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The obligate intracellularChlamydiaceaedo not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensivede novosynthesis and uncontrolled release of immunogenic peptidoglycan subunits inChlamydia trachomatis. Our data indicate that the homolog of aBacillusNlpC/P60 protein is widely conserved amongChlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitorsin vitroand in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.Author SummaryFree-living bacteria are wrapped by a peptidoglycan cell wall. This envelope provides shape and protects from osmotic stress, while also triggering human immune defense. Chlamydial pathogens live inside human cells and dispensed with a cell wall in their isotonic niche. Instead, they produce a transient peptidoglycan ring that is essentially involved in cell division. Recycling of peptidoglycan is likely important for chlamydiae to maintain a complete cycle of peptidoglycan ring synthesis and cell division. Moreover, the process may help to save energy and to protect from recognition through the immune system. Despite a potentially central role in cell viability and pathogenicity almost nothing is known about recycling of peptidoglycan in chlamydiae. The minimalist organisms lack all enzymes known to catalyze the process in model organismEscherichia coli. Here, we found an NlpC/P60 enzyme to serve a critical step in decomposing peptidoglycan ring derived peptides inChlamydia trachomatis. Our data indicate that the peptidase recycles energy cost intensive components and breaks down the minimal recognition motif of innate immune factor NOD1. We also identified a natural lead compound to inhibit the NlpC/P60 enzyme opening the way for innovative anti-chlamydial drug development at the intersection of energy recovery, cell division, and immune evasion.

The human pathogeniHaemophilus influenzae/icauses respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria includingiH. influenzae/i. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficientiH. influenzae/icompared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in "foci" in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA foriH. influenzae/ipeptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.

SummaryFrancisella tularensis is a Gram-negative, intracellular bacterium that causes the zoonotic disease tularemia. Intracellular pathogens, including F. tularensis, have evolved mechanisms to survive in the harsh environment of macrophages and neutrophils, where they are exposed to cell envelope-damaging molecules. The bacterial cell wall, primarily composed of peptidoglycan (PG), maintains cell morphology, structure, and membrane integrity. Intracellular Gram-negative bacteria protect themselves from macrophage and neutrophil killing by recycling and repairing damaged PG – a process that involves over 50 different PG synthesis and recycling enzymes. Here, we identified a PG recycling enzyme, L,D-carboxypeptidase A (LdcA), of F. tularensis that is responsible for converting PG tetrapeptide stems to tripeptide stems. Unlike E. coli LdcA and most other orthologs, F. tularensis LdcA does not localize to the cytoplasm and also exhibits L,D-endopeptidase activity, converting PG pentapeptide stems to tripeptide stems. Loss of F. tularensis LdcA led to altered cell morphology and membrane integrity, as well as attenuation in a mouse pulmonary infection model and in primary and immortalized macrophages. Finally, an F. tularensis ldcA mutant protected mice against virulent Type A F. tularensis SchuS4 pulmonary challenge.

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of β-lactamase induction in the presence of β-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of meso-diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE-MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [13C,15N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a β-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE-MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20%.

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNAGly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNAGly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these ‘non-proteogenic’ tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of ‘proteogenic’ aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNAGly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.

Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE–MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE–MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.

