VISLISI trial, a prospective clinical study allowing identification of a new metalloprotease and putative virulence factor from Staphylococcus lugdunensis.

OBJECTIVE: Staphylococcus lugdunensis is a coagulase-negative staphylococcus that displays an unusually high virulence rate close to that of Staphylococcus aureus. It also shares phenotypic properties with S. aureus and several studies found putative virulence factors. The objective of the study was to describe the clinical manifestations of S. lugdunensis infections and investigate putative virulence factors. METHOD: We conducted a prospective study from November 2013 to March 2016 at the University Hospital of Strasbourg. Putative virulence factors were investigated by clumping factor detection, screening for proteolytic activity, and sequence analysis using tandem nano-liquid chromatography-mass spectrometry. RESULTS: In total, 347 positive samples for S. lugdunensis were collected, of which 129 (37.2%) were from confirmed cases of S. lugdunensis infection. Eighty-one of these 129 patients were included in the study. Bone and prosthetic joints (PJI) were the most frequent sites of infection (n=28; 34.6%) followed by skin and soft tissues (n=23; 28.4%). We identified and purified a novel protease secreted by 50 samples (61.7%), most frequently associated with samples from deep infections and PJI (pr 0.97 and pr 0.91, respectively). Protease peptide sequencing by nano-liquid chromatography-mass spectrometry revealed a novel protease bearing 62.42% identity with ShpI, a metalloprotease secreted by Staphylococcus hyicus. CONCLUSION: This study confirms the pathogenicity of S. lugdunensis, particularly in bone and PJI. We also identified a novel metalloprotease called lugdulysin that may contribute to virulence.

A reinvestigation provides no evidence for sugar residues on structural proteins of poleroviruses and argues against a role for glycosylation of virus structural proteins in aphid transmission.

Poleroviruses are strictly transmitted by aphids. Glycosylation of Turnip yellows virus (TuYV) was previously reported and this modification was supposed to be required for aphid transmission. Using different approaches based on (i) a lectin-binding assay, (ii) use of specific complex glycan antibodies and (iii) mass spectrometry, we found no evidence that the structural proteins of TuYV and Cucurbit aphid-borne yellow virus (CABYV) carry glycan residues. Moreover, mutation of each of the four potential N-glycosylation sites of the structural protein sequences of CABYV indicated that, unless more than one site on the structural protein is glycosylated, N-glycosylation is not involved in aphid transmission. These results did not corroborate the previous hypothesis for the role of glycosylation in aphid transmission. They, however, revealed the presence of a glycosylated plant protein in purified polerovirus suspensions, whose function in aphid transmission should be further investigated.

Chfr interacts and colocalizes with TCTP to the mitotic spindle.

Chfr is a checkpoint protein that plays an important function in cell cycle progression and tumor suppression, although its exact role and regulation are unclear. Previous studies have utilized overexpression of Chfr to determine the signaling pathway of this protein in vivo. In this study, we demonstrate, by using three different antibodies against Chfr, that the endogenous and highly overexpressed ectopic Chfr protein is localized and regulated differently in cells. Endogenous and lowly expressed ectopic Chfr are cytoplasmic and localize to the spindle during mitosis. Higher expression of ectopic Chfr correlates with a shift in the localization of this protein to the nucleus/PML bodies, and with a block of cell proliferation. In addition, endogenous and lowly expressed ectopic Chfr is stable throughout the cell cycle, whereas when highly expressed, ectopic Chfr is actively degraded during S-G2/M phases in an autoubiquitination and proteasome-dependent manner. A two-hybrid screen identified TCTP as a possible Chfr-interacting partner. Biochemical analysis with the endogenous proteins confirmed this interaction and identified beta-tubulin as an additional partner for Chfr, supporting the mitotic spindle localization of Chfr. The Chfr-TCTP interaction was stable throughout the cell cycle, but it could be diminished by the complete depolymerization of the microtubules, providing a possible mechanism where Chfr could be the sensor that detects microtubule disruption and then activates the prophase checkpoint.

Antimicrobial chromogranins and proenkephalin-A-derived peptides: Antibacterial and antifungal activities of chromogranins and proenkephalin-A-derived peptides.

The secretory granules from adrenal medullary chromaffin cells contain a complex mixture of low-molecular mass constituents such as catecholamines, ascorbate, nucleotides, calcium, peptides, and several high-molecular mass water-soluble proteins including chromogranins and proenkephalin-A. These proteins are sequestered into secretory granules in which processing yields a large variety of peptides. These fragments are released into the extracellular space upon cell stimulation and are recovered in blood, lymph, cerebrospinal fluid, and synovial fluid. Some of them have biological activity on cells in an autocrine, paracrine, or endocrine fashion. In addition, we have shown that peptides with antimicrobial activity are present with the secretory chromaffin granules and demonstrated that they are released from stimulated chromaffin cells. We have shown that posttranslational modifications modulate the antimicrobial activities. For some peptides, using confocal laser microscopy, we have examined the interaction of the rhodaminated peptides with biological membranes. In addition, we have shown that chromofungin, the antifungal peptide corresponding to chromogranin A(47-66), can bind calmodulin in the presence of calcium and induce inhibition of calcineurin, a calmodulin-dependent enzyme. Because these antibacterial peptides are colocalized with catecholamines, they may be activated during stress, playing a role as a first protective barrier against bacterial infection, and thus act as factors of the innate immunity shortly after infection and before the induction and mobilization of an adaptative immune system.

Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains.

The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor. In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant [rho degrees ] with EDTA and two non-ionic surfactants (Hecameg and HTAC). The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives. However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells. The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity. The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the [rho degrees ] mutant. Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE. Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase. Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively. The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition.

Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor.

Numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence which belongs to the cathelicidin family of proteins. The three-dimensional structure of this cathelicidin motif, which contains two disulfide bonds, has not yet been reported. The cathelicidin motif (ProS) of the protegrin-3 precursor was overexpressed in Escherichia coli as a His-tagged protein. The His(6) tag was removed by thrombin cleavage. ProS was purified to homogeneity and single crystals were obtained by the hanging-drop vapour-diffusion method at pH 3-4. Preliminary X-ray diffraction analysis indicated that these crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 51.42, c = 134.25 A. These crystals diffracted beyond 2.75 A (1.9 A at ESRF) and contain one molecule per asymmetric unit.

A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.

A comparison between two fluorescent metal chelates for staining proteins separated by electrophoresis has been carried out. One of these chelates is ruthenium II tris (bathophenanthroline disulfonate) and the other is commercial Sypro Ruby. Both can be efficiently detected either with UV tables or with commercial laser fluorescence scanners. The sensitivity and homogeneity of the stains and the interference with mass spectrometry analysis have been investigated. It appears that both stains perform similarly for protein detection, while ruthenium II tris (bathophenanthroline disulfonate) performs better for mass spectrometry analyses and as cost-effectiveness ratio. However, Sypro Ruby is easier to use as a stain.

The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p.

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.

Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp. PCC 7120.

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.

Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast.

As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology. In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest. Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown. Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae. The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S. cerevisiae separated on 2-D gels. This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S. cerevisiae proteins.

Biochemical characterization of the helper component of Cauliflower mosaic virus.

The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% alpha-helical. Most alpha-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.

Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels.

Changes in gene expression occurring during differentiation of human monocytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase in mitochondrial function and in the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary.

Processing of proenkephalin-A in bovine chromaffin cells. Identification of natural derived fragments by N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

A large variety of proenkephalin-A-derived peptides (PEAPs) are present in bovine adrenal medulla secretory granules that are cosecreted with catecholamines upon stimulation of chromaffin cells. In the present paper, after reverse phase high performance liquid chromatography of intragranular soluble material, PEAPs were immunodetected with antisera raised against specific proenkephalin-A (PEA) sequences (PEA63-70 and PEA224-237) and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Thirty PEAPs were characterized in addition to enkephalins and whole PEA, indicating that preferential proteolytic attacks occurred at both N- and C-terminal regions. A similar approach was used to characterize PEA-derived fragments exocytotically released into the extracellular space that showed five additional minor PEAPs. Among all these naturally generated peptides, enkelytin, the antibacterial bisphos- phorylated C-terminal peptide (PEA209-237), was predominantly generated, as shown by MALDI-TOF mass spectrometry analysis, which constituted an efficient method for its identification. Finally, the data on PEA intragranular and extracellular processing in adrenal medulla are discussed in regard to the known enzymatic processing mechanisms. We note the high conservation of the cleavage points in evolutionarily diverse organisms, highlighting an important biological function for the released PEAPs.

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp.

Penaeidins are 5.5- to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.

Phosphorylation and O-glycosylation sites of human chromogranin A (CGA79-439) from urine of patients with carcinoid tumors.

Because of their water-soluble properties, chromogranins (CGs) and chromogranin-derived fragments are released together with catecholamines from adrenal chromaffin cells during stress situations and can be detected in the blood by radiochemical and enzyme assays. It is well known that chromogranins can serve as immunocytochemical markers for neuroendocrine tissues and as a diagnostic tool for neuroendocrine tumors. In 1993, large CGA-derived fragments have been shown to be excreted into the urine in patients with carcinoid tumors and the present study deals with the characterization of the post-translational modifications (phosphorylation and O-glycosylation) located along the largest natural CGA-derived fragment CGA79-439. Using mild proteolysis of peptidic material, high performance liquid chromatography, sequencing, and mass spectrometry analysis, six post-translational modifications were detected along the C-terminal CGA-derived fragment CGA79-439. Three O-linked glycosylation sites were located in the core of the protein on Thr163, Thr165, and Thr233, consisting in di-, tri-, and tetrasaccharides. Three phosphorylation sites were located in the middle and C-terminal domain, on serine residues Ser200, Ser252, and Ser315. These modified sites were compared with sequences of others species and discussed in relation with the post-translational modifications that we have reported previously for bovine CGA.

Antibacterial peptides are present in chromaffin cell secretory granules.

1. Antibacterial activity has recently been associated with the soluble matrix of bovine chromaffin granules. Furthermore, this activity was detected in the contents secreted from cultured chromaffin cells following stimulation. 2. The agents responsible for the inhibition of Gram+ and Gram- bacteria growth are granular peptides acting in the micromolar range or below. In secretory granules, these peptides are generated from cleavage of chromogranins and proenkephalin A and are released together with catecholamines into the circulation. 3. Secretolytin and enkelytin are the best characterized; these two peptides share sequence homology and similar antibacterial activity with insect cecropins and intestinal diazepam-binding inhibitor. For some of the peptides derived from chromogranin A, posttranslational modifications were essential since antibacterial activity was expressed only when peptides were phosphorylated and/or glycosylated. 4. The significance of this activity is not yet understood. It may be reminiscent of some primitive defense mechanism or may serve as a first barrier to bacteria infection during stress, as these peptides are secreted along with catecholamines.

Phosphorylation and O-glycosylation sites of bovine chromogranin A from adrenal medullary chromaffin granules and their relationship with biological activities.

Bovine adrenal medullary chromogranin A, the major soluble component of chromaffin granules, is a phosphorylated glycoprotein. In the present work, phosphorylation and glycosylation sites were determined using mild proteolysis, peptide separation, microsequencing, and mass analysis by electrospray and matrix-assisted laser desorption ionization time-of-flight techniques. Seven post-translational modification sites were detected. Two O-linked glycosylation sites, each consisting of the trisaccharide NeuAcalpha2-3Galbeta1-3GalNAcalpha1, were located in the middle part of the protein, on Ser186 and on Thr231. The former residue is present in the antibacterial peptide named chromacin. Four phosphorylation sites were located on serine residues at positions Ser81 in the N-terminal region of the protein and Ser307, Ser372, and Ser376 in the C-terminal end. One additional phosphorylation site was found on the tyrosine residue at position Tyr173, the N-terminal amino acid of chromacin. With the exception of the phosphorylation on Tyr173, all of the other post-translational modifications are located on highly conserved chromogranin A regions, implying some biological importance.

Antibacterial activity of glycosylated and phosphorylated chromogranin A-derived peptide 173-194 from bovine adrenal medullary chromaffin granules.

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

The C-terminal bisphosphorylated proenkephalin-A-(209-237)-peptide from adrenal medullary chromaffin granules possesses antibacterial activity.

The chromaffin granules have been shown to be an excellent model to study the processing of proenkephalin-A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin I and the occurrence of the C-terminal chromogranin B-derived peptide 614-626 which was shown to have antibacterial activity [Strub, J.M., Garcia-Sablone, P., Looning, K., Taupenot, L., Hubert, P., Van Dorsselaer, A., Aunis, D. & Metz-Boutigue, M.H. (1995) Eur. J. Biochem. 229, 356-368]. We also observed that this new antibacterial activity present in chromaffin granules was associated with other endogenous protein-derived fragments yet to be characterized. The present study reports the isolation and characterization of a peptide which possesses antibacterial activity and which corresponds to the C-terminal 209-237 sequence of proenkephalin-A. A detailed study using microsequencing and matrix-assisted-laser-desorption time-of-flight mass spectrometry (MALD-TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM) with post-translational modifications. Endogenous bisphosphorylated proenkephalin-A-(209-237) was active on Micrococcus luteus and Bacillus megaterium killing bacteria in the 0.2 - 0.4 microM range but was inactive in similar conditions towards Escherichia coli. Enkelytin shares sequence and structural similarities with the antibacterial C-terminal domain of diazepam-binding inhibitor. According to this similarity, a prediction of secondary structure is proposed for enkelytin and discussed in relationship to its biological activity.