Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation ß-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA-deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach toward diagnosis and treatment of ß-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus, high-level, broad-spectrum resistance to ß-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA. This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to ß-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA, as seen by increased bacterial survival in the presence of ß-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and GGDEF domain protein containing phosphodiesterase (gdpP) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of ß-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by Methicillin-Resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to Next Generation ß-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance is classically mediated by PBP2a, a Penicillin-Binding Protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as Methicillin-Resistant Lacking mec (MRLM) are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance towards NGBs at levels comparable to that of MRSAs. Our study, provides a fresh new perspective about alternative mechanisms of NGBs resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of ß-lactam resistant S. aureus infections.

Cell division protein FtsK coordinates bacterial chromosome segregation and daughter cell separation in Staphylococcus aureus.

Unregulated cell cycle progression may have lethal consequences and therefore, bacteria have various mechanisms in place for the precise spatiotemporal control of cell cycle events. We have uncovered a new link between chromosome replication/segregation and splitting of the division septum. We show that the DNA translocase domain-containing divisome protein FtsK regulates cellular levels of a peptidoglycan hydrolase Sle1, which is involved in cell separation in the bacterial pathogen Staphylococcus aureus. FtsK interacts with a chaperone (trigger factor, TF) and establishes a FtsK-dependent TF concentration gradient that is higher in the septal region. Trigger factor binds Sle1 and promotes its preferential export at the septal region, while also preventing Sle1 degradation by the ClpXP proteolytic machinery. Upon conditions that lead to paused septum synthesis, such as DNA damage or impaired DNA replication/segregation, TF gradient is dissipated and Sle1 levels are reduced, thus halting premature septum splitting.

The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity.

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

A molecular link between cell wall biosynthesis, translation fidelity, and stringent response in Streptococcus pneumoniae.

Survival in the human host requires bacteria to respond to unfavorable conditions. In the important Gram-positive pathogen Streptococcus pneumoniae, cell wall biosynthesis proteins MurM and MurN are tRNA-dependent amino acyl transferases which lead to the production of branched muropeptides. We demonstrate that wild-type cells experience optimal growth under mildly acidic stressed conditions, but ΔmurMN strain displays growth arrest and extensive lysis. Furthermore, these stress conditions compromise the efficiency with which alanyl-tRNAAla synthetase can avoid noncognate mischarging of tRNAAla with serine, which is toxic to cells. The observed growth defects are rescued by inhibition of the stringent response pathway or by overexpression of the editing domain of alanyl-tRNAAla synthetase that enables detoxification of tRNA misacylation. Furthermore, MurM can incorporate seryl groups from mischarged Seryl-tRNAAlaUGC into cell wall precursors with exquisite specificity. We conclude that MurM contributes to the fidelity of translation control and modulates the stress response by decreasing the pool of mischarged tRNAs. Finally, we show that enhanced lysis of ΔmurMN pneumococci is caused by LytA, and the murMN operon influences macrophage phagocytosis in a LytA-dependent manner. Thus, MurMN attenuates stress responses with consequences for host-pathogen interactions. Our data suggest a causal link between misaminoacylated tRNA accumulation and activation of the stringent response. In order to prevent potential corruption of translation, consumption of seryl-tRNAAla by MurM may represent a first line of defense. When this mechanism is overwhelmed or absent (ΔmurMN), the stringent response shuts down translation to avoid toxic generation of mistranslated/misfolded proteins.

eHooke: A tool for automated image analysis of spherical bacteria based on cell cycle progression.

Fluorescence microscopy is a critical tool for cell biology studies on bacterial cell division and morphogenesis. Because the analysis of fluorescence microscopy images evolved beyond initial qualitative studies, numerous images analysis tools were developed to extract quantitative parameters on cell morphology and organization. To understand cellular processes required for bacterial growth and division, it is particularly important to perform such analysis in the context of cell cycle progression. However, manual assignment of cell cycle stages is laborious and prone to user bias. Although cell elongation can be used as a proxy for cell cycle progression in rod-shaped or ovoid bacteria, that is not the case for cocci, such as Staphylococcus aureus. Here, we describe eHooke, an image analysis framework developed specifically for automated analysis of microscopy images of spherical bacterial cells. eHooke contains a trained artificial neural network to automatically classify the cell cycle phase of individual S. aureus cells. Users can then apply various functions to obtain biologically relevant information on morphological features of individual cells and cellular localization of proteins, in the context of the cell cycle.

Assembly of Peptidoglycan Fragments-A Synthetic Challenge.

