Publications
102 results found
Beetham CM, Schuster CF, Kviatkovski I, et al., 2024, Histidine transport is essential for the growth of Staphylococcus aureus at low pH, PLoS Pathogens, ISSN: 1553-7366
Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH. We identified 31 genes that were essential for the growth of S. aureus at pH 4.5 and confirmed the importance of many of them through follow up experiments using mutant strains inactivated for individual genes. Most of the genes identified code for proteins with functions in cell wall assembly and maintenance. These data suggest that the cell wall has a more important role than previously appreciated in promoting bacterial survival when under acid stress. We also identified several novel processes previously not linked to the acid stress response in S. aureus. These include aerobic respiration and histidine transport, the latter by showing that one of the most important genes, SAUSA300_0846, codes for a previously uncharacterized histidine transporter. We further show that under acid stress, the expression of the histidine transporter gene is increased in WT S. aureus. In a S. aureus SAUSA300_0846 mutant strain expression of the histidine biosynthesis genes is induced under acid stress conditions allowing the bacteria to maintain cytosolic histidine levels. This strain is, however, unable to maintain its cytosolic pH to the same extent as a WT strain, revealing an important function specifically for histidine transport in the acid stress response of S. aureus.
Rismondo J, Gründling A, 2024, Type I Lipoteichoic Acid (LTA) Purification by Hydrophobic Interaction Chromatography and Structural Analysis by 2D Nuclear Magnetic Resonance (NMR) Spectroscopy., Methods Mol Biol, Vol: 2727, Pages: 107-124
Type I lipoteichoic acid (LTA) is a glycerol phosphate polymer found in the cell envelope of diverse Gram-positive bacteria. The glycerol phosphate backbone is often further decorated with D-alanine and/or sugar residues. Here, we provide details of a 1-butanol extraction and purification method of type I LTA by hydrophobic interaction chromatography. The protocol has been adapted from methods originally described by Fischer et al. (Eur J Biochem 133:523-530, 1983) and further optimized by Morath et al. (J Exp Med 193:393-397, 2001). We also present information on a 2D nuclear magnetic resonance (NMR) analysis method to gain chemical and structural information of the purified LTA material.
Millership C, Gründling A, 2024, Type I Lipoteichoic Acid (LTA) Detection by Western Blot., Methods Mol Biol, Vol: 2727, Pages: 95-106
Type I lipoteichoic acid (LTA) is a glycerol phosphate polymer found in the cell envelope of diverse Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes. The polymer is linked by a lipid anchor to the outer leaflet of the bacterial membrane and in some bacteria can also be shed and detected in the culture supernatant. Here, we describe a simple and rapid western blot method for the detection of Type I LTA in bacterial cell extracts and culture supernatant fractions using a polyglycerol phosphate specific monoclonal LTA antibody.
Zeden MS, Schuster CF, Gründling A, 2023, Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli., Cold Spring Harb Protoc, Vol: 2023
We present a protocol for the generation of a gene-deletion allelic-exchange plasmid and its recovery in Escherichia coli for the purpose of constructing an in-frame gene deletion in Staphylococcus aureus Here, we present detailed methodologies for (i) the primer design (using the S. aureus tagO gene as our specific example); (ii) PCR amplification of the required gene fragments; (iii) preparation of the cloning vector (using the S. aureus allelic-exchange vector pIMAY* as an example); (iv) the Gibson assembly cloning method; (v) introduction of the plasmid into E. coli; (vi) confirmation of the plasmid insert in E. coli by colony PCR; and, finally, (vii) confirmation of the insert by sequencing. We also consider the long-term storage of the E. coli strains containing the desired plasmid.
Zeden MS, Schuster CF, Gründling A, 2023, Preparation of Electrocompetent Staphylococcus aureus Cells and Plasmid Transformation., Cold Spring Harb Protoc, Vol: 2023
This protocol is part of a series of methodologies for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. Having previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, we now present details of the next steps in this method-the preparation of electrocompetent S. aureus cells and introduction of the tagO mutant plasmid DNA into the S. aureus cells by electroporation. Colonies containing the plasmid can then be selected on chloramphenicol plates at a low temperature permissive for plasmid replication.
