Adherence Protein Research Articles

IL-8 antagonist, CXCL8(3-72)K11R/G31P coupled with probiotic exhibit variably enhanced therapeutic potential in ameliorating ulcerative colitis

Inflammatory bowel disease (IBD) remains a major health challenge due in part to unsafe and limited treatment options, hence there is the need for alternatives. CXCL8/interleukin 8 (IL-8) is elevated in inflammation, and binds preferentially to G protein-couple receptors (GPCRs) CXCR1/2 of the CXC chemokine family to initiate cascades of downstream inflammatory signals. A mutant CXCL8 protein, CXCL8(3-72)K11R/G31P (G31P), competitively and selectively binds to CXCR1/2, making CXCL8 redundant. We explore the therapeutic potential of G31P in dextran sulfate sodium (DSS) induced ulcerative colitis (UC), and the corresponding effect if G31P treatment is augmented with Lactobacillus acidophilus (LACT). The treatment options administered significantly reduced TNF-α, IFN-γ, IL-1β, IL-6, and IL-8, but maintained elevated levels of IL-10. CD68 and F4/80 expressions were down-regulated and showed restricted infiltration to inflamed colon, while IL-17F levels were insignificantly different from the DSS treated mice. Also, we observed up-regulation of IL-17A in G31P + LACT but not G31P treated mice if compared with Control group. The treatments ameliorated colonic fibrosis by reducing VEGF, TGF-β, MMP-2 and MMP-9. In addition, we observed elevated levels of E-cadherin, and marginal up-regulation of occludin, suggesting the role of the treatments in regulating tight intestinal junction and adherence proteins. Mechanism-wise, G31P interferes with AKT and ERK signaling pathways. Our study suggests that G31P confers protection in IBD, particularly UC, and when G31P treatment is augmented with Lactobacillus acidophilus, the protection is variably enhanced.

Interaction between the Staphylococcus aureus extracellular adherence protein Eap and its subdomains with platelets

S. aureus associated bacteremia can lead to severe infections with high risk of mortality (e.g. sepsis, infective endocarditis). Many virulence factors and adhesins of S. aureus are known to directly interact with platelets. Extracellular adherence protein, Eap, one of the most important virulence factors in S. aureus mediated infections is a multi-tandem domain protein and has been shown to interact with almost all cell types in the human circulatory system. By using amine reactive fluorescent N-hydroxysuccinimidyl (NHS)-ester dyes and by direct detection with primary fluorescently conjugated anti-histidine (His-tag) antibodies against detect N-terminal His6, we show Eap subdomain Eap D3D4 specifically interacts and rapidly activates human platelets. Furthermore, we validate our finding by using site directed directional immobilization of Eap D3D4 through N-terminal His6 on nickel (II)-nitrilotriacetic acid (Ni-NTA) functionalized bacteriomimetic microbead arrays to visualize real-time platelet activation through calcium release assay. These methods offer an easily adoptable protocols for screening of S.aureus derived virulence factors and adhesins with platelets.

Role of glutamine in the mediation of E-cadherin, p120-catenin and inflammation in ventilator-induced lung injury.

