens contributes to study pathways differentiating between sexes, and the results shed light on the evolution and differences in development between primitive hemimetabolous insects and more advanced holometabolous insects. The data are discussed in relation to potential function(s) in development and sex specificity.

Microproteomic Profiling of High-Grade Squamous Intraepithelial Lesion of the Cervix: Insight into Biological Mechanisms of Dysplasia and New Potential Diagnostic Markers.

PURPOSE: High-grade squamous intraepithelial lesion (HSIL) is a known precursor for squamous cell carcinoma of uterine cervix. Although it is known that SILs are associated to infection by human papillomavirus, downstream biological mechanisms are still poorly described. In this study, we compared the microproteomic profile of HSIL to normal tissues: ectocervix (ectoC) and endocervix (endoC). EXPERIMENTAL DESIGN: Tissue regions of endoC, ectoC, and HSlL were collected by laser microdissection (3500 cells each) from five patients. Samples were processed and analyzed using our recently developed laser microdissection-based microproteomic method. Tissues were compared in order to retrieve HSIL's proteomic profile. Potentially interesting proteins for pathology were stained by immunohistochemistry. RESULTS: We identified 3072 proteins among the fifteen samples and 2386 were quantified in at least four out of the five biological replicates of at least one tissue type. We found 236 proteins more abundant in HSIL. Gene ontology enrichments revealed mechanisms of DNA replication and RNA splicing. Despite the squamous nature of HSIL, a common signature between HSIL and endoC could be found. Finally, potential new markers could support diagnosis of dysplasia in SILs. CONCLUSION AND CLINICAL RELEVANCE: This microproteomic investigation of HSIL gives insights into the biology of cervical precancerous lesions.

Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay.

Disulfide connectivity in peptides bearing at least two intramolecular disulfide bonds is highly important for the structure and the biological activity of the peptides. In that context, analytical strategies allowing a characterization of the cysteine pairing are of prime interest for chemists, biochemists, and biologists. For that purpose, this study evaluates the potential of MALDI in-source decay (ISD) for characterizing cysteine pairs through the systematic analysis of identical peptides bearing two disulfide bonds, but not the same cysteine connectivity. Three different matrices have been tested in positive and/or in negative mode (1,5-DAN, 2-AB and 2-AA). As MALDI-ISD is known to partially reduce disulfide bonds, the data analysis of this study rests firstly on the deconvolution of the isotope pattern of the parent ions. Moreover, data analysis is also based on the formed fragment ions and their signal intensities. Results from MS/MS-experiments (MALDI-ISD-MS/MS) constitute the last reference for data interpretation. Owing to the combined use of different ISD-promoting matrices, cysteine connectivity identification could be performed on the considered peptides. Graphical Abstract ᅟ.

Jug r 6 is the allergenic vicilin present in walnut responsible for IgE cross-reactivities to other tree nuts and seeds.

Walnuts are ranked high in the list of the culprit foods inducing severe allergic reactions. Jug r 2 has been identified as a major allergen in common walnut by cDNA cloning from a somatic cell line. So far, studies were performed on the allergenic activity of recombinant Jug r 2, yet there is still no evidence about the physicochemical characteristics of the natural allergen. Therefore, we aimed to purify and deeply characterize natural Jug r 2 and to assess IgE cross-reactivity among vicilins from different tree nuts. Extensive mass spectrometry analysis of the obtained purified vicilin allowed identification of the protein sequence that displayed only 44% identity to Jug r 2. The newly identified vicilin (Jug r 6) was recognized by IgE of 26% in walnut allergic patients' sera tested. In contrast to Jug r 2, Jug r 6 displayed a remarkable level of cross-reactivity when tested with homologues from hazelnut, sesame and pistachio. It is the first report showing the necessity of proteomic studies to improve allergy component resolved diagnosis.

G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets.

G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in vivo in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite Plasmodium falciparum, which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, P. falciparum parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has a rapid initial rate of kill and is active against ring stages as well as replicative stages of intraerythrocytic development. We show that several G-quadruplex-stabilizing drugs, including quarfloxin, can suppress the transcription of a G-quadruplex-containing reporter gene in P. falciparum but that quarfloxin does not appear to disrupt the transcription of rRNAs, which was proposed as its mode of action in both human cells and trypanosomes. These data suggest that quarfloxin has potential for repositioning as an antimalarial with a novel mode of action. Furthermore, G-quadruplex biology in P. falciparum may present a target for development of other new antimalarial drugs.