The biogenesis of bacterial cell-envelope polysaccharides requires the translocation, across the plasma membrane, of sugar sub-units that are produced inside the cytoplasm. To this end, the hydrophilic sugars are anchored to a lipid phosphate carrier (undecaprenyl phosphate (C55-P)), yielding membrane intermediates which are translocated to the outer face of the membrane. Finally, the glycan moiety is transferred to a nascent acceptor polymer, releasing the carrier in the “inactive” undecaprenyl pyrophosphate (C55-PP) form. Thus, C55-P is generated through the dephosphorylation of C55-PP, itself arising from either de novo synthesis or recycling. Two types of integral membrane C55-PP phosphatases were described: BacA enzymes and a sub-group of PAP2 enzymes (type 2 phosphatidic acid phosphatases). The human pathogen Helicobacter pylori does not contain BacA homologue but has four membrane PAP2 proteins: LpxE, LpxF, HP0350 and HP0851. Here, we report the physiological role of HP0851, renamed HupA, via multiple and complementary approaches ranging from a detailed biochemical characterization to the assessment of its effect on cell envelope metabolism and microbe-host interactions. HupA displays a dual function as being the main C55-PP pyrophosphatase (UppP) and phosphatidylglycerol phosphate phosphatase (PGPase). Although not essential in vitro, HupA was essential in vivo for stomach colonization. In vitro, the remaining UppP activity was carried out by LpxE in addition to its lipid A 1-phosphate phosphatase activity. Both HupA and LpxE have crucial roles in the biosynthesis of several cell wall polysaccharides and thus constitute potential targets for new therapeutic strategies., Author summary Helicobacter pylori colonizes the human’s gastric mucosa and infects around 50% of the world’s population. This pathogen is responsible for chronic gastritis, peptic ulcers and in worst cases leads to gastric cancer. It has been classified as a class I carcinogen by the World Health Organization in 1994. Here, we show that HP0851, renamed HupA, is the major undecaprenyl pyrophosphate (C55-PP) phosphatase (UppP) and the major phosphatidylglycerol phosphate phosphatase (PGPase). This enzyme is also involved in cationic antimicrobial peptide (CAMP) resistance to which H. pylori hupA mutant shows an increased sensitivity (4 fold). This mutant was unable to colonize the stomach in mouse model of infection showing that even if hupA was not essential in vitro, this gene was essential in vivo. Both HupA and LpxE have crucial roles in the biosynthesis of several cell wall polysaccharides and thus constitute potential targets for new therapeutic strategies.

Clostridium difficile 630 possesses a cryptic but functional gene cluster vanG Cd homologous to the vanG operon of Enterococcus faecalis . Expression of vanG Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso-DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanG Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli . In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, cbrA (formerly yidS), was shown to protect E. coli cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway. IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C55-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.

Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C55-P). C55-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C55-P are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C55-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an induced-fit model of action.

The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background. Fil: Di Gregorio, Sabrina Noelia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernandez, Silvina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Cuirolo, Arabela Ximena. Université de Liège; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Verlaine, Olivier. Université de Liège; Bélgica Fil: Amoroso, Ana. Université de Liège; Bélgica Fil: Mengin Lecreulx, Dominique. Université Paris Sud; Francia Fil: Famiglietti, Angela María Rosa. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Joris, Bernard. Université de Liège; Bélgica Fil: Mollerach, Marta Eugenia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination. IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell., Author summary c-di-AMP has recently emerged as an essential molecule in Gram-positive bacteria, controlling osmotic stress resistance and virulence. The molecule has also been linked to antibiotic resistance/sensitivity, as alterations in its levels have been shown to modulate the effect of cell wall-targeting antibiotics. c-di-AMP is produced and degraded by dedicated enzymes and the activities of these enzymes are tightly regulated in the cell for optimal bacterial growth. In the human pathogen Staphylococcus aureus, c-di-AMP is produced by the di-adenylate cyclase enzyme DacA, which is thought to form a complex and be regulated by YbbR and GlmM, two other conserved proteins. There are ongoing efforts to devise strategies to inhibit di-adenylate cyclase enzymes, as inhibition of c-di-AMP production will negatively impact bacterial growth and also re-sensitize methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. Here, we investigated the activity and regulation of the S. aureus c-di-AMP cyclase DacA. We determined the atomic structures of the S. aureus DacA and GlmM enzymes and show that GlmM readily binds to DacA in vitro and blocks its activity by masking its active site. With this, our work provides important insight into possible ways to ablate c-di-AMP production in bacterial cells.