Background:Ventilator-induced lung injury (VILI) is commonly associated with barrier dysfunction and inflammation reaction. Glutamine could ameliorate VILI, but its role has not been fully elucidated. This study examined the relationship between inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, and IL-10) and adherens junctions (E-cadherin, p120-catenin), which were ameliorated by glutamine in VILI, both in vitro and in vivo.Methods:For the in vivo study, 30 healthy C57BL/6 mice weighing 25–30 g were randomly divided into five groups with random number table (n = 6 in each group): control (Group C); low tidal volume (Group L); low tidal volume + glutamine (Group L + G); high tidal volume (Group H); and high tidal volume + glutamine (Group H + G). Mice in all groups, except Group C, underwent mechanical ventilation for 4 h. For the in vitro study, mouse lung epithelial 12 (MLE-12) cells pretreated with glutamine underwent cyclic stretching at 20% for 4 h. Cell lysate and lung tissue were obtained to detect the junction proteins, inflammatory cytokines, and lung pathological changes by the Western blotting, cytokine assay, hematoxylin and eosin staining, and immunofluorescence.Results:In vivo, compared with Group C, total cell counts (t = −28.182, P < 0.01), the percentage of neutrophils (t = −28.095, P < 0.01), IL-6 (t = −28.296, P < 0.01), and TNF-α (t = −19.812, P < 0.01) in bronchoalveolar lavage (BAL) fluid, lung injury scores (t = −6.708, P < 0.01), and the wet-to-dry ratio (t = −15.595, P < 0.01) were increased in Group H; IL-10 in BAL fluid (t = 9.093, P < 0.01) and the expression of E-cadherin (t = 10.044, P < 0.01) and p120-catenin (t = 13.218, P < 0.01) were decreased in Group H. Compared with Group H, total cell counts (t = 14.844, P < 0.01), the percentage of neutrophils (t = 18.077, P < 0.01), IL-6 (t = 18.007, P < 0.01), and TNF-α (t = 10.171, P < 0.01) in BAL fluid were decreased in Group H + G; IL-10 in BAL fluid (t = −7.531, P < 0.01) and the expression of E-cadherin (t = −14.814, P < 0.01) and p120-catenin (t = −9.114, P < 0.01) were increased in Group H + G. In vitro, compared with the nonstretching group, the levels of IL-6 (t = −21.111, P < 0.01) and TNF-α (t = −15.270, P < 0.01) were increased in the 20% cyclic stretching group; the levels of IL-10 (t = 5.450, P < 0.01) and the expression of E-cadherin (t = 17.736, P < 0.01) and p120-catenin (t = 16.136, P < 0.01) were decreased in the 20% cyclic stretching group. Compared with the stretching group, the levels of IL-6 (t = 11.818, P < 0.01) and TNF-α (t = 8.631, P < 0.01) decreased in the glutamine group; the levels of IL-10 (t = −3.203, P < 0.05) and the expression of E-cadherin (t = −13.567, P < 0.01) and p120-catenin (t = −10.013, P < 0.01) were increased in the glutamine group.Conclusions:High tidal volume mechanical ventilation and 20% cyclic stretching could cause VILI. Glutamine regulates VILI by improving cytokines and increasing the adherens junctions, protein E-cadherin and p120-catenin, to enhance the epithelial barrier function.

Hepatocyte specific ablation of β and γ‐catenin leads to altered hepatocyte polarity and microvillae association

Liver is the largest organ in a vertebrate body which is structurally and functionally complex and is considered second to the brain in its complexity. It is highly heterogeneous in nature and consists of various cell types. Hepatocytes are the predominant type of cells present in liver which is responsible for its many important functions including bile production, detoxification, cholesterol synthesis etc. Like any other epithelial cell, hepatocytes are polarized towards the apico‐basal axes. Polarization of hepatocytes is necessary for many functions and requires carefully orchestrated cooperation between cell adhesion molecules, extracellular matrix, cell junctions, cytoskeleton, and intracellular trafficking machinery. Loss of hepatocyte polarity is associated with various liver diseases including cholestasis.Recently, we have shown that absence of adherent junctional protein β and γ catenin in the liver leads to cholestatic liver disease. However, the molecular mechanism that leads to cholestasis in the absence of β‐ γ‐catenin is not known. Therefore, we developed a hepatocyte specific conditional knockout of β and γ catenin to further characterize their role in this disease. TEM and SEM analysis of β‐ γ‐catenin knockout liver showed aberrant tight junctional and adherent junctional structurers. Permeability assay confirmed that barrier function of tight junctions is completely lost in these mice resulting in mixing of the bile with the blood. We also found that bile canaliculi, which is apically located in hepatocytes designated for bile storage is strongly dilated with significant reduction in microvilli inside them. Further analysis revealed that loss of β‐ γ‐catenin leads to reduced expression of HNF4α target genes. Remarkably, we found that loss of HNF4α phenocopies β‐ γ‐catenin knockout liver causing aberrant tight and adherent junctional structure. Studies designed to address the molecular mechanism behind the regulation of β; γ‐catenin in maintaining hepatocyte polarity and association of β; γ‐catenin and HNF4α in promoting hepatocyte polarity are currently underway. Profound understanding of the regulation of hepatocyte polarity by both β ‐and γ‐catenin will be useful in the context of liver development and pathophysiology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diet-dependent changes in the intestinal DNA methylome after introduction of enteral feeding in preterm pigs.