Zeden MS, Schuster CF, Gründling A, 2023, Staphylococcus aureus Colony Polymerase Chain Reaction., Cold Spring Harb Protoc, Vol: 2023
Here, we describe a protocol for a colony polymerase chain reaction (PCR) method for Staphylococcus aureus The methodology involves the preparation of small S. aureus lysates by using the enzyme lysostaphin to degrade the peptidoglycan layer. These lysates are prepared using a small patch of bacteria grown on LB agar plates, and the lysates can subsequently be used for PCR analyses.
Zeden MS, Schuster CF, Gründling A, 2023, Allelic-Exchange Procedure in Staphylococcus aureus., Cold Spring Harb Protoc, Vol: 2023
This protocol continues a series of methods for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for S. aureus We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, then introduced into electrocompetent S. aureus cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (pheS* gene) and confers chloramphenicol resistance to S. aureus As a specific example, we present the construction of strain RN4220*ΔtagO from strain RN4220 carrying the pIMAY*-ΔtagO plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.
Zeden MS, Schuster CF, Gründling A, 2023, Allelic Exchange: Construction of an Unmarked In-Frame Deletion in Staphylococcus aureus., Cold Spring Harb Protoc, Vol: 2023
Here we describe an allelic-exchange procedure for the construction of an unmarked gene deletion in the bacterium Staphylococcus aureus As a practical example, we outline the construction of a tagO gene deletion in S. aureus using the allelic-exchange plasmid pIMAY*. We first present the general principles of the allelic-exchange method, along with information on counterselectable markers. Furthermore, we summarize relevant cloning procedures, such as the splicing by overhang extension (SOE) polymerase chain reaction (PCR) and Gibson assembly methods, and we conclude by giving some general consideration to performing genetic modifications in S. aureus.
Gründling A, Salipante SJ, 2023, Oligonucleotide Design and Construction of a Gene-Targeting CRISPR-Cas9 Plasmid in Escherichia coli for Generating a Gene-Deletion Strain in Staphylococcus aureus., Cold Spring Harb Protoc
Gene deletions can be constructed in Staphylococcus aureus using recombineering in combination with a CRISPR-Cas9 counterselection approach. The method involves first designing the recombineering oligonucleotides and generating the relevant plasmids, and then introducing these elements into S. aureus to generate the desired gene deletion. Here, we describe the first part of this workflow, oligonucleotide design and plasmid generation. To better illustrate the method and oligonucleotide design, the construction of a 55-bp out-of-frame deletion in the S. aureus geh gene will be presented as a specific example. To this end, we describe the use of geh gene-specific recombineering oligonucleotides and the construction of a geh gene-targeting CRISPR-Cas9 plasmid. The protocol is divided into three parts: (1) design of the gene-specific targeting spacer oligonucleotides for introduction into the CRISPR-Cas9 plasmid pCas9-counter, (2) design of 90-mer recombineering oligonucleotides to generate a 55-bp out-of-frame gene deletion, and (3) construction of the gene-targeting CRISPR-Cas9 plasmid pCas9-geh, plasmid recovery in Escherichia coli, and confirmation by colony PCR and sequencing. The method can easily be adapted to design deletions for other S. aureus genes.
Zeden MS, Gründling A, 2023, Small-Scale Illumina Library Preparation Using the Illumina Nextera XT DNA Library Preparation Kit., Cold Spring Harb Protoc
Here, we describe a protocol for a scaled-down version of a genomic DNA (gDNA)-fragmentation and tagmentation reaction using the Illumina Nextera XT DNA Library Preparation Kit. Using Staphylococcus aureus as an example, which has a genome size of ∼3 Mb, we show how 24 different samples can be pooled for a typical paired-end Illumina high-throughput sequencing run using the MiSeq Reagent V2 300-cycle kit, with which it is possible to sequence 5.1 Gb of DNA. As part of the protocol, a DNA size-selection method using a standard DNA agarose gel-extraction procedure and a final sample quality-control step using a Bioanalyzer are described.