Significance Permanent infections with beneficial bacteria are widespread in nature and are believed to play a pivotal role in evolution. How hosts’ immune functions are regulated to maintain cooperative bacteria while preventing them from constantly activating the host immune system is a key question in understanding host–bacterial associations’ sustainability. In insects, beneficial bacteria are often confined within specialized host cells, the bacteriocytes, which avoid direct contact between the host’s immune system and bacteria. Here, we report an additional mechanism that prevents bacteria-released immunogenic molecules (i.e., peptidoglycan fragments) from escaping the bacteriocytes. We show that two peptidoglycan-cleaving enzymes are specifically produced in the bacteriocytes, where they cleave bacterial peptidoglycan into nonimmunogenic fragments, thereby preventing continuous and damageable host systemic immune activation., Long-term intracellular symbiosis (or endosymbiosis) is widely distributed across invertebrates and is recognized as a major driving force in evolution. However, the maintenance of immune homeostasis in organisms chronically infected with mutualistic bacteria is a challenging task, and little is known about the molecular processes that limit endosymbiont immunogenicity and host inflammation. Here, we investigated peptidoglycan recognition protein (PGRP)-encoding genes in the cereal weevil Sitophilus zeamais’s association with Sodalis pierantonius endosymbiont. We discovered that weevil pgrp-lb generates three transcripts via alternative splicing and differential regulation. A secreted isoform is expressed in insect tissues under pathogenic conditions through activation of the PGRP-LC receptor of the immune deficiency pathway. In addition, cytosolic and transmembrane isoforms are permanently produced within endosymbiont-bearing organ, the bacteriome, in a PGRP-LC–independent manner. Bacteriome isoforms specifically cleave the tracheal cytotoxin (TCT), a peptidoglycan monomer released by endosymbionts. pgrp-lb silencing by RNAi results in TCT escape from the bacteriome to other insect tissues, where it chronically activates the host systemic immunity through PGRP-LC. While such immune deregulations did not impact endosymbiont load, they did negatively affect host physiology, as attested by a diminished sexual maturation of adult weevils. Whereas pgrp-lb was first described in pathogenic interactions, this work shows that, in an endosymbiosis context, specific bacteriome isoforms have evolved, allowing endosymbiont TCT scavenging and preventing chronic endosymbiont-induced immune responses, thus promoting host homeostasis.

International audience; Peptidoglycan recognition proteins (PGRPs) constitute the primary means of bacterial recognition in insects. Recent work in the model organism Drosophila has revealed the mechanisms by which the complement of PGRPs refine the sensitivity of different tissues to bacterial elicitors, permitting the persistence of commensal bacteria in the gut whilst maintaining vigilance against bacterial infection. Here, we use in vivo knockdowns and in vitro pull-down assays to investigate the role of the three major isoforms of the transmembrane receptor of the Imd pathway, PGRPLC, in basal immunity in the Anopheles coluzzii mosquito midgut. Our results indicate that the mosquito midgut is regionalized in its expression of immune effectors and of PGRPLC1. We show that PGRPLC1 and PGRPLC3 are pulled down with polymeric DAP-type peptidoglycan, while PGRPLC2 and PGRPLC3 co-precipitate in the presence of TCT, a peptidoglycan monomer. These data suggest that, as found in Drosophila, discrimination of polymeric and monomeric PGN by Anopheles PGRPLC participates in the regulation of the Imd pathway.

Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination.IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.

International audience; Conjugation of RNA with multiple partners to obtain mimics of complex biomolecules is limited by the identification of orthogonal reactions. Here, lipid-carbohydrate-peptidyl-RNA conjugates were obtained by post-functionalization reactions, solid-phase synthesis, and enzymatic steps, to generate molecules mimicking the substrates of FmhB, an essential peptidoglycan synthesis enzyme of Staphylococcus aureus. Mimics of Gly-tRNAGly and lipid intermediate II (undecaprenyl-diphospho-disaccharide-pentapeptide) were combined in a single "bi-substrate" inhibitor (IC50 =56 nm). The synthetic route was exploited to generate substrates and inhibitors containing d-lactate residue (d-Lac) instead of d-Ala at the C-terminus of the pentapeptide stem, a modification responsible for vancomycin resistance in the enterococci. The substitution impaired recognition of peptidoglycan precursors by FmhB. The associated fitness cost may account for limited dissemination of vancomycin resistance genes in S. aureus.