To examine how enteral feeding affects the intestinal epigenome and gene expression just after preterm birth. Intestinal tissue from preterm pigs, modeling preterm infants, was collected at birth and 5days after gradual introduction of infant formula or bovine colostrum. The intestinal tissue was analyzed by reduced representation bisulfite sequencing and real-time qPCR. Relative to colostrum, formula increased bacterial epithelial adherence and lipopolysaccharide binding protein (LBP)expression, which was regulated by promoter methylation. Diet-dependent changes in DNA methylation and/or mRNA expression were related to innate immune response, hypoxia, angiogenesis and epithelial-mesenchymal transition pathways (e.g., TTC38,IL8,C3,HIF1A and VEGFR1). Epigenetic changes may mediate important effects of the first feeding on intestinal development in preterm neonates.

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection.

Development of a Microfluidic Open Interface with Flow Isolated Desorption Volume for the Direct Coupling of SPME Devices to Mass Spectrometry.

Technologies that efficiently integrate the sampling and sample preparation steps with direct introduction to mass spectrometry (MS), providing simple and sensitive analytical workflows as well as capabilities for automation, can generate a great impact in a vast variety of fields, such as in clinical, environmental, and food-science applications. In this study, a novel approach that facilitates direct coupling of Bio-SPME devices to MS using a microfluidic design is presented. This technology, named microfluidic open interface (MOI), which operates under the concept of flow-isolated desorption volume, consists of an open-to-ambient desorption chamber (V ≤ 7 μL) connected to an ionization source. Subsequently, compounds of interest are transported to the ionization source by means of the self-aspiration process intrinsic of these interfaces. Thus, any ionization technology that provides a reliable and constant suction, such as electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), or inductively coupled plasma ionization (ICP), can be hyphenated to MOI. Using this setup, the desorption chamber is used to release target compounds from the coating, while the isolation of the flow enables the ionization source to be continuously fed with solvent, all without the necessity of employment of additional valves. As a proof of concept, the design was applied to an ESI-MS/MS system for experimental validation. Furthermore, numerical simulations were undertaken to provide a detailed understanding of the fluid flow pattern inside the interface, then used to optimize the system for better efficiency. The analytical workflow of the developed Bio-SPME-MOI-MS setup consists of the direct immersion of SPME fibers into the matrix to extract/enrich analytes of interest within a short period of time, followed by a rinsing step with water to remove potentially adhering proteins, salts, and/or other interfering compounds. Next, the fiber is inserted into the MOI for desorption of compounds of interest. Finally, the volume contained in the chamber is drained and moved toward the electrospray needle for ionization and direct introduction to MS. Aiming to validate the technology, the fast determination of selected immunosuppressive drugs (e.g., tacrolimus, cyclosporine, sirolimus, and everolimus) from 100 μL of whole blood was assessed. Limits of quantitation in the subppb range were obtained for all studied compounds. Good linearity (r2 ≥ 0.99) and excellent precision, with (8%) and without (14%) internal standard correction, were attained.

Greetings from the endoplasmic reticulum (ER): escaping ER thiol isomerases regulate thrombosis

Greetings from the endoplasmic reticulum (ER): escaping ER thiol isomerases regulate thrombosis

1H, 15N, and 13C resonance assignments of the third domain from the S. aureus innate immune evasion protein Eap.

