scholarly journals Regulated cleavage of glycan strands by the murein hydrolase SagB in S. aureus involves a direct interaction with LyrA (SpdC)

2021 ◽  
Author(s):  
Stephanie Willing ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Protein Trafficking ◽  
Transmembrane Domain ◽  
Protein A ◽  
Direct Interaction ◽  
Staphylococcal Protein A ◽  
Lipid Ii ◽  
Molecular Ruler ◽  
Membrane Bound ◽  
Insight Into

LyrA (SpdC), a homologue of eukaryotic CAAX proteases that act on prenylated substrates, has been implicated in the assembly of several pathways of the envelope of Staphylococcus aureus. We described earlier the Lysostaphin resistance (Lyr) and Staphylococcal protein A display (Spd) phenotypes associated with loss of the lyrA (spdC) gene. However, a direct contribution to the assembly of pentaglycine crossbridges, the target of lysostaphin cleavage in S. aureus peptidoglycan, or of Staphylococcal protein A attachment to peptidoglycan could not be attributed directly to LyrA (SpdC). These two processes are catalyzed by the Fem factors and Sortase A, respectively. To gain insight into the function of LyrA (SpdC), here we use affinity chromatography and LC-MS/MS analysis and report that LyrA interacts with SagB. SagB cleaves glycan strands of peptidoglycan to achieve physiological length. Similar to sagB peptidoglycan, lyrA peptidoglycan contains extended glycan strands. Purified lyrA peptidoglycan can still be cleaved to physiological length by SagB in vitro. LyrA does not modify or cleave peptidoglycan, it also does not modify or stabilize SagB. The membrane bound domain of LyrA is sufficient to support SagB activity but predicted ‘CAAX enzyme’ catalytic residues in this domain are dispensable. We speculate that LyrA exerts its effect on bacterial prenyl substrates, specifically undecaprenol-bound peptidoglycan substrates of SagB, to help control glycan length. Such an activity also explains the Lyr and Spd phenotypes observed earlier. IMPORTANCE Peptidoglycan is assembled on the trans side of the plasma membrane from lipid II precursors into glycan chains that are crosslinked at stem peptides. In S. aureus, SagB, a membrane-associated N-acetylglucosaminidase, cleaves polymerized glycan chains to their physiological length. Deletion of sagB is associated with longer glycan strands in peptidoglycan, altered protein trafficking and secretion in the envelope, and aberrant excretion of cytosolic proteins. It is not clear whether SagB, with its single transmembrane segment, serves as the molecular ruler of glycan chains or whether other factors modulate its activity. Here, we show that LyrA (SpdC), a protein of the CAAX type II prenyl endopeptidase family, modulates SagB activity via interaction though its transmembrane domain.

scholarly journals An In Vitro Study of the Effect of Viburnum opulus Extracts on Key Processes in the Development of Staphylococcal Infections

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1758
Author(s):  
Urszula Wójcik-Bojek ◽  
Joanna Rywaniak ◽  
Przemysław Bernat ◽  
Anna Podsędek ◽  
Dominika Kajszczak ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Protein A ◽  
In Vitro Study ◽  
Alternative Methods ◽  
Staphylococcal Protein A ◽  
Viburnum Opulus ◽  
Staphylococcal Infections ◽  
Diet Supplements ◽  
Drug Resistant Strains