International audience; The bacterial cell wall peptidoglycan contains unusual Land D amino acids assembled in branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by L,D-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the L,D-transpeptidase Ldt$_{Mt2}$ from M. tuberculosis. Accordingly, construction of a conditional mutant established the essentiality of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.

Peptidoglycan (PG) is a highly cross-linked, protective mesh-like sacculus that surrounds the bacterial cytoplasmic membrane. Expansion of PG is tightly coupled to growth of a bacterial cell and requires hydrolases to cleave the cross-links for insertion of nascent PG material. In Escherichia coli, a proteolytic system comprising the periplasmic PDZ-protease Prc and the lipoprotein adaptor NlpI contributes to PG enlargement by regulating cellular levels of MepS, a cross-link-specific hydrolase. Here, we demonstrate how NlpI binds Prc to facilitate the degradation of its substrate MepS by structural and mutational analyses. An NlpI homodimer binds two molecules of Prc and forms three-sided MepS-docking cradles using its tetratricopeptide repeats. Prc forms a monomeric bowl-shaped structure with a lid-like PDZ domain connected by a substrate-sensing hinge that recognizes the bound C terminus of the substrate. In summary, our study reveals mechanistic details of protein degradation by the PDZ-protease Prc bound to its cognate adaptor protein., MepS is a peptidoglycan (PG) cross-link specific hydrolase needed for cell wall expansion and its cellular levels must be tightly regulated. Here the authors present the structure of the MepS degrading protease Prc bound to its adaptor NlpI and propose a model how the NlpI-Prc complex mediates MepS degradation.

A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.

Phages play critical roles in the spread of virulence factors and control of bacterial populations through their predation of bacteria. An essential step in the phage lifecycle is genome entry, where the infecting phage must productively interact with the components of the bacterial cell envelope in order to transmit its genome out of the viral particle and into the host cell cytoplasm. In this study, we characterize this process for the Escherichia coli phage HK97. We have discovered that HK97 genome injection requires the activities of the inner membrane glucose transporter protein, PtsG, and the periplasmic chaperone, FkpA. The requirements for PtsG and FkpA are determined by the sequence of the phage tape measure protein (TMP). We also identify a region of the TMP that mediates inhibition of phage genome injection by the HK97 superinfection exclusion protein, gp15. This region of the TMP also determines the PtsG requirement, and we show that gp15-mediated inhibition requires PtsG. Based on these data, we present a model for the in vivo genome injection process of phage HK97 and postulate a mechanism by which the inhibitory action of gp15 is reliant upon PtsG.

International audience; The straightforward synthesis of 5'-methylene-[1,4]-triazole-substituted aminoribosyl uridines is described. Two families of compounds were synthesized from a unique epoxide which was regioselectively opened by acetylide ions (for compounds II) or azide ions (for compounds III). Sequential diastereoselective glycosylation with a ribosyl fluoride derivative, Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) with various complementary azide and alkyne partners afforded the targeted compounds after final deprotection. The biological activity of the 16 resulting compounds together with that of 14 previously reported compounds I, lacking the 5' methylene group, was evaluated on the MraY transferase activity. Out of the 30 tested compounds, 18 compounds revealed MraY inhibition with IC50 ranging from 15 to 150 μM. A molecular modeling study was performed to rationalize the observed structure-activity relationships (SAR), which allowed us to correlate the activity of the most potent compounds with an interaction involving Leu191 of MraYAA. The antibacterial activity was also evaluated and seven compounds exhibited a good activity against Gram-positive bacterial pathogens with MIC ranging from 8 to 32 μg mL(-1), including the methicillin resistant Staphylococcus aureus (MRSA).