Gründling A, Ji Q, 2023, Identification of Editable Sites, Spacer Oligonucleotide Design, and Generation of the Gene-Targeting CRISPR-nCas9 Plasmid for Gene Disruption in Staphylococcus aureus Using the CRISPR-nCas9 and Cytidine Deaminase System., Cold Spring Harb Protoc
Methods for gene disruption are essential for functional genomics, and there are multiple approaches for altering gene function in bacteria. One of these methods involves introducing a premature stop codon in a gene of interest, which can be achieved by using the CRISPR-nCas9-cytidine deaminase system. The approach involves the mutation of editable cytidines to thymidines, with the goal of generating a novel stop codon that ultimately results in a nonfunctional gene product. The workflow involves two major sections, one for the identification of editable cytidines, the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid, and the construction of the gene-targeting CRISPR-nCas9 cytosine deaminase plasmids, and one for the actual introduction of the mutation in the species of interest. Here, we describe the steps for the first part. To better illustrate the method and oligonucleotide design, we describe the construction of Staphylococcus aureus RN4220 geh mutants with C to T base changes at two different positions, leading to the construction of strains RN4220-geh(160stop) and RN4220-geh(712stop). We outline the steps for (1) the identification of editable cytidines within genes using the CRISPR-CBEI toolkit website, and (2) the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid pnCasSA-BEC, followed by (3) the construction of the gene-targeting (in this example, geh gene-targeting) CRISPR-nCas9 cytosine deaminase plasmids pnCasSA-BEC-gehC160T and pnCasSA-BEC-gehC712T using the Golden Gate assembly method, plasmid recovery in Escherichia coli, and confirmation by colony PCR and sequencing. The method can be easily adapted to construct gene-inactivation mutants in other S. aureus genes.
Gründling A, Ji Q, 2023, Introduction of a CRISPR-nCas9 Gene-Targeting Plasmid into Staphylococcus aureus for Gene Disruption., Cold Spring Harb Protoc
Methods for gene disruption are essential for functional genomics, and there are multiple approaches for altering gene function in bacteria. One of these methods involves introducing a premature stop codon in a gene of interest, which can be achieved by using the CRISPR-nCas9-cytidine deaminase system. The approach involves the mutation of editable cytidines to thymidines, with the goal of generating a novel stop codon that ultimately results in a nonfunctional gene product. The workflow involves two major sections, one for the identification of editable cytidines, the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid, and the construction of the gene-targeting CRISPR-nCas9 cytosine deaminase plasmids, and one for the actual introduction of the mutation in the species of interest. Here, we describe the steps for the second part. Specifically, we describe (1) how to introduce the gene-targeting pnCasSA-BEC plasmid into Staphylococcus aureus, (2) how the gene inactivation in S. aureus can be confirmed by PCR and sequencing, and (3) how, following successful gene inactivation, the strain can be cured of the pnCasSA-BEC plasmid. To better illustrate the method, and as specific example, two different geh gene-inactivation mutations are generated here in S. aureus RN4220. The protocol, however, can easily be adapted to generate other gene-inactivating mutations.
Gründling A, Ji Q, Salipante SJ, 2023, Using CRISPR-Cas9-Based Methods for Genome Editing in Staphylococcus aureus., Cold Spring Harb Protoc
Chromosomal mutations and targeted gene deletions and inactivations in Staphylococcus aureus are typically generated using the allelic exchange method. In recent years, however, more rapid methods have been developed, often using CRISPR-Cas9-based systems. Here, we describe recently developed CRISPR-Cas9-based plasmid systems for use in S. aureus, and discuss their use for targeted gene mutation and inactivation. First, we describe how a CRISPR-Cas9 counterselection strategy can be combined with a recombineering strategy to generate gene deletions in S. aureus We then introduce dead Cas9 (dCas9) and Cas9 nickase (nCas9) enzymes, and discuss how the nCas9 enzyme fused to different nucleoside deaminases can be used to introduce specific base changes in target genes. We then discuss how the nCas9-deaminase fusion enzymes can be used for targeted gene inactivation via the introduction of premature stop codons or by mutating the start codon. Together, these tools highlight the power and potential of CRISPR-Cas9-based methods for genome editing in S. aureus.