Staphylococcus aureus is still one of the leading causes of both hospital- and community-acquired infections. Due to the very high percentage of drug-resistant strains, the participation of drug-tolerant biofilms in pathological changes, and thus the limited number of effective antibiotics, there is an urgent need to search for alternative methods of prevention or treatment for S. aureus infections. In the present study, biochemically characterized (HPLC/UPLC–QTOF–MS) acetonic, ethanolic, and water extracts from fruits and bark of Viburnum opulus L. were tested in vitro as diet additives that potentially prevent staphylococcal infections. The impacts of V. opulus extracts on sortase A (SrtA) activity (Fluorimetric Assay), staphylococcal protein A (SpA) expression (FITC-labelled specific antibodies), the lipid composition of bacterial cell membranes (LC-MS/MS, GC/MS), and biofilm formation (LIVE/DEAD BacLight) were assessed. The cytotoxicity of V. opulus extracts to the human fibroblast line HFF-1 was also tested (MTT reduction). V. opulus extracts strongly inhibited SrtA activity and SpA expression, caused modifications of S. aureus cell membrane, limited biofilm formation by staphylococci, and were non-cytotoxic. Therefore, they have pro-health potential. Nevertheless, their usefulness as diet supplements that are beneficial for the prevention of staphylococcal infections should be confirmed in animal models in the future.

scholarly journals The Synergistic Effect of Nicotine and Staphylococcus aureus on Peri-Implant Infections

2021 ◽  
Vol 9 ◽  
Author(s):  
Yao Hu ◽  
Wen Zhou ◽  
Chengguang Zhu ◽  
Yujie Zhou ◽  
Qiang Guo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Synergistic Effects ◽  
Protein A ◽  
Treated Group ◽  
Combination Group ◽  
Staphylococcal Protein A ◽  
Receptor Activator ◽  
And Staphylococcus Aureus

Smoking is considered a key risk factor for implant survival; however, how it interacts with the pathogens in peri-implant infections is not clear. Here, we identified that nicotine, the key component of cigarette smoking, can interact with Staphylococcus aureus and synergistically induce peri-implant infections in a rat osteolysis model. The nicotine–S. aureus combination group increased the gross bone pathology, osteolysis, periosteal reactions, and bone resorption compared to the nicotine or S. aureus single treated group (p < 0.05). Nicotine did not promote the proliferation of S. aureus both in vitro and in vivo, but it can significantly upregulate the expression of staphylococcal protein A (SpA), a key virulence factor of S. aureus. The nicotine–S. aureus combination also synergistically activated the expression of RANKL (receptor activator of nuclear factor-kappa B ligand, p < 0.05) to promote the development of peri-implant infections. The synergistic effects between nicotine and S. aureus infection can be a new target to reduce the peri-implant infections.

scholarly journals Lymphocyte transformation induced by autologous cells. V. generation of immunologic memory and specificity during the autologous mixed lymphocyte reaction

1977 ◽  
Vol 146 (6) ◽  
pp. 1833-1838 ◽  
Author(s):  
ME Weksler ◽  
R Kozak
Keyword(s):  
T Lymphocytes ◽  
Lymphocyte Proliferation ◽  
Mixed Lymphocyte Reaction ◽  
Protein A ◽  
Lymphocyte Transformation ◽  
Cell Mediated Immunity ◽  
Staphylococcal Protein A ◽  
Autologous Mixed Lymphocyte Reaction ◽  
Proliferation In Vitro

Lymphocyte proliferation in vitro may follow antigen recognition and serve as a correlate of cell-mediated immunity. Lymphocyte proliferation can also be simulated by nonimmune mechanisms as, for example, following culture with plant lectin, lipopolysaccharides, or staphylococcal protein A (1). The autologous mixed lymphocyte reaction (MLR) refers to the proliferation of T lymphocytes cultured with autologous mon-T lymphocytes (2,3). The purpose of this study was to determine whether lymphocyte proliferation in the autologous MLR results from immune or nonimmune mechanisms. We have shown that the autologous MLR has two classical attributes of an immune phenomenon: memory and specificity.