Staphylococcus aureus is a widespread and persistent pathogen of humans and livestock. The bacterium expresses a wide variety of virulence proteins, many of which serve to disrupt the host's innate immune system from recognizing and clearing bacteria with optimal efficiency. The extracellular adherence protein (Eap) is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement system (Woehl et al in J Immunol 193:6161-6171, 2014) and Neutrophil Serine Proteases (NSPs) (Stapels et al in Proc Natl Acad Sci USA 111:13187-13192, 2014). The third domain of Eap, Eap3, is an ~ 11kDa protein that was recently shown to bind complement component C4b (Woehl et al in Protein Sci 26:1595-1608, 2017) and therefore play an essential role in inhibiting the classical and lectin pathways of complement (Woehl et al in J Immunol 193:6161-6171, 2014). Since structural characterization of Eap3 is still incomplete, we acquired a series of 2D and 3D NMR spectra of Eap3 in solution. Here we report the backbone and side-chain 1H, 15N, and 13C resonance assignments of Eap3 and its predicted secondary structure via the TALOS-N server. The assignment data have been deposited in the BMRB data bank under accession number 27087.

Epithelial-Mesenchymal Transition (EMT) and Prostate Cancer.

Typically the normal epithelial cells are a single layer, held tightly by adherent proteins that prevent the mobilization of the cells from the monolayer sheet. During prostate cancer progression, the epithelial cells can undergo epithelial-mesenchymal transition or EMT, characterized by morphological changes in their phenotype from cuboidal to spindle-shaped. This is associated with biochemical changes in which epithelial cell markers such as E-cadherin and occludins are down-regulated, which leads to loss of cell-cell adhesion, while mesenchymal markers such as vimentin and N-cadherin are up-regulated, thereby allowing the cells to migrate or metastasize to different organs. The EMT transition can be regulated directly and indirectly by multiple molecular mechanisms including growth factors and cytokines such as transforming growth factor-beta (TGF-β), epidermal growth factor (EGF) and insulin-like growth factor (IGF), and signaling pathways such as mitogen-activated protein kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3K). This signaling subsequently induces expression of various transcription factors like Snail, Twist, Zeb1/2, that are also known as master regulators of EMT. Various markers associated with EMT have been reported in prostate cancer patient tissue as well as a possible association with health disparities. There has been consideration to therapeutically target EMT in prostate cancer patients by targeting the EMT signaling pathways.

Decreased Expression of Claudin 1, 3, 4, 5 and 7: A New Prognostic Marker in Colon Carcinoma

Background and objective: Claudins are members of a large family of tissue specific adherent proteins which have been suggested as tumor markers. In this study we analyzed the protein expression of claudin 1, 3, 4, 5 and 7, their clinical significance and association with tumor growth pattern in colon carcinoma. Methods: Immunohistochemical staining (IHC) was used to detect the expression of claudin 1, 3, 4, 5 and 7 in samples diagnosed with colon carcinoma as well as the adjacent normal mucosa. Complexity index (CI) was calculated using images of cytokeratin-8 stained slides of the tumours using Photoshop CS, Fovea Pro, and Image J computer programs. The results from the IHC and CI were correlated to clinicopathological parameters as well as 5-years survival of the patients diagnosed with colon carcinoma. Results: Significantly high staining intensity was observed in normal mucosa as compared to colon cancer tissue for claudin 4 (p=0.031) and 7 (p=0.011) and number of stained cells for claudin 4 (p=0.001). A significant association was also observed between weaker expression at the invasive front of the tumor tissue and heterogenous expression of claudin 1 (p=0.000), claudin 3(p=0.003), claudin 5(p=0.001) and claudin 7(p=0.000). There was no significant correlation between the expression of claudins, CI and clinicopathological parameters. Similarly, no significant association was found between claudin expression and 5-year survival of the patients diagnosed with colon carcinoma. Conclusion: Altered expression of claudin 1, 3, 4, 5 and 7 in colon carcinoma cells may play a promoting role in colon carcinoma development and is inversely proprtional to higher expression at invasive front of the tumor. Claudin protein expression can be used as a tumor marker since the expression generally is weaker in tumor tissue compared to the normal mucosa.