Zeden MS, Gründling A, 2023, Agar Plate-Based Method for the Selection of Antibiotic-Resistant Bacterial Strains., Cold Spring Harb Protoc
Identifying the molecular mechanisms underlying antibiotic resistance is important, as it can reveal key information on the mode of action of a drug and provide insights for the development of novel or improved antimicrobials. Here, we describe an agar-based method for the selection of bacterial strains with increased antibiotic resistance, and how the increase in resistance can be confirmed by a spot-plating assay. As a specific example, we describe the selection of Staphylococcus aureus strains with increased resistance to oxacillin; however, the protocol can be easily adapted and used with other bacteria and antibiotics.
Zeden MS, Gründling A, 2023, Preparation of Staphylococcus aureus Genomic DNA Using a Chloroform Extraction and Ethanol Precipitation Method, Followed by Additional Cleanup and Quantification Steps., Cold Spring Harb Protoc
In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus strains using a chloroform extraction and ethanol precipitation method. This gDNA-isolation method is well-suited for downstream whole-genome sequencing applications when working with S. aureus strains that contain plasmids, as only a small amount of plasmid DNA is isolated along with the gDNA. Similar to other gDNA isolation methods for Gram-positive bacteria, the first step in the procedure is a mechanical lysis (e.g., using a bead beating grinder) or an enzymatic lysis step. In this protocol, the peptidoglycan layer of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves pentaglycine cross-bridges within the peptidoglycan of S. aureus. After this lysis step, gDNA can be purified using similar procedures as those used for Gram-negative bacteria. We include additional cleanup and quantification procedures in the final steps of this protocol, in case the aim is to use the gDNA for genome-sequencing projects. By modifying the bacterial lysis step, the procedure can be easily adapted to isolate gDNA from other bacteria.
Zeden MS, Gründling A, 2023, Bacterial Whole-Genome-Resequencing Analysis: Basic Steps Using the CLC Genomics Workbench Software., Cold Spring Harb Protoc
In this protocol, we describe the basic steps for bacterial genome resequencing analysis using the QIAGEN CLC Genomics Workbench software. More specifically, we present how a reference genome sequence can be generated from Illumina reads of a wild-type reference bacterial strain and how this reference genome sequence can then be used to identify genomic alterations in mutant strains. As specific examples, Illumina reads from the Staphylococcus aureus RN4220 strain will be used to generate a consensus reference genome based on the publicly available S. aureus NCTC8325 genome sequence. The generated RN4220 consensus reference genome will subsequently be used to identify genomic mutations in an RN4220 mutant strain with increased oxacillin resistance (OxaR strain).
Zeden MS, Gründling A, 2023, Preparation of Staphylococcus aureus Genomic DNA Using Promega Nuclei Lysis and Protein Precipitation Solutions, Followed by Additional Cleanup and Quantification Steps., Cold Spring Harb Protoc
In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus using the Promega Nuclei Lysis and Protein Precipitation solutions. Gram-positive bacteria such as S. aureus are harder to lyse than Gram-negative bacteria. Hence, the first step in the procedure for isolating gDNA from Gram-positive bacteria consists of a mechanical lysis step (e.g., using a bead beating grinder or homogenizer) or an enzymatic lysis step. For the method described here, the peptidoglycan layer of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves the pentaglycine cross-bridges within the peptidoglycan of S. aureus. After this lysis step, the gDNA can be purified using procedures similar to those used for Gram-negative bacteria. We include additional cleanup and quantification procedures in the final steps of this protocol, in case the gDNA is subsequently used for genome-sequencing projects. By modifying the bacterial lysis step, the procedure can be easily adapted to isolate gDNA from other bacteria.