Peptidoglycan (PGN) is a major constituent of most bacterial cell walls that is recognized as a primary target of the innate immune system. The availability of pure PGN molecules has become key to different biological studies. This review aims to (1) provide an overview of PGN biosynthesis, focusing on the main biosynthetic intermediates; (2) focus on the challenges for chemical synthesis posed by the unique and complex structure of PGN; and (3) cover the synthetic routes of PGN fragments developed to date. The key difficulties in the synthesis of PGN molecules mainly involve stereoselective glycosylation involving NAG derivatives. The complex synthesis of the carbohydrate backbone commonly involves multistep sequences of chemical reactions to install the lactyl moiety at the O-3 position of NAG derivatives and to control enantioselective glycosylation. Recent advances are presented and synthetic routes are described according to the main strategy used: (i) based on the availability of starting materials such as glucosamine derivatives; (ii) based on a particular orthogonal synthesis; and (iii) based on the use of other natural biopolymers as raw materials.

A top-down chemo-enzymatic approach towards N-acetylglucosamine-N-acetylmuramic oligosaccharides: Chitosan as a reliable template.

An unprecedented approach towards oligosaccharides containing N-acetylglucosamine-N-acetylmuramic (NAG-NAM) units was developed. These novel bacterial cell wall surrogates were obtained from chitosan via a top down approach involving both chemical and enzymatic reactions. The chemical modification of chitosan using a molecular clamp based strategy, allowed obtaining N-acetylglucosamine-N-acetylmuramic (NAG-NAM) containing oligomers. Intercalation of NAM residues was confirmed through the analysis of oligosaccharide fragments from enzymatic digestion and it was found that this route affords NAG-NAM containing oligosaccharides in 33% yield. These oligosaccharides mimic the carbohydrate basic skeleton of most bacterial cell surfaces. The oligosaccharides prepared are biologically relevant and will serve as a platform for further molecular recognition studies with different receptors and enzymes of both bacterial cell wall and innate immune system. This strategy combining both chemical modification and enzymatic digestion provides a novel and simple route for an easy access to bacterial cell wall fragments - biologically important targets.

The pentaglycine bridges of Staphylococcus aureus peptidoglycan are essential for cell integrity.

Bacterial cells are surrounded by cell wall, whose main component is peptidoglycan (PG), a macromolecule that withstands the internal turgor of the cell. PG composition can vary considerably between species. The Gram-positive pathogen Staphylococcus aureus possesses highly crosslinked PG due to the presence of cross bridges containing five glycines, which are synthesised by the FemXAB protein family. FemX adds the first glycine of the cross bridge, while FemA and FemB add the second and the third, and the fourth and the fifth glycines, respectively. Of these, FemX was reported to be essential. To investigate the essentiality of FemAB, we constructed a conditional S. aureus mutant of the femAB operon. Depletion of femAB was lethal, with cells appearing as pseudomulticellular forms that eventually lyse due to extensive membrane rupture. This deleterious effect was mitigated by drastically increasing the osmolarity of the medium, indicating that pentaglycine crosslinks are required for S. aureus cells to withstand internal turgor. Despite the absence of canonical membrane targeting domains, FemA has been shown to localise at the membrane. To study its mechanism of localisation, we constructed mutants in key residues present in the putative transferase pocket and the α6 helix of FemA, possibly involved in tRNA binding. Mutations in the α6 helix led to a sharp decrease in protein activity in vivo and in vitro but did not impair correct membrane localisation, indicating that FemA activity is not required for localisation. Our data indicates that, contrarily to what was previously thought, S. aureus cells do not survive in the absence of a pentaglycine cross bridge.

Revisiting Anti-tuberculosis Therapeutic Strategies That Target the Peptidoglycan Structure and Synthesis.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is one of the leading cause of death by an infectious diseases. The biosynthesis of the mycobacterial cell wall (CW) is an area of increasing research significance, as numerous antibiotics used to treat TB target biosynthesis pathways of essential CW components. The main feature of the mycobacterial cell envelope is an intricate structure, the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex responsible for its innate resistance to many commonly used antibiotics and involved in virulence. A hallmark of mAGP is its unusual peptidoglycan (PG) layer, which has subtleties that play a key role in virulence by enabling pathogenic species to survive inside the host and resist antibiotic pressure. This dynamic and essential structure is not a target of currently used therapeutics as Mtb is considered naturally resistant to most ß-lactam antibiotics due to a highly active ß-lactamase (BlaC) that efficiently hydrolyses many ß-lactam drugs to render them ineffective. The emergence of multidrug- and extensive drug-resistant strains to the available antibiotics has become a serious health threat, places an immense burden on health care systems, and poses particular therapeutic challenges. Therefore, it is crucial to explore additional Mtb vulnerabilities that can be used to combat TB. Remodeling PG enzymes that catalyze biosynthesis and recycling of the PG are essential to the viability of Mtb and are therefore attractive targets for novel antibiotics research. This article reviews PG as an alternative antibiotic target for TB treatment, how Mtb has developed resistance to currently available antibiotics directed to PG biosynthesis, and the potential of targeting this essential structure to tackle TB by attacking alternative enzymatic activities involved in Mtb PG modifications and metabolism.

Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis.

Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively. The tubulin homologue FtsZ is the first protein recruited to the division site, where it assembles in filaments-forming the Z ring-that undergo treadmilling and recruit later divisome proteins. The rate of treadmilling in B. subtilis controls the rates of both peptidoglycan synthesis and cell division. The actin homologue MreB forms discrete patches that move circumferentially around the cell in tracks perpendicular to the long axis of the cell, and organize the insertion of new cell wall during elongation. Cocci such as Staphylococcus aureus possess only one type of peptidoglycan synthesis machinery, which is diverted from the cell periphery to the septum in preparation for division. The molecular cue that coordinates this transition has remained elusive. Here we investigate the localization of S. aureus peptidoglycan biosynthesis proteins and show that the recruitment of the putative lipid II flippase MurJ to the septum, by the DivIB-DivIC-FtsL complex, drives peptidoglycan incorporation to the midcell. MurJ recruitment corresponds to a turning point in cytokinesis, which is slow and dependent on FtsZ treadmilling before MurJ arrival but becomes faster and independent of FtsZ treadmilling after peptidoglycan synthesis activity is directed to the septum, where it provides additional force for cell envelope constriction.

Hydrolysis of peptidoglycan is modulated by amidation of meso-diaminopimelic acid and Mg2+ in Bacillus subtilis.

The ability of excess Mg2+ to compensate the absence of cell wall related genes in Bacillus subtilis has been known for a long time, but the mechanism has remained obscure. Here, we show that the rigidity of wild-type cells remains unaffected with excess Mg2+ , but the proportion of amidated meso-diaminopimelic (mDAP) acid in their peptidoglycan (PG) is significantly reduced. We identify the amidotransferase AsnB as responsible for mDAP amidation and show that the gene encoding it is essential without added Mg2+ . Growth without excess Mg2+ causes ΔasnB mutant cells to deform and ultimately lyse. In cell regions with deformations, PG insertion is orderly and indistinguishable from the wild-type. However, PG degradation is unevenly distributed along the sidewalls. Furthermore, ΔasnB mutant cells exhibit increased sensitivity to antibiotics targeting the cell wall. These results suggest that absence of amidated mDAP causes a lethal deregulation of PG hydrolysis that can be inhibited by increased levels of Mg2+ . Consistently, we find that Mg2+ inhibits autolysis of wild-type cells. We suggest that Mg2+ helps to maintain the balance between PG synthesis and hydrolysis in cell wall mutants where this balance is perturbed in favor of increased degradation.

Analysis of Cell Wall Teichoic Acids in Staphylococcus aureus.

Most bacterial cells are surrounded by a surface composed mainly of peptidoglycan (PGN), a glycopolymer responsible for ensuring the bacterial shape and a telltale molecule that betrays the presence of bacteria to the host immune system. In Staphylococcus aureus, as in most gram-positive bacteria, peptidoglycan is concealed by covalently linked molecules of wall teichoic acids (WTA)-phosphate rich molecules made of glycerol and ribitol phosphates which may be tailored by different amino acids and sugars.In order to analyze and compare the composition of WTA produced by different S. aureus strains, we describe methods to: (1) quantify the total amount of WTA present at the bacterial cell surface, through the determination of the inorganic phosphate present in phosphodiester linkages of WTA; (2) identify which sugar constituents are present in the assembled WTA molecules, by detecting the monosaccharides, released by acid hydrolysis, through an high-performance anion exchange chromatography analysis coupled with pulsed amperometric detection (HPAEC-PAD) and (3) compare the polymerization degree of WTA found at the cell surface of different S. aureus strains, through their different migration in a polyacrylamide gel electrophoresis (PAGE).

Cell shape dynamics during the staphylococcal cell cycle.

Staphylococcus aureus is an aggressive pathogen and a model organism to study cell division in sequential orthogonal planes in spherical bacteria. However, the small size of staphylococcal cells has impaired analysis of changes in morphology during the cell cycle. Here we use super-resolution microscopy and determine that S. aureus cells are not spherical throughout the cell cycle, but elongate during specific time windows, through peptidoglycan synthesis and remodelling. Both peptidoglycan hydrolysis and turgor pressure are required during division for reshaping the flat division septum into a curved surface. In this process, the septum generates less than one hemisphere of each daughter cell, a trait we show is common to other cocci. Therefore, cell surface scars of previous divisions do not divide the cells in quadrants, generating asymmetry in the daughter cells. Our results introduce a need to reassess the models for division plane selection in cocci.

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release.

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.

L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane.

Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for L-rhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection.

Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance.

Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG) synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins), when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus.

Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria.

The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 5' end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal.

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries.

Thioridazine induces major changes in global gene expression and cell wall composition in methicillin-resistant Staphylococcus aureus USA300.

Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant Staphylococcus aureus (MRSA) by ß-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and ß-lactams is not fully understood. In the present study, we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan (PGN) synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs PGN biosynthesis at a stage that precedes transpeptidation by penicillin-binding proteins (PBPs). In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced ß-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal PGN precursors with pentaglycine branches, the correct substrate of S. aureus PBPs. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards ß-lactam antibiotics.