Roles of Low-Molecular-Weight Penicillin-Binding Proteins in Bacillus subtilis Spore Peptidoglycan Synthesis and Spore Properties

ABSTRACT The peptidoglycan cortex of endospores of Bacillusspecies is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted byd,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparentd,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while thedacA and dacC products have no significant role in spore peptidoglycan formation, the dacB anddacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of adacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.

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Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli

10.1101/2021.02.12.430937 ◽

2021 ◽

Author(s):

Henri Voedts ◽

Delphine Dorchêne ◽

Adam Lodge ◽

Waldemar Vollmer ◽

Michel Arthur ◽

...

Keyword(s):

Turgor Pressure ◽

Cross Linking ◽

Penicillin Binding Proteins ◽

Peptidoglycan Synthesis ◽

Dual Specificity ◽

Common Motif ◽

Cross Links ◽

Hydrolysis Of ◽

Controlled Hydrolysis

ABSTRACTBacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify amongst eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

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Multiple Low-Reactivity Class B Penicillin-Binding Proteins Are Required for Cephalosporin Resistance in Enterococci

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02273-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 1

Author(s):

Dušanka Djorić ◽

Jaime L. Little ◽

Christopher J. Kristich

Keyword(s):

Binding Proteins ◽

Cross Linking ◽

Intrinsic Resistance ◽

Content Type ◽

Penicillin Binding Proteins ◽

Enterococcal Infection ◽

Peptidoglycan Synthesis ◽

Cephalosporin Resistance

ABSTRACT Enterococcus faecalis and Enterococcus faecium are commensals of the gastrointestinal tract of most terrestrial organisms, including humans, and are major causes of health care-associated infections. Such infections are difficult or impossible to treat, as the enterococcal strains responsible are often resistant to multiple antibiotics. One intrinsic resistance trait that is conserved among E. faecalis and E. faecium is cephalosporin resistance, and prior exposure to cephalosporins is one of the most well-known risk factors for acquisition of an enterococcal infection. Cephalosporins inhibit peptidoglycan biosynthesis by acylating the active-site serine of penicillin-binding proteins (PBPs) to prevent the PBPs from catalyzing cross-linking during peptidoglycan synthesis. For decades, a specific PBP (known as Pbp4 or Pbp5) that exhibits low reactivity toward cephalosporins has been thought to be the primary PBP required for cephalosporin resistance. We analyzed other PBPs and report that in both E. faecalis and E. faecium, a second PBP, PbpA(2b), is also required for resistance; notably, the cephalosporin ceftriaxone exhibits a lethal effect on the ΔpbpA mutant. Strikingly, PbpA(2b) exhibits low intrinsic reactivity with cephalosporins in vivo and in vitro. Unlike the Δpbp5 mutant, the ΔpbpA mutant exhibits a variety of phenotypic defects in growth kinetics, cell wall integrity, and cellular morphology, indicating that PbpA(2b) and Pbp5(4) are not functionally redundant and that PbpA(2b) plays a more central role in peptidoglycan synthesis. Collectively, our results shift the current understanding of enterococcal cephalosporin resistance and suggest a model in which PbpA(2b) and Pbp5(4) cooperate to coordinately mediate peptidoglycan cross-linking in the presence of cephalosporins.

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Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs

10.1101/2021.08.06.454924 ◽

2021 ◽

Author(s):

Heiner Atze ◽

Filippo Rusconi ◽

Michel Arthur

Keyword(s):

Mass Spectrometry ◽

Bacterial Cell ◽

Isotope Labeling ◽

Side Wall ◽

Cross Linking ◽

Heavy Isotope ◽

Penicillin Binding Proteins ◽

Peptidoglycan Synthesis ◽

Cross Links ◽

Cell Wall Expansion

Antibiotics of the β-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate β-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this unprecedented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a β-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.

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Double-Cross-Linked Networks Based on Methacryloyl Mucin

Polymers ◽

10.3390/polym13111706 ◽

2021 ◽

Vol 13 (11) ◽

pp. 1706

Author(s):

Elena Olăreț ◽

Brîndușa Bălănucă ◽

Andra Mihaela Onaș ◽

Jana Ghițman ◽

Horia Iovu ◽

...

Keyword(s):

Mechanical Properties ◽

Aqueous Solution ◽

Chemical Modification ◽

Structural Modification ◽

Aqueous Media ◽

Cross Linking ◽

Ph Values ◽

Acid Aqueous Solution ◽

Reaction Media ◽

Double Cross

Mucin is a glycoprotein with proven potential in the biomaterials field, but its use is still underexploited for such applications. The present work aims to produce a synthesis of methacryloyl mucin single-network (SN) hydrogels and their double-cross-linked-network (DCN) counterparts. Following the synthesis of the mucin methacryloyl derivative, various SN hydrogels are prepared through the photopolymerization of methacrylate bonds, using reaction media with different pH values. The SN hydrogels are converted into DCN systems via supplementary cross-linking in tannic acid aqueous solution. The chemical modification of mucin is described, and the obtained product is characterized; the structural modification of mucin is assessed through FTIR spectroscopy, and the circular dichroism and the isoelectric point of methacryloyl mucin is evaluated. The affinity for aqueous media of both SN and DCN hydrogels is estimated, and the mechanical properties of the systems are assessed, both at macroscale through uniaxial compression and rheology tests and also at microscale through nanoindentation tests.