Changes in Circulating Immune Complexes in Tumour Patient Serum after in Vitro or ex Vivo Affinity Chromatography of Blood Plasma or whole Blood over Immunoglobulinbinding Staphylococcal Protein A-Sepharose

1984 ◽  
Vol 7 (1) ◽  
pp. 23-26 ◽  
Author(s):  
L. Håkansson ◽  
J. Hed ◽  
L. Baldetorp ◽  
S. Eneström ◽  
S. Jonsson ◽  
...  
Keyword(s):  
Blood Plasma ◽  
Immune Complexes ◽  
Whole Blood ◽  
Ex Vivo ◽  
Protein A ◽  
Circulating Immune Complexes ◽  
Staphylococcal Protein A ◽  
Lower Affinity ◽  
Tumour Patient

Circulating immune complexes (CIC) were determined in tumour patient sera using three methods. One is based on PEG-precipitation, one on C1q-reactivity, and one on protein A-reactivity. About 25-30% of the sera were positive in at least one of the tests. Incubation of serum with protein A-Sepharose in vitro removed PEG-precipitable CIC from most sera, whereas C1q-reactive CICs had a much lower affinity to protein A. The protein A-reactive complexes showed considerable variation in their binding to protein A-Sepharose, and in some sera the amount of these CICs was actually increased. Similar changes in protein A-reactive CIC were also found during ex vivo treatment of tumour patients with immune adsorption. It is proposed that the binding of immune complexes to protein A can result in remodelling of protein A itself. Results from ultracentrifugation and fractionated PEG-precipitation support this hypothesis.

PRTX-100 Inhibits Platelet Phagocytosis In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1081-1081 ◽  
Author(s):  
Chris Yatko ◽  
Christopher Herrem ◽  
Samia Siddiqui ◽  
Victor S. Sloan
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Option ◽  
Protein A ◽  
Flow Cytometric Analysis ◽  
Human Platelets ◽  
Staphylococcal Protein A ◽  
Human Monocytes ◽  
Peripheral Blood Mononuclear ◽  
Vitro Assay

Abstract Background: In idiopathic thrombocytopenic purpura (ITP), autoantibodies bind to platelets which are then phagocytosed by monocytes/macrophages and removed by the reticuloendothelial system. PRTX-100 (Staphylococcal protein A) is being investigated for the treatment of ITP. Objective: To assess the effect of PRTX-100 on phagocytosis of platelets in an in vitro assay. Methods: Human monocytes were isolated from whole blood peripheral blood mononuclear cells (PBMCs) by adherence and cultured for 6 days in RPMI + 5% human serum. 48 hours prior to phagocytosis assay, PRTX-100 was added at 250, 25, and 2.5ng/ml. Human platelets were labeled with a fluorescent (PerCP) lipophilic dye and opsonized with an antibody to MHC Class I (W632). 2×10−5 monocytes were co-cultured with 2×10−7 labeled platelets for 1 hour at 37 ° C. All conditions were performed in triplicate. After an hour, phycoerythrin (PE) labeled anti-CD61 antibody was added to assess surface bound platelets versus ingested platelets. Phagocytosis was determined by flow cytometric analysis. The monocyte population was gated upon by forward and side scatter properties, then verified by staining with CD14-FITC. Percent phagocytosis was calculated as the fraction of ingested platelets (PerCP +/CD61−) to the total PerCP population (PerCP +/CD61−) + ( PerCP+/CD61+) within the gated monocyte population. Results: PRTX-100 inhibits the phagocytosis of W632 opsonized platelets by human monocytes. Phagocytosis of W632 opsonized platelets was 40%, while phagocytosis in the presence of PRTX-100 at concentrations of 250, 25, and 2.5ng/ml was 18.3%, 23%, and 24.3%, respectively. Phagocytosis at 250ng/ml and 25ng/ml was significantly different from control phagocytosis with p values of 0.014 and 0.001 respectively by Student’s t test. Conclusions: PRTX-100 inhibits the phagocytosis of platelets by monocytes, the effector limb of ITP. Prevention of platelet phagocytosis is an important treatment goal in ITP. PRTX-100 has been shown to be generally safe and well-tolerated in a phase I study in healthy volunteers (J Clin Pharmacol, in press). PRTX -100 is a promising therapeutic option for ITP and deserves further study. Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets

scholarly journals The transmembrane domain of diphtheria toxin improves molecular conjugate gene transfer

