scholarly journals O-acetylation controls the glycosylation of bacterial serine-rich repeat glycoproteins.

2020 ◽  
pp. jbc.RA120.016116
Author(s):  
Ravin Seepersaud ◽  
Alexander C. Anderson ◽  
Barbara A. Bensing ◽  
Biswa P Choudhury ◽  
Anthony J. Clarke ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Regulatory Mechanism ◽  
Human Platelets ◽  
Bacterial Virulence ◽  
Wild Type ◽  
Post Translational Modification ◽  
Gram Positive Bacteria ◽  
Wide Range ◽  
Platelet Binding

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec (aSec) system. While all aSec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin GspB. Since these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues, and that O-acetylation prevented Glc deposition. Whereas streptococci expressing non-acetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to wild-type levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, since O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.

scholarly journals Subinhibitory Antimicrobial Concentrations: A Review of In Vitro and In Vivo Data

1992 ◽  
Vol 3 (4) ◽  
pp. 193-201 ◽  
Author(s):  
George G Zhanel ◽  
Daryl J Hoban ◽  
Godfrey KM Harding
Keyword(s):  
Antimicrobial Activity ◽  
Animal Studies ◽  
Molecular Level ◽  
Bacterial Virulence ◽  
Antibacterial Effects ◽  
Wide Range ◽  
Immune Defences

Antimicrobial activity is not an ‘all or none’ effect. An increase in the rate and extent of antimicrobial action is usually observed over a wide range of antimicrobial concentrations. Subinhibitory antimicrobial concentrations are well known to produce significant antibacterial effects, and various antimicrobials at subinhibitory concentrations have been reported to inhibit the rate of bacterial growth. Bacterial virulence may be increased or decreased by subinhibitory antimicrobial concentrations by changes in the ability of bacteria to adhere to epithelial cells or by alterations in bacterial susceptibility to host immune defences. Animal studies performed in rats, hamsters and rabbits demonstrate decreased bacterial adherence, reduced infectivity and increased survival of animals treated with subinhibitory antimicrobial concentrations compared to untreated controls. The major future role of investigation of subinhibitory antimicrobial concentrations will be to define more fully, at a molecular level, how antimicrobials exert their antibacterial effects.

scholarly journals Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits

2021 ◽  
Vol 17 (3) ◽  
pp. e1009116
Author(s):  
Allison N. Dammann ◽  
Anna B. Chamby ◽  
Andrew J. Catomeris ◽  
Kyle M. Davidson ◽  
Hervé Tettelin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amniotic Fluid ◽  
Deletion Strain ◽  
Growth Defect ◽  
Wild Type ◽  
Human Amniotic Fluid ◽  
Wide Range ◽  
Pregnant Mice ◽  
Group B

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

scholarly journals Targeting mutant p53-expressing tumours with a T cell receptor-like antibody specific for a wild-type antigen

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lionel Low ◽  
Angeline Goh ◽  
Joanna Koh ◽  
Samantha Lim ◽  
Cheng-I Wang
Keyword(s):  
Cell Receptor ◽  
Class I ◽  
Human Antibody ◽  
Mutant P53 ◽  
Wild Type ◽  
Class I Mhc ◽  
Type Antigen ◽  
Wide Range

AbstractAccumulation of mutant p53 proteins is frequently found in a wide range of cancers. While conventional antibodies fail to target intracellular proteins, proteosomal degradation results in the presentation of p53-derived peptides on the tumour cell surface by class I molecules of the major histocompatibility complex (MHC). Elevated levels of such p53-derived peptide-MHCs on tumour cells potentially differentiate them from healthy tissues. Here, we report the engineering of an affinity-matured human antibody, P1C1TM, specific for the unmutated p53125-134 peptide in complex with the HLA-A24 class I MHC molecule. We show that P1C1TM distinguishes between mutant and wild-type p53 expressing HLA-A24+ cells, and mediates antibody dependent cellular cytotoxicity of mutant p53 expressing cells in vitro. Furthermore, we show that cytotoxic PNU-159682-P1C1TM drug conjugates specifically inhibit growth of mutant p53 expressing cells in vitro and in vivo. Hence, p53-associated peptide-MHCs are attractive targets for the immunotherapy against mutant p53 expressing tumours.

scholarly journals Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design

2019 ◽  
Vol 32 (12) ◽  
pp. 555-564
Author(s):  
Magdalena Wójcik ◽  
Susana Vázquez Torres ◽  
Wim J Quax ◽  
Ykelien L Boersma
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Sortase A ◽  
Wild Type ◽  
Protein Thermostability ◽  
Consensus Analysis ◽  
Gram Positive Bacteria ◽  
Covalently Linked

Abstract Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme’s stability while at the same time enhancing the enzyme’s activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.

scholarly journals Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis

2001 ◽  
Vol 183 (11) ◽  
pp. 3391-3398 ◽  
Author(s):  
Vicente Monedero ◽  
Oscar P. Kuipers ◽  
Emmanuel Jamet ◽  
Josef Deutscher
Keyword(s):  
Lactococcus Lactis ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
Gram Positive Bacteria ◽  
In Vivo Experiments ◽  
Regulatory Functions

ABSTRACT In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methylβ-d-thiogalactoside (TMG) and 2-deoxy-d-glucose (2-DG). In vivo experiments with theptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

Dynamics of GPIIb/IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 929-936 ◽  
Author(s):  
Dipti Patel ◽  
Heikki Väänänen ◽  
Markéta Jiroušková ◽  
Thomas Hoffmann ◽  
Carol Bodian ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Wild Type Mouse ◽  
Poisson Model ◽  
Human Platelets ◽  
Wild Type ◽  
Initial Adhesion ◽  
Platelet Deposition ◽  
Coated Surfaces