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Localization of the Transglutaminase Cross-Linking Sites in the Bacillus subtilis Spore Coat Protein GerQ

Journal of Bacteriology ◽

10.1128/jb.01116-06 ◽

2006 ◽

Vol 188 (21) ◽

pp. 7609-7616 ◽

Cited By ~ 36

Author(s):

Alicia Monroe ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Coat Protein ◽

Cross Linking ◽

Spore Coat ◽

Bacillus Subtilis Spore ◽

Binding Partners ◽

Lysine Residues ◽

Cross Links ◽

Hydrolysis Of ◽

Spore Coat Protein

ABSTRACT The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an ε-(γ-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.

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Insights into the role of lipoteichoic acids in Bacillus subtilis- a new function for MprF

10.1101/2021.12.12.472321 ◽

2021 ◽

Author(s):

Aurelie Guyet ◽

Amirah Alofi ◽

Richard A Daniel

Keyword(s):

Bacillus Subtilis ◽

Cell Envelope ◽

Cellular Level ◽

Penicillin Binding Proteins ◽

Class A ◽

Peptidoglycan Synthesis ◽

A Cell ◽

Antimicrobial Peptide Resistance ◽

Lipoteichoic Acids

In Bacillus subtilis, the cell is protected from the environment by a cell envelope, which comprises of layers of peptidoglycan that maintain the cell shape and anionic teichoic acids polymers whose biological function remains unclear. In B. subtilis, loss of all Class A Penicillin-Binding Proteins (aPBPs) which function in peptidoglycan synthesis is conditionally lethal. Here we show that this lethality is associated with an alteration of the lipoteichoic acids (LTA) and the accumulation of the major autolysin LytE in the cell wall. We provide the first evidence that the length and abundance of LTA acts to regulate the cellular level of LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF which acts to modulate LTA biosynthesis in B. subtilis and in the pathogen Staphylococcus aureus. This finding has implications for our understanding of antimicrobial peptide resistance (particularly daptomycin) in clinically relevant bacteria and MprF-associated virulence in pathogens, such as methicillin resistant S. aureus.

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Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

eLife ◽

10.7554/elife.61525 ◽

2021 ◽

Vol 10 ◽

Author(s):

Victor M Hernández-Rocamora ◽

Natalia Baranova ◽

Katharina Peters ◽

Eefjan Breukink ◽

Martin Loose ◽

...

Keyword(s):

Real Time ◽

Lipid Bilayers ◽

High Throughput Screening ◽

Binding Proteins ◽

Cell Envelope ◽

Penicillin Binding Proteins ◽

Peptidoglycan Biosynthesis ◽

Class A ◽

Peptidoglycan Synthesis ◽

Osmotic Lysis

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

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TOLAK UKUR FUNGSI HATI BERDASARKAN DERAJAT FIBROSIS PENYAKIT HATI KRONIS (Liver Function Parameters based on Degree of Liver Fibrosis in Chronic Liver Disease)

INDONESIAN JOURNAL OF CLINICAL PATHOLOGY AND MEDICAL LABORATORY ◽

10.24293/ijcpml.v21i1.1260 ◽

2018 ◽

Vol 21 (1) ◽

pp. 57

Author(s):

Rahmafitria Rahmafitria ◽

Mutmainnah Mutmainnah ◽

Ibrahim Abdul Samad

Keyword(s):

Liver Disease ◽

Liver Fibrosis ◽

Liver Function ◽

Chronic Liver Disease ◽

Total Bilirubin ◽

Chronic Liver ◽

Low Degree ◽

Significant Difference ◽

The Mean ◽

High Degree

Evaluating the degree of liver fibrosis degree is invasive as well as uncomfortable, therefore, non invasive examinations such as liverfunction tests and elastography (Fibro Scan) as a predictor‘s device of liver fibrosis degree are necessary. The aim of this study was toknow the differences of liver function parameters based on the fibrosis degree in patients with chronic liver disease. This study was a crosssectional design using data from chronic liver disease patients treated at the Dr. Wahidin Sudirohusodo Hospital. The elasticity of the liverwas measured using a fibro scan device during June 2010–July 2011. The analysis was carried out by ANOVA test on various parametersof liver function particularly on the fibrosis degree in chronic liver disease. In this study PT, albumin, total bilirubin and platelet countshowed a significant difference of 0.019, 0.009, 0.017 and 0.000 respectively. The mean values of PT and total bilirubin were significantlyhigher in the high degree of fibrosis compared to those with medium and low degree of fibrosis in the chronic liver disease patients. Basedon this study, the mean albumin levels and platelet count were significantly lower in the high degree of fibrosis compared with the mediumand low degree of fibrosis, however, no significant differences in AST, ALT, APTT and GGT were found.