Genotypic variations between wild-type and small colony variant of Staphylococcus aureus in prosthetic valve infectious endocarditis: a comparative genomic and transcriptomic analysis

Staphylococcus aureus small colony variants (SCVs) can cause persistent infections. However, the genomes and transcriptomes of S. aureus SCVs remain poorly understood. A pair of isogenic wild-type and SCV methicillin-resistant S. aureus (MRSA) strains (IE1 and IE2, respectively) were isolated from a patient with prosthetic valve infectious endocarditis. The SCV strain IE2 grew more slowly than the wild-type strain, and serum killing and mouse lethality assays revealed that the virulence of SCV strain IE2 was decreased. Whole-genome sequencing of the SCV and wild-type strains revealed 15 mutations in nine genes associated with metabolism, virulence and DNA repair, including serine/threonine-protein kinase PrkC (prkC), glycerol-3-phosphate acyltransferase (plsY), 2-deoxyribose-5-phosphate aldolase (deoC), extracellular adherence protein (eap), iron compound ABC uptake transporter substrate-binding protein (sstD), RecU Holliday junction resolvase (recU), excinuclease ABC subunit B (uvrB), type I restriction–modification system, M subunit (hsdM) and smooth muscle caldesmon. Sequencing of RNA transcripts revealed that expression levels of 321 genes were upregulated and 582 genes were downregulated in SCV strain IE2 compared with IE1. Most of the differentially expressed genes were involved in metabolism. Expression levels of several genes involved in the pathways to which plsY, deoC, eap and sstD belonged were changed, associated with the metabolism and virulence of S. aureus. In conclusion, the reduced growth rate and decreased virulence of MRSA SCV strains may be related to mutations in and downregulation of genes associated with metabolism and virulence, especially plsY, deoC, eap and sstD.

Stenotrophomonas maltophilia Serine Protease StmPr1 Induces Matrilysis, Anoikis, and Protease-Activated Receptor 2 Activation in Human Lung Epithelial Cells.

Stenotrophomonas maltophilia is an emerging, opportunistic nosocomial pathogen that can cause severe disease in immunocompromised individuals. We recently identified the StmPr1 and StmPr2 serine proteases to be the substrates of the Xps type II secretion system in S. maltophilia strain K279a. Here, we report that a third serine protease, StmPr3, is also secreted in an Xps-dependent manner. By constructing a panel of protease mutants in strain K279a, we were able to determine that StmPr3 contributes to the previously described Xps-mediated rounding and detachment of cells of the A549 human lung epithelial cell line as well as the Xps-mediated degradation of fibronectin, fibrinogen, and the cytokine interleukin-8 (IL-8). We also determined that StmPr1, StmPr2, and StmPr3 account for all Xps-mediated effects toward A549 cells and that StmPr1 contributes the most to Xps-mediated activities. Thus, we purified StmPr1 from the S. maltophilia strain K279a culture supernatant and evaluated the protease's activity toward A549 cells. Our analyses revealed that purified StmPr1 behaves more similarly to subtilisin than to trypsin. We also determined that purified StmPr1 likely induces cell rounding and detachment of A549 cells by targeting cell integrin-extracellular matrix connections (matrilysis) as well as adherence and tight junction proteins for degradation. In this study, we also identified anoikis as the mechanism by which StmPr1 induces the death of A549 cells and found that StmPr1 induces A549 IL-8 secretion via activation of protease-activated receptor 2. Altogether, these results suggest that the degradative and cytotoxic activities exhibited by StmPr1 may contribute to S. maltophilia pathogenesis in the lung by inducing tissue damage and inflammation.