Laser Microdissection-Based Microproteomics of Formalin-Fixed and Paraffin-Embedded (FFPE) Tissues.

Laser microdissection-based proteomics on formalin-fixed and paraffin-embedded tissues is usually performed from relatively large tissue areas or pools of multiple tissue pieces. However, several molecular pathology studies require working on very limited amounts of tissue. This is for example the case when very early cancer lesions have to be handled. Hereby, we present a method for the processing of very small pieces of formalin-fixed and paraffin-embedded tissues for proteomic purposes. This approach is designed in order to avoid sample loss during technical processing and to optimize the digestion of tissue areas containing as little as 2700 cells.

MALDI Imaging-Guided Microproteomic Analyses of Heterogeneous Breast Tumors-A Pilot Study.

Matrix-assisted laser desorption/ionization (MALDI) imaging is an ideal tool to study intratumor heterogeneity (ITH) and its implication in prognostic stratification of patients. However, there are some drawbacks concerning protein identification. On the other hand, laser microdissection (LMD)-based microproteomics allows retrieving thousands of protein identifications from small tissue pieces. As a proof of concept, the authors combine these two complementary approaches to analyze heterogeneous regions in breast tumors. Invasive ductal breast cancer FFPE tissue sections from five patients are analyzed by MALDI imaging and the dataset is processed by segmentation. Heterogeneous regions within tumors are processed by LMD-based microproteomics, in duplicates. Liquid chromatography-tandem mass spectrometry data are classified by hierarchical clustering. Heterogeneous tissue regions are discriminated on the basis of their actual molecular heterogeneity. The dataset is correlated with MALDI imaging to identify m/z values discriminating heterogeneous regions. The molecular characterization of cell clones in tumors related to bad patient outcome could have great impact for pathology. A combined application of LMD-based microproteomics and MALDI imaging for ITH studies is presented.

Reaction Product Variability and Biological Activity of the Lactoperoxidase System Depending on Medium Ionic Strength and pH, and on Substrate Relative Concentration.

The potential of ions produced in water by the lactoperoxidase system against plant pests has shown promising results. We tested the bioactivity of ions produced by the lactoperoxidase oxidation of I- and SCN- in several buffers or in tap water and characterized the ions produced. In vitro biological activity was tested against Penicillium expansum, the causal agent of mold in fruits, and the major cause of patulin contamination of fruit juices and compotes. In buffers, the ionic concentration was increased 3-fold, and pathogen inhibition was obtained down to the 1:15 dilution. In tap water, the ionic concentration was weaker, and pathogen inhibition was obtained only down to the 1:3 dilution. Acidic buffer increased ion concentrations as compared to less acidic (pH 5.6 or 6.2) or neutral buffers, as do increased ionic strength. 13 C-labelled SCN- and MS showed that different ions were produced in water and in buffers. In specific conditions the ion solution turned yellow and a product was formed, probably diiodothiocyanate (I2 SCN- ), giving an intense signal at 49.7 ppm in 13 C-NMR. The formation of the signal was unambiguously favored in acidic media and disadvantaged or inhibited in neutral or basic conditions. It was enhanced at a specific SCN- : I- ratio of 1:4.5, but decreased when the ratio was 1:2, and was inhibited at ratio SCN- >I- . We demonstrated that the formation of the signal required the interaction between I2 and SCN- , and MS showed the presence of I2 SCN- .

MALDI Imaging Combined with Laser Microdissection-Based Microproteomics for Protein Identification: Application to Intratumor Heterogeneity Studies.

Matrix-assisted laser desorption ionization (MALDI) imaging is widely used for in situ proteomic mapping and finds multiple applications in pathology. However, low fragmentation yields in MALDI avoid an optimal identification of peptides from tissues. On the other hand, LMD-based microproteomic analyses allow for the identification of hundreds to thousands of proteins from small tissue regions. Herein, we present the combination of MALDI imaging and LMD-based microproteomic approaches for parallel identification. We illustrate the workflow with an application to intratumor heterogeneity studies.

In-Depth Glyco-Peptidomics Approach Reveals Unexpected Diversity of Glycosylated Peptides and Atypical Post-Translational Modifications in Dendroaspis angusticeps Snake Venom.

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S-cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments.