Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics

Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics

Schistosoma mansoni venom allergen-like proteins:Phylogenetic relationships, stage-specific transcription and tissue localization as predictors of immunological cross-reactivity

O artigo encontra-se disponível para download no site do Editor.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2019-07-15T18:23:01Z No. of bitstreams: 1 Farias, L.P. Schistosoma mansoni venom...2019.pdf: 1118803 bytes, checksum: 1ddd953840abbbd5d56675c8d6c4fa6e (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-07-15T18:39:31Z (GMT) No. of bitstreams: 1 Farias, L.P. Schistosoma mansoni venom...2019.pdf: 1118803 bytes, checksum: 1ddd953840abbbd5d56675c8d6c4fa6e (MD5)Made available in DSpace on 2019-07-15T18:39:31Z (GMT). No. of bitstreams: 1 Farias, L.P. Schistosoma mansoni venom...2019.pdf: 1118803 bytes, checksum: 1ddd953840abbbd5d56675c8d6c4fa6e (MD5) Previous issue date: 2019Welcome Trust (UK) (WT084273/Z/07/Z) to KFH, Fundação Butantan, Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil) to LPF and LLC (2012/23124-4), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to LCCL and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001, and by fellowships from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) to LPF (2008/57946-5) and HKF (2007/07685-8) and from CNPq to MIK (160861/2017-9). We thank Dra. Eliana Nakano and Ms. Patricia A. Miyasato for supplying the parasite stages and to Alexsander Seixas de Souza for confocal microscopy (FAPESP 00/11624-5) imaging support, all from Instituto Butantan, Brazil.Instituto Butantan. Centro de Biotecnologia. São Paulo, SP, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.Aberystwyth University. Institute of Biological. Environmental and Rural Sciences. Aberystwyth, UK.Aberystwyth University. Institute of Biological. Environmental and Rural Sciences. Aberystwyth, UK.Instituto Butantan. Centro de Biotecnologia. São Paulo, SP, Brasil.Aberystwyth University. Institute of Biological. Environmental and Rural Sciences. Aberystwyth, UK.Aberystwyth University. Institute of Biological. Environmental and Rural Sciences. Aberystwyth, UK.Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.Instituto Butantan. Centro de Biotecnologia. São Paulo, SP, Brasil / Universidade de São Paulo. Pós-Graduação Interunidades em Biotecnologia. São Paulo, SP, Brasil.Leiden University Medical Centre. Center for Proteomics and Metabolomics. RC Leiden, The Netherlands.Leiden University Medical Centre. Department of Parasitology. RC Leiden, The Netherlands.Instituto Butantan. Centro de Biotecnologia. São Paulo, SP, Brasil.Aberystwyth University. Institute of Biological. Environmental and Rural Sciences. Aberystwyth, UK.Schistosoma mansoni venom allergen-like proteins (SmVALs) are part of a diverse protein superfamily partitioned into two groups (group 1 and group 2). Phylogenetic analyses of group 1 SmVALs revealed that members could be segregated into subclades (A-D); these subclades share similar gene expression patterns across the parasite lifecycle and immunological cross-reactivity. Furthermore, whole-mount in situ hybridization demonstrated that the phylogenetically, transcriptionally and immunologically-related SmVAL4, 10, 18 and 19 (subclade C) were all localized to the pre-acetabular glands of immature cercariae. Our results suggest that SmVAL group 1 phylogenetic relationships, stage-specific transcriptional profiles and tissue localization are predictive of immunological cross-reactivity

Furin is a chemokine-modifying enzyme: in vitro and in vivo processing of CXCL10 generates a C-terminally truncated chemokine retaining full activity

Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the total spectrum of chemokine variants. We have recently shown that the native chemokine CXCL10 is processed at the C terminus, thereby shedding the last four amino acids. The present study was performed to elucidate the mechanism in vivo and in vitro and to study the biological activity of this novel isoform of CXCL10. Using a combination of protein purification and mass spectrometric techniques, we show that the production of C-terminally truncated CXCL10 by primary keratinocytes is inhibited in vivo by a specific inhibitor of pro-protein convertases (e.g. furin) but not by inhibition of matrix metalloproteinases. Moreover, CXCL10 is processed by furin in vitro, which is abrogated by a mutation in the furin recognition site. Using GTRγS binding, C

Proteomic Analysis of the Dysferlin Protein Complex Unveils Its Importance for Sarcolemmal Maintenance and Integrity

Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity

A functional Campylobacter jejuni maf4 gene results in novel glycoforms on flagellin and altered autoagglutination behaviour

Flagellin of Campylobacter jejuni is extensively modified with (derivatives of) pseudaminic acid. The flagellar glycosylation locus contains several genes with homopolymeric G-tracts prone to slipped-strand mispairing, some of which belong to the maf gene family. We investigated the function of the putative phase-variable maf4 gene of C. jejuni strain 108. A constructed maf4 mutant displayed unaltered flagella assembly and bacterial motility. 2D-PAGE analysis revealed that the flagellin of strain 108 migrated at a more acidic pI than the protein of the Maf4 mutant. MS-MS in combination with high-resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron MS (MALDI-FT-ICR-MS) on flagellin-derived glycopeptides showed that the flagellins of the mutant lacked two previously unidentified modifications of pseudaminic acid. These glycoforms carried additional CO(2) and C(2)H(2)O(2) groups, consistent with the more acidic pI of the wild-type flagellin. Phenotypically, the maf4 mutant displayed strongly delayed bacterial autoagglutination. Collectively, our results suggest that the presence of a functional Maf4 expands the flagellin glycan repertoire with novel glycoforms of pseudaminic acid and, in the event of phase variation, alters the population behaviour of C. jejuni

Proteomic Analysis of Uveal Melanoma Reveals Novel Potential Markers Involved in Tumor Progression

PURPOSE. Patient survival in uveal melanoma may benefit from earlier recognition of potential metastases to the liver, but as yet, proper markers indicating metastases are not available. Identification of metastasis markers would therefore be of great value. The proteins that are expressed in two cell lines originating from two liver metastases were compared with the proteins expressed in a cell line obtained from the primary uveal melanoma of the same patient, to identify proteins that play a role in tumor progression as well as proteins that are expressed specifically in metastases. METHODS. Protein analysis was performed by using two-dimensional gel electrophoresis. A subset of proteins was subsequently identified with mass spectrometry. RESULTS. A set of 24 proteins was differentially expressed in both of the two metastatic cell lines compared with the cell line derived from the primary tumor. These proteins were subdivided into groups according to cellular function, with important roles in tumor development. CONCLUSIONS. Tumor progression and development of metastases is a multicomplex system. Comparing protein expression in two cell lines derived from metastases with a cell line derived from a primary uveal melanoma from the same patient identified proteins involved in tumor progression, and proteins specifically expressed in the metastases, which have the potential of becoming clinically useful biomarkers. (Invest Ophthalmol Vis Sci. 2006;47:786 -793) DOI:10.1167/iovs.05-0314 T he most prevalent primary intraocular malignancy in adults is the melanoma originating from the uveal tract, affecting approximately 7 per 1 million people in the Western world each year. Understanding the molecular changes in gene and protein expression that are responsible for the development and progression of uveal melanoma would be an important step toward the identification of biomarkers that are indicative of metastatic melanoma. High-throughput technologies in genomics 7,8 and proteomics 9 offer the potential to find such previously unidentified alterations in malignancies. In recent years, genomics has increased our insight in gene expression profiles in uveal melanoma 10 -12 and has provided potential clinically important screening markers. However, alterations at the RNA level may not be reflected in changes at the protein level. The application of proteomics is a powerful screening method for alterations in protein expression and posttranslational modifications. Recently, our group revealed that comparison of protein profiles of aqueous humor from eyes containing a uveal melanoma with eyes undergoing cataract surgery, could separate patients and control subjects on the basis of two proteins. 13 To identify proteins that are associated with uveal melanoma metastases, we focused, in this study, on the differential protein expression of primary and metastatic uveal melanoma analyzing three cell lines representing a primary uveal melanoma and two of its metastases, using two-dimensional poly acrylamide gel electrophoresis (2-D-PAGE) and mass spectrometry (MS). We hypothesized that downregulation of proteins with growth-inhibitory capacity would be seen during progression from primary tumor to metastasis, whereas similarly, growth-stimulating proteins would show upregulation in the metastasis-derived cell lines. Markers that are specifically upregulated in metastases may be potential markers of metastases at a stage at which such metastases are not yet clinically recognizable by, for example, echography. Because the two metastatic cell lines were obtained from two different metastases in the same patient, they served as an internal control. Twenty-four differentially expressed proteins were identified, of which most play a role in tumor-promoting cellular mechanisms. MATERIALS AND METHODS Cell Culture Three cell lines were included in the study. One was derived from a primary uveal melanoma (Mel 270) and two (Omm 1.3 and -1.5) were derived from liver metastases from this same primary tumor. All cell lines were kindly provided by Bruce R. Ksander (Schepens Eye Institute, Boston, MA). The research complied with the tenets of the Declaration of Helsinki. From th

Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

During inflammation, the endothelium mediates rolling and firm adhesion of activated leukocytes. Integrin-mediated adhesion to endothelial ligands of the Ig-superfamily induces intracellular signaling in endothelial cells, which promotes leukocyte transendothelial migration. We identified the actin cross-linking molecule filamin B as a novel binding partner for intracellular adhesion molecule-1 (ICAM-1). Immune precipitation as well as laser scanning confocal microscopy confirmed the specific interaction and co-localization of endogenous filamin B with ICAM-1. Importantly, clustering of ICAM-1 promotes the ICAM-1-filamin B interaction. To investigate the functional consequences of filamin B binding to ICAM-1, we used small interfering RNA to reduce filamin B expression in ICAM-1-GFP expressing HeLa cells. We found that filamin B is required for the lateral mobility of ICAM-1 and for ICAM-1-induced transmigration of leukocytes. Reducing filamin B expression in primary human endothelial cells resulted in reduced recruitment of ICAM-1 to endothelial docking structures, reduced firm adhesion of the leukocytes to the endothelium, and inhibition of transendothelial migration. In conclusion, this study identifies filamin B as a molecular linker that mediates ICAM-1-driven transendothelial migratio