Gründling A, Salipante SJ, 2023, Introduction of a Recombineering Oligonucleotide and a CRISPR-Cas9 Gene-Targeting Plasmid into Staphylococcus aureus for Generating a Gene-Deletion Strain., Cold Spring Harb Protoc
Gene deletions can be generated in Staphylococcus aureus using recombineering in combination with a CRISPR-Cas9 counterselection approach. The method involves first designing the recombineering oligonucleotides and generating the relevant plasmids, and then introducing these elements into S. aureus to generate the desired gene deletion. Here, we describe the second part of this workflow; the introduction of the gene-targeting plasmid and the recombineering oligonucleotide(s) into S. aureus to generate the gene-deletion strain. Specifically, we outline the steps to (1) generate the S. aureus recipient strain for the recombineering CRISPR-Cas9 counterselection method by introducing plasmid pCN-EF2132tet, (2) introduce the recombineering oligonucleotide(s) and gene-targeting plasmid into the pCN-EF2132tet plasmid-containing S. aureus strain, (3) confirm the gene deletion in S. aureus by colony PCR and sequencing, and (4) curate the plasmids following successful gene deletion. To illustrate the method, we give a specific example of how to generate a 55-bp deletion in the geh gene of S. aureus strain RN4220. The protocol, however, can be easily adapted to other strain backgrounds and to generate deletions in other genes.
Zeden MS, Gründling A, 2023, Selection of Antibiotic-Resistant Bacterial Strains and Identification of Genomic Alterations by Whole-Genome Sequencing: Using Staphylococcus aureus and Oxacillin Resistance as an Example., Cold Spring Harb Protoc
Here, we discuss methods for the selection of antibiotic-resistant bacteria and the use of high-throughput whole-genome sequencing for the identification of the underlying mutations. We comment on sample requirements and the choice of specific DNA preparation methods depending on the strain used and briefly introduce a workflow we use for the selection of Staphylococcus aureus strains with increased oxacillin resistance and identification of genomic alterations.
Nolan AC, Zeden MS, Kviatkovski I, et al., 2023, Purine Nucleosides Interfere with c-di AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To β-Lactam Antibiotics, MBIO, Vol: 14, ISSN: 2150-7511
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Schulz LM, Rothe P, Halbedel S, et al., 2022, Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes, The Cell Surface, Vol: 8, Pages: 1-16, ISSN: 2468-2330
The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.
Schulz LM, Rothe P, Halbedel S, et al., 2022, Imbalance of peptidoglycan biosynthesis alters the cell surface charge of <i>Listeria monocytogenes</i>
<jats:title>ABSTRACT</jats:title><jats:p>The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen <jats:italic>Listeria monocytogenes</jats:italic> is intrinsically resistant towards lysozyme, mainly due to deacetylation and <jats:italic>O</jats:italic>-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An <jats:italic>eslB</jats:italic> mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less <jats:italic>O</jats:italic>-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of <jats:italic>eslB</jats:italic> phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the <jats:italic>eslB</jats:italic> mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-<jats:italic>N</jats:italic>-acetylglucosamine (UDP-Glc<jats:italic>N</jats:italic>Ac) production or distribution.</jats:p>
Nolan AC, Zeden MS, Campbell C, et al., 2022, Purine nucleosides interfere with c-di-AMP levels and act as adjuvants to re-sensitise MRSA to β-lactam antibiotics
<jats:title>Abstract</jats:title><jats:p>Elucidating the complex mechanisms controlling <jats:italic>mecA</jats:italic>/PBP2a-mediated β-lactam resistance in methicillin resistant <jats:italic>Staphylococcus aureus</jats:italic> (MRSA) has the potential to identify new drug targets with therapeutic potential. Here, we report that mutations that interfere with <jats:italic>de novo</jats:italic> purine synthesis (<jats:italic>pur</jats:italic> operon), purine transport (NupG, PbuG and PbuX) and the nucleotide salvage pathway (DeoD2, Hpt) increased β-lactam resistance in MRSA strain JE2. Extrapolating from these findings, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis were shown to significantly reduce MRSA β-lactam resistance. In contrast adenosine, which is fluxed to ATP, significantly increased oxacillin resistance, whereas inosine, which can be fluxed to ATP and GTP via hypoxanthine, only marginally reduced the oxacillin MIC. Increased oxacillin resistance of the <jats:italic>nupG</jats:italic> mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which in turn is required to reduce β-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including <jats:italic>nupG</jats:italic> and <jats:italic>hpt</jats:italic>. Microscopic analysis revealed that guanosine significantly increased cell size, a phenotype also associated with reduced levels of c-di-AMP. Consistent with this, guanosine significantly reduced levels of c-di-AMP, and inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on β-lactam susceptibility. PBP2a expression was unaffected in <jats:italic>nupG</jats:italic> or <jats:italic>deoD2</jats:
Chee Wezen X, Chandran A, Eapen RS, et al., 2022, Structure-based discovery of lipoteichoic acid synthase inhibitors., Journal of Chemical Information and Modeling, Vol: 62, Pages: 2586-2599, ISSN: 1549-9596
Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate β-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.