1997 ◽  
Vol 321 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Krishna J. FISHER ◽  
James M. WILSON
Keyword(s):  
Gene Transfer ◽  
Diphtheria Toxin ◽  
Transmembrane Domain ◽  
Transfection Efficiency ◽  
Bacterial Protein ◽  
Protein Fusion ◽  
Membrane Perturbation ◽  
Membrane Bound ◽  
Rna Transcript

Vectors based on the formation of a soluble DNA–polycation complex are being developed for the treatment of human diseases. These complexes are rapidly taken up by receptor-mediated endocytosis, but are inefficiently delivered to the nucleus owing to entrapment in membrane-bound vesicles. In this study we introduced the transmembrane domain of diphtheria toxin into a DNA–polycation conjugate complex in an effort to increase gene transfer by membrane perturbation. The transmembrane domain of diphtheria toxin was expressed in Escherichia coli as a maltose-binding protein fusion and chemically coupled to high-molecular-mass poly-l-lysine. Incorporation of this conjugate into a traditional complex formed with a luciferase-containing plasmid with an asialo-orosomucoid–polycation conjugate significantly increased transfection efficiency in vitro in a manner proportional to the amount of diphtheria toxin incorporated. The delivery of luciferase RNA transcript was similarly increased when complexed with similar polycation conjugates. This study uses the structural biology of a bacterial protein to improve polycation-based gene delivery.

scholarly journals Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain

2021 ◽  
Author(s):  
Ian Daniel Wolff ◽  
Jeremy Alden Hollis ◽  
Sarah Marie Wignall
Keyword(s):  
Adaptor Protein ◽  
Direct Interaction ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Interaction Domain ◽  
Microtubule Binding ◽  
C Elegans ◽  
Insight Into

During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force. Instead, the kinesin-12 family motor KLP-18 performs this function. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.

scholarly journals Prevalence of fosfomycin resistance and gene mutations in clinical isolates of methicillin-resistant Staphylococcus aureus

2020 ◽  
Author(s):  
Yi-Chien Lee ◽  
Pao-Yu Chen ◽  
Jann-Tay Wang ◽  
Shan-Chwen Chang
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Agents ◽  
Genetic Relatedness ◽  
Protein A ◽  
Gene Mutations ◽  
Resistance Rate ◽  
Staphylococcal Protein A ◽  
Methicillin Resistant ◽  
Fosfomycin Resistance

Abstract Background: Fosfomycin exhibits excellent in vitro activity against multidrug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Increasing fosfomycin resistance among clinical MRSA isolates was reported previously, but little is known about the genetic mechanisms of fosfomycin resistance.Methods: All MRSA isolates, collected in 2002 and 2012 by the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program, were used in this study. Susceptibility to various antimicrobial agents, including fosfomycin, was determined by broth microdilution. Genetic determinants of fosfomycin resistance, including fosB carriage and murA, glpT and uhpT mutations, were investigated using PCR and sequencing of amplicons. Staphylococcal protein A (spa) typing was also performed to determine the genetic relatedness of MRSA isolates.Results: A total of 969 MRSA strains, 495 in the year 2002 and 474 in the year 2012, were analyzed. The overall in vitro susceptibility was 8.2% to erythromycin, 18.0% to clindamycin, 29.0% to tetracycline, 44.6% to ciprofloxacin, 57.5% to trimethoprim/sulfamethoxazole, 86.9% to rifampicin, 92.9% to fosfomycin and 100% to linezolid and vancomycin. A significant increase in the fosfomycin resistance rate was observed from 3.4% in 2002 to 11.0% in 2012. Of 68 fosfomycin-resistant MRSA isolates, 12 harbored the fosB gene, and expression of murA, uhpT, and glpT mutations was noted in 11, 59, and 66 isolates, respectively. Combination of mutations of uhpT and glpT genes (58 isolates) was the most prevalent resistant mechanism. The vast majority of the fosfomycin-resistant MRSA isolates belonged to spa type t002.Conclusions: An increased fosfomycin resistance rate of MRSA isolates was observed in our present study, mostly due to mutations in the glpT and uhpT genes. Clonal spread probably contributed to the increased fosfomycin resistance.