Abstract The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22°C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of “vanguard” platelets, “follower” platelets attached to the spread-out vanguard platelets. Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets. Thus, thrombi formed as a concerted process rather than as sequential processes. Treatment of human platelets with monoclonal antibody (mAb) 7E3 (anti–GPIIb/IIIa (αIIbβ3) + αVβ3) or tirofiban (anti–GPIIb/IIIa) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi. Similar results were obtained with Glanzmann thrombasthenia platelets. Wild-type mouse platelets in the presence of mAb 1B5 (anti–GPIIb/IIIa) and platelets from β3-null mice behaved like human platelets in the presence of 7E3 or tirofiban. Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model, but those obtained with 7E3- and tirofiban-treated human platelets, 1B5-treated mouse platelets, or β3-null platelets demonstrated a more uniform deposition than predicted. Thus, in this model system, absence or blockade of GPIIb/IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition.

scholarly journals The Flagellar Motility ofChlamydomonas pf25 Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions

2006 ◽  
Vol 17 (1) ◽  
pp. 227-238 ◽  
Author(s):  
Chun Yang ◽  
Pinfen Yang
Keyword(s):  
Regulatory Proteins ◽  
Flagellar Motility ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Wide Range ◽  
Anchoring Protein ◽  
Radial Spokes ◽  
Dependent Protein ◽  
Cilia And Flagella

Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas.

scholarly journals Identification of a putative DNA-binding protein in Arabidopsis that acts as a susceptibility hub and interacts with multiple Pseudomonas syringae effectors

2020 ◽  
Author(s):  
Karl Schreiber ◽  
Jennifer D Lewis
Keyword(s):  
Pseudomonas Syringae ◽  
Transcriptional Profiling ◽  
Effector Proteins ◽  
Ribosomal Protein Genes ◽  
Loss Of Function ◽  
Wild Type ◽  
In Planta ◽  
Arabidopsis Protein ◽  
Wide Range

Phytopathogens use secreted effector proteins to suppress host immunity and promote pathogen virulence, and there is increasing evidence that the host-pathogen interactome comprises a complex network. In an effort to identify novel interactors of the Pseudomonas syringae effector HopZ1a, we performed a yeast two-hybrid screen that identified a previously uncharacterized Arabidopsis protein that we designate HopZ1a Interactor 1 (ZIN1). Additional analyses in yeast and in planta revealed that ZIN1 also interacts with several other P. syringae effectors. We show that an Arabidopsis loss-of-function zin1 mutant is less susceptible to infection by certain strains of P. syringae, while overexpression of ZIN1 results in enhanced susceptibility. Functionally, ZIN1 exhibits topoisomerase-like activity in vitro. Transcriptional profiling of wild-type and zin1 Arabidopsis plants inoculated with P. syringae indicated that while ZIN1 regulates a wide range of pathogen-responsive biological processes, the list of genes more highly expressed in zin1 versus wild-type plants was particularly enriched for ribosomal protein genes. Altogether, these data illuminate ZIN1 as a potential susceptibility hub that interacts with multiple effectors to influence the outcome of plant-microbe interactions.

scholarly journals Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

2020 ◽  
Author(s):  
Shejuan An ◽  
Jeannie Camarillo ◽  
Tina Huang ◽  
Daphne Li ◽  
Juliette Morris ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radiation Treatment ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Response To Treatment ◽  
Wild Type ◽  
Post Translational Modification ◽  
Pontine Glioma ◽  
Epigenetic Signatures ◽  
The Impact

Abstract Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles.Methods: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n=12) and cell lines (n=7).Results: We found 29.2% and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

scholarly journals Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

2021 ◽  
Author(s):  
◽  
Jack Alexander Sissons
Keyword(s):  
Single Gene ◽  
Specific Protein ◽  
Rapid Identification ◽  
Phosphopantetheinyl Transferase ◽  
Post Translational Modification ◽  
Diverse Range ◽  
Peptide Motifs ◽  
Wide Range

<p>Throughout all domains of life, phosphopantetheinyl transferase (PPTase) enzymes catalyse a post-translational modification that is important in both primary and secondary metabolism; the transfer of a phosphopantetheine (PPant) group derived from Coenzyme A to specific protein domains within large, multi-modular biosynthetic enzymes, thereby activating each module for biosynthesis. The short peptide motif of the protein to which this group is attached is known as a ‘tag’, and can be fused to other proteins, making them also substrates for post-translational modification by a PPTase. Additionally, it has been demonstrated that PPTases can utilise a diverse range of CoA analogues, such as biotin-linked or click-chemistry capable CoA derivatives, as substrates for tag attachment. Together, these characteristics make post-translational modification by PPTases an attractive system for many different biotechnological applications. Perhaps the most significant application is in vivo and in vitro site-specific labelling of proteins, for which current technologies are hindered by cumbersome fusion protein requirements, toxicity of the process, or limited reporter groups that can be attached. Confoundingly, most PPTases exhibit a high degree of substrate promiscuity which limits the number of PPTase-tag pairs that can be used simultaneously, and therefore the number of protein targets that can be simultaneously labelled. To address this, directed evolution at a single gene level was used in an attempt to generate multiple PPTase variants that have non-overlapping tag specificity which have applications in orthogonal labelling. Furthermore, assays for the rapid identification, characterisation and evolution of short, novel peptide motifs that are recognised by PPTases has further diversified the labelling toolkit. These developments have enhanced the utility of the PPTase system and potentially have a wide range of applications in a number of fields.</p>

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