Pathania M, Tosi T, Millership C, et al., 2021, Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM, Journal of Biological Chemistry, Vol: 297, Pages: 1-15, ISSN: 0021-9258
Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.
Grundling A, Collet J-F, 2021, Editorial overview: "All in all, it is not just another brick in the wall": new concepts and mechanisms on how bacteria build their wall, CURRENT OPINION IN MICROBIOLOGY, Vol: 62, Pages: 110-113, ISSN: 1369-5274
Rismondo J, Gillis A, Grundling A, 2021, Modifications of cell wall polymers in Gram-positive bacteria by multi-component transmembrane glycosylation systems, Current Opinion in Microbiology, Vol: 60, Pages: 24-33, ISSN: 1369-5274
Secondary cell wall polymers fulfil diverse and important functions within the cell wall of Gram-positive bacteria. Here, we will provide a brief overview of the principles of teichoic acid and complex secondary cell wall polysaccharide biosynthesis pathways in Firmicutes and summarize the recently revised mechanism for the decoration of teichoic acids with d-alanines. Many cell wall polymers are decorated with glycosyl groups, either intracellularly or extracellularly. The main focus of this review will be on the extracellular glycosylation mechanism and recent advances that have been made in the identification of enzymes involved in this process. Based on the proteins involved, we propose to rename the system to multi-component transmembrane glycosylation system in place of three-component glycosylation system.
Wu C-H, Rismondo J, Morgan RML, et al., 2021, Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth, Journal of Biological Chemistry, Vol: 296, Pages: 1-14, ISSN: 0021-9258
UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.
Rismondo J, Schulz LM, Yacoub M, et al., 2021, EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes., Journal of Bacteriology, Vol: 203, Pages: 1-16, ISSN: 0021-9193
Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only af
Zhang K, Raju C, Zhong W, et al., 2021, Cationic glycosylated block Co-β-peptide acts on the cell wall of gram-positive bacteria as anti-biofilm agents, ACS Applied Bio Materials, Vol: 4, Pages: 3749-3761, ISSN: 2576-6422
Antimicrobial resistance is a global threat. In addition to the emergence of resistance to last resort drugs, bacteria escape antibiotics killing by forming complex biofilms. Strategies to tackle antibiotic resistance as well as biofilms are urgently needed. Wall teichoic acid (WTA), a generic anionic glycopolymer present on the cell surface of many Gram-positive bacteria, has been proposed as a possible therapeutic target, but its druggability remains to be demonstrated. Here we report a cationic glycosylated block co-β-peptide that binds to WTA. By doing so, the co-β-peptide not only inhibits biofilm formation, it also disperses preformed biofilms in several Gram-positive bacteria and resensitizes methicillin-resistant Staphylococcus aureus to oxacillin. The cationic block of the co-β-peptide physically interacts with the anionic WTA within the cell envelope, whereas the glycosylated block forms a nonfouling corona around the bacteria. This reduces physical interaction between bacteria-substrate and bacteria-biofilm matrix, leading to biofilm inhibition and dispersal. The WTA-targeting co-β-peptide is a promising lead for the future development of broad-spectrum anti-biofilm strategies against Gram-positive bacteria.
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