Highly sensitive and accurate detection of Vibrio cholera O1 OmpW gene by fluorescence DNA biosensor based on gold and magnetic nanoparticles

Abstract Ultrasensitive DNA based fluorescent assay is used for the determination of Vibrio cholera O1 OmpW gene. OmpW as a surface adherence protein plays a major role in virulence of bacteria with high gene expression. In the present study, two nanoparticles including gold nanoparticles (AuNPs) and magnetic nanoparticles (MNPs) were used. MNPs were functionalized with the first target-specific DNA probe1, which can recognize one end of the target DNA sequence (t DNA) and the AuNPs were functionalized with the second target-specific DNA FAM-probe 2 which can recognize the other end of the target gene. After hybridization of probes with the target DNA, the sandwich structure was formed as MNPs-probe1-target DNA-FAM-probe2-AuNPs by applying the magnetic field to separate the sandwich complex from no hybridized materials. Dithiothreitol (DTT) was used to decompose the sandwich structures and release the probes from target DNA and nanoparticles. Then, fluorescence emission of the released FAM probe 2 on surface AuNPs was detected by fluorescence spectrophotometry. The resulting nanomaterial system demonstrated excellent sensitivity (LOD = 2.34 ng mL−1) with good linear relationship (R2 = 0.9989) between target DNA and great selectivity. In conclusion, the present method has a good potential as a rapid detector of bacteria and other infection.

Shiga Toxin-Producing Serogroup O91 Escherichia coli Strains Isolated from Food and Environmental Samples.

Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identical or nearly identical to those of the clinical O91:H21 strains that have caused severe diseases. Multilocus sequence typing and clustered regularly interspaced short palindromic repeat analyses showed that the O91:H21 strains clustered within the STEC 1 clonal group but the other O91 serotype strains were phylogenetically diverse.IMPORTANCE This study showed that food and environmental O91:H21 strains have similar genotypic profiles and Shiga toxin subtypes and are phylogenetically related to the O91:H21 strains that have caused hemolytic-uremic syndrome, suggesting that these strains may also have the potential to cause severe illness.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

Backbone and side-chain 1H, 15N, and 13C resonance assignments of a novel Staphylococcal inhibitor of myeloperoxidase

The bacterium Staphylococcus aureus produces an array of anti-inflammatory molecules that prevent the innate immune system from recognizing it as a pathogen and clearing it from the host. In the acute phase of inflammation, our immune system relies on neutrophils to clear invading bacteria. Recently, novel classes of secreted proteins from S. aureus, including the Extracellular Adherence Protein (EAP) family (Stapels et al., Proc Natl Acad Sci USA 111:13187-13192, 2014) and the Staphylococcal Peroxidase Inhibitor (SPIN), (unpublished work) have been identified as highly selective inhibitors acting on Neutrophil Serine Proteases (NSPs) and myeloperoxidase (MPO) respectively. SPIN is a protein found only in Staphylococci, with no sequence homology to any known proteins. Solution NMR structural studies of SPIN are therefore expected to provide a deeper understanding of its interaction with MPO. In this study, we report the backbone and side-chain 1H, 15N, and 13C resonance assignments of SPIN. Furthermore, using the chemical shifts of these resonances, we predicted the secondary structure of SPIN in solution via the TALOS-N server. The assignment data has been deposited in the BMRB data bank under Accession No. 27069.

A new data processing routine facilitating the identification of surface adhered proteins from bacterial conditioning films via QCM-D/MALDI-ToF/MS.