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Preparation and preclinical evaluation of aceclofenac loaded pectinate mucoadhesive microspheres

Drugs and Therapy Studies ◽

10.4081/dts.2012.e8 ◽

2012 ◽

Vol 2 (1) ◽

pp. 8 ◽

Cited By ~ 1

Author(s):

Santanu Chakraborty ◽

Priyanka Nayak ◽

Bala Murali Krishna ◽

Madhusmruti Khandai ◽

Ashoke Kumar Ghosh

Keyword(s):

Particle Size ◽

Drug Release ◽

Polymer Concentration ◽

Research Work ◽

In Vivo Studies ◽

Systemic Absorption ◽

Cross Linking ◽

Significant Difference

The aim of the present research work was to fabricate aceclofenac loaded pectinate microspheres by ionic gelation method and evaluate the effect of different cross-linking agents and polymer concentration on particle size, encapsulation efficacy and drug release behavior. It was also investigated that whether this pectinate dosage form was able to target the drug release in intestinal region and prevent the different side effect associated with the drug in stomach or not. It was observed that particle size, encapsulation efficacy and in vitro drug release were largely depended on polymer concentration and cross-linking agents. It was also observed that pectinate microspheres showed excellent pH depended mucoadhesive properties and they were able to restrict the drug release in stomach. <em>In vitro</em> drug release study showed that alminium-pectinate microspheres have more sustaining property as compared to barium-pectinate microspheres. Holm-Sidak multiple comparison analysis suggested a significant difference in measured t<sub>50%</sub> values among all the formulations with same cross-linking agent. In vivo studies revealed that the anti inflammatory and analgesic effects induced by pectinate microspheres were significantly high and prolonged as compared to pure drug. So, pectinate microspheres can be an excellent carrier for targeting the delivery of aceclofenac as well as help in improving the patient compliance by prolonging the systemic absorption.

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Corneal biomechanical outcome of collagen cross-linking in keratoconic patients evaluated by Corvis ST

European Journal of Ophthalmology ◽

10.1177/1120672120944798 ◽

2020 ◽

pp. 112067212094479

Author(s):

Mahmoud Jabbarvand ◽

Zahra Moravvej ◽

Kianoush Shahraki ◽

Hessam Hashemian ◽

Hamed Ghasemi ◽

...

Keyword(s):

Amplitude Ratio ◽

Corneal Thickness ◽

Case Series ◽

Cross Linking ◽

Corvis St ◽

Significant Difference ◽

Biomechanical Changes ◽

The Mean ◽

Prospective Case Series ◽

Collagen Cross Linking

Purpose: A 6-month evaluation of the topographic and biomechanical changes induced by corneal collagen cross-linking (CXL) in keratoconic eyes using Pentacam and Corvis ST. Design: Longitudinal prospective case series. Methods: In this study, 67 eyes of 67 patients with progressive keratoconus (KCN) treated with “Epithelium-off” CXL were evaluated. Patients with stages 1 or 2 of KCN and a corneal thickness of at least 400 μm at the thinnest point were included. Standard ophthalmologic examinations were carried out for all patients. The topographic and biomechanical measurements of the cornea were obtained by Pentacam (Oculus Optikgeräte GmbH, Wetzlar, Germany) and Corvis ST (Oculus Optikgeräte GmbH, Wetzlar, Germany) preoperatively and 6-month postoperatively. Results: The mean age of the participants was 21.68 ± 4.23 years. There was significant difference in mean spherical equivalent (SE) before and 6 months after CXL. Uncorrected and best corrected visual acuity improved postoperatively, although not statistically significant. The mean and maximum keratometry showed a significant decrease 6 months after CXL (0.93 ± 0.38 D and 1.43 ± 0.62 D, respectively p < 0.001). Among Corvis ST parameters, first applanation length and velocity (AL1 and AV1) showed statistically significant changes. The radius at highest concavity changed significantly (0.13 ± 0.37 mm mean increase after CXL; p < 0.001). A significant increase was observed in stiffness parameter A1 (SP-A1; p < 0.001) and significant decreases were noted in integrated radius (IR) and deformation amplitude ratio (DAR; p < 0.001). Conclusion: Analyzing biomechanical changes after corneal cross-linking can provide basis for efficient KCN treatment. Corvis ST parameters demonstrated changes in corneal biomechanical characteristics indicative of stiffing after CXL.

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