Regeneration of isthmic tissue is the result of a specific and direct interaction between rhombomere 1 and midbrain

Development ◽  
1999 ◽  
Vol 126 (18) ◽  
pp. 3981-3989 ◽  
Author(s):  
C. Irving ◽  
I. Mason
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Small Proportion ◽  
Direct Interaction ◽  
Good Evidence ◽  
Fibroblast Growth ◽  
Fibroblast Growth Factor 8 ◽  
Insight Into

The midbrain-hindbrain boundary, or isthmus, is the source of signals that are responsible for regional specification of both the midbrain and anterior hindbrain. Fibroblast growth factor 8 (Fgf8) is expressed specifically at the isthmus and there is now good evidence that it forms at least part of the patterning signal. In this study, we use Fgf8 as a marker for isthmic cells to examine how interactions between midbrain and hindbrain can regenerate isthmic tissue and, thereby, gain insight into the normal formation and/or maintenance of the isthmus. We show that Fgf8-expressing tissue with properties of the isthmic organiser is generated when midbrain and rhombomere 1 tissue are juxtaposed but not when midbrain contacts any other rhombomere. The use of chick/quail chimeras shows that the isthmic tissue is largely derived from rhombomere 1. In a few cases a small proportion of the Fgf8-positive cells were of midbrain origin but this appears to be the result of a local respecification to a hindbrain phenotype, a process mimicked by ectopic FGF8. Studies in vitro show that the induction of Fgf8 is the result of a direct planar interaction between the two tissues and involves a diffusible signal.

scholarly journals Efficacy of Azithromycin in a Mouse Pneumonia Model against Hospital-Acquired Methicillin-Resistant Staphylococcus aureus

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Yu Yamashita ◽  
Kentaro Nagaoka ◽  
Hiroki Kimura ◽  
Masaru Suzuki ◽  
Satoshi Konno ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Lavage Fluid ◽  
Suppressive Effect ◽  
The Other ◽  
Staphylococcal Protein A ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACT The use of macrolides against pneumonia has been reported to improve survival; however, little is known about their efficacy against methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. In this study, we investigated the effect of azithromycin (AZM) and compared it with that of vancomycin (VCM) and daptomycin (DAP) in a murine model of MRSA pneumonia. Mice were infected with MRSA by intratracheal injection and then treated with AZM, VCM, or DAP. The therapeutic effect of AZM, in combination or not with the other drugs, was compared in vivo, whereas the effect of AZM on MRSA growth and toxin mRNA expression was evaluated in vitro. In vivo, the AZM-treated group showed significantly longer survival and fewer bacteria in the lungs 24 h after infection than the untreated group, as well as the other anti-MRSA drug groups. No significant decrease in cytokine levels (interleukin-6 [IL-6] and macrophage inflammatory protein-2 [MIP-2]) in bronchoalveolar lavage fluid or toxin expression levels (α-hemolysin [Hla] and staphylococcal protein A [Spa]) was observed following AZM treatment. In vitro, AZM suppressed the growth of MRSA in late log phase but not in stationary phase. No suppressive effect against toxin production was observed following AZM treatment in vitro. In conclusion, contrary to the situation in vitro, AZM was effective against MRSA growth in vivo in our pneumonia model, substantially improving survival. The suppressive effect on MRSA growth at the initial stage of pneumonia could underlie the potential mechanism of AZM action against MRSA pneumonia.

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