Conditioning films are an important factor in the initiation and development of microbial biofilms, which are the leading cause of chronic infections associated with medical devices. Here, we analyzed the protein content of conditioning films formed after exposure to supernatants of cultures of the human pathogen Pseudomonas aeruginosa PAO1. Adhesion of substances from the supernatant was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) sensor chips modified with the commonly used implant material titanium dioxide (TiO2). Attached proteins were identified after on-chip digestion using matrix-assisted laser desorption/ionization (MALDI) time of flight (ToF) mass spectrometry (MS), and a new data processing tool consisting of an XML-database with theoretical tryptic peptides of every PAO1 protein and PHP scripts. Sub-databases containing only proteins, that we found in all replicates, were created and used for MS/MS precursor selection. The obtained MS/MS peaklists were then matched against theoretical fragmentations of the expected peptide sequences to verify protein identification. Using this approach we were able to identify 40 surface-associated proteins. In addition to extracellular proteins such as adhesins, a number of intra-cellular proteins were identified which may be involved in conditioning film formation, suggesting an as-yet unidentified role for these proteins, possibly after cell lysis. Graphical Abstract Flowchart of the method.

Anesthesia and Surgery Impair Blood-Brain Barrier and Cognitive Function in Mice.

Blood–brain barrier (BBB) dysfunction, e.g., increase in BBB permeability, has been reported to contribute to cognitive impairment. However, the effects of anesthesia and surgery on BBB permeability, the underlying mechanisms, and associated cognitive function remain largely to be determined. Here, we assessed the effects of surgery (laparotomy) under 1.4% isoflurane anesthesia (anesthesia/surgery) for 2 h on BBB permeability, levels of junction proteins and cognitive function in both 9- and 18-month-old wild-type mice and 9-month-old interleukin (IL)-6 knockout mice. BBB permeability was determined by dextran tracer (immunohistochemistry imaging and spectrophotometric quantification), and protein levels were measured by Western blot and cognitive function was assessed by using both Morris water maze and Barnes maze. We found that the anesthesia/surgery increased mouse BBB permeability to 10-kDa dextran, but not to 70-kDa dextran, in an IL-6-dependent and age-associated manner. In addition, the anesthesia/surgery induced an age-associated increase in blood IL-6 level. Cognitive impairment was detected in 18-month-old, but not 9-month-old, mice after the anesthesia/surgery. Finally, the anesthesia/surgery decreased the levels of β-catenin and tight junction protein claudin, occludin and ZO-1, but not adherent junction protein VE-cadherin, E-cadherin, and p120-catenin. These data demonstrate that we have established a system to study the effects of perioperative factors, including anesthesia and surgery, on BBB and cognitive function. The results suggest that the anesthesia/surgery might induce an age-associated BBB dysfunction and cognitive impairment in mice. These findings would promote mechanistic studies of postoperative cognitive impairment, including postoperative delirium.

Water Infused Surface Protection as an Active Mechanism for Fibrin Sheath Prevention in Central Venous Catheters.

Protein adhesion in central venous catheters (CVCs) leads to fibrin sheath formation, the precursor to thrombotic and biofilm-related CVC failures. Advances in material properties and surface coatings do not completely prevent fibrin sheath formation and post-formation treatment options are limited and expensive. We propose water infused surface protection (WISP), an active method for prevention of fibrin sheath formation on CVCs, which creates a blood-free boundary layer on the inner surface of the CVC, limiting blood contact with the CVC lumen wall. A hollow fiber membrane (HFM) in a benchtop device served as a CVC testing model to demonstrate the WISP concept. Porcine blood was pumped through the HFM while phosphate buffered saline (PBS) was infused through the HFM wall, creating the WISP boundary layer. Protein adherences on model CVC surfaces were measured and imaged. Analytical and finite volume lubrication models were used to justify the assumption of a blood-free boundary layer. We found a 92.2% reduction in average adherent protein density when WISP is used, compared with our model CVC without WISP flow. Lubrication models matched our experimental pressure drop measurements suggesting that a blood-free boundary layer was created. The WISP technique also provides a novel strategy for drug administration for biofilm treatment. Reduction in adherent protein indicates a restriction on long-term fibrin sheath and biofilm formation making WISP a promising technology which improves a wide range of vascular access treatments.