The activity of the PrkC protein kinase is regulated in a sophisticated manner in Bacillus subtilis cells. In spores, in the presence of muropeptides, PrkC stimulates dormancy exit. The extracellular region containing PASTA domains binds peptidoglycan fragments to probably enhance the intracellular kinase activity. During exponential growth, the cell division protein GpsB interacts with the intracellular domain of PrkC to stimulate its activity. In this paper, we have reinvestigated the regulation of PrkC during exponential and stationary phases. We observed that, during exponential growth, neither its septal localization nor its activity are influenced by the addition of peptidoglycan fragments or by the deletion of one or all PASTA domains. However, Dynamic Light Scattering experiments suggest that peptidoglycan fragments bind specifically to PrkC and induce its oligomerization. In addition, during stationary phase, PrkC appeared evenly distributed in the cell wall and the deletion of one or all PASTA domains led to a non-activated kinase. We conclude that PrkC activation is not as straightforward as previously suggested and that regulation of its kinase activity via the PASTA domains and peptidoglycan fragments binding occurs when PrkC is not concentrated to the bacterial septum, but all over the cell wall in non-dividing bacillus cells.

International audience; Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C-55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C-55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.

International audience; Members of the GH73 glycosidase family cleave the β-1,4-glycosidic bond between the N-acetylglucosaminyl (GlcNAc) and N-acetylmuramyl (MurNAc) moieties in bacterial peptidoglycan. A catalytic mechanism has been proposed for members FlgJ, Auto, AcmA and Atl(WM) and the structural analysis of FlgJ and Auto revealed a conserved α/β fold reminiscent of the distantly related GH23 lysozyme. Comparison of the active site residues reveals variability in the nature of the catalytic general base suggesting two distinct catalytic mechanisms: an inverting mechanism involving two distant glutamate residues and a substrate-assisted mechanism involving anchimeric assistance by the C2-acetamido group of the GlcNAc moiety. Herein, we present the biochemical characterization and crystal structure of TM0633 from the hyperthermophilic bacterium Thermotoga maritima. TM0633 adopts the α/β fold of the family and displays β-N-acetylglucosaminidase activity on intact peptidoglycan sacculi. Site-directed mutagenesis identifies Glu34, Glu65 and Tyr118 as important residues for catalysis. A thorough bioinformatic analysis of the GH73 sequences identified five phylogenetic clusters. TM0633, FlgJ and Auto belong to a group of three clusters that conserve two carboxylate residues involved in a classical inverting acid-base mechanism. Members of the other two clusters lack a conserved catalytic general base supporting a substrate-assisted mechanism. Molecular modeling of representative members from each cluster suggests that variability in length of the β-hairpin region above the active site confers ligand-binding specificity and modulates the catalytic mechanisms within the GH73 family.

During the biogenesis of bacterial cell-wall polysaccharides, such as peptidoglycan, cytoplasmic synthesized precursors should be trafficked across the plasma membrane. This essential process requires a dedicated lipid, undecaprenyl-phosphate that is used as a glycan lipid carrier. The sugar is linked to the lipid carrier at the inner face of the membrane and is translocated toward the periplasm, where the glycan moiety is transferred to the growing polymer. Undecaprenyl-phosphate originates from the dephosphorylation of its precursor undecaprenyl-diphosphate, with itself generated by de novo synthesis or by recycling after the final glycan transfer. Undecaprenyl-diphosphate is de novo synthesized by the cytosolic cis-prenyltransferase undecaprenyl-diphosphate synthase, which has been structurally and mechanistically characterized in great detail highlighting the condensation process. In contrast, the next step toward the formation of the lipid carrier, the dephosphorylation step, which has been overlooked for many years, has only started revealing surprising features. In contrast to the previous step, two unrelated families of integral membrane proteins exhibit undecaprenyl-diphosphate phosphatase activity: BacA and members of the phosphatidic acid phosphatase type 2 super-family, raising the question of the significance of this multiplicity. Moreover, these enzymes establish an unexpected link between the synthesis of bacterial cell-wall polymers and other biological processes. In the present review, the current knowledge in the field of the bacterial lipid carrier, its mechanism of action, biogenesis, recycling, regulation, and future perspective works are presented.

International audience; Activation of the innate immune response in Metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor PGRP-LC and by the intracellular receptor PGRP-LE. Here, we show that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria.

Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld -carboxypeptidase A (LdcA) catabolizes the bond between the last two l - and d -amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA ( Na LdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The Na LdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.

The synthesis of the peptide stem of bacterial peptidoglycan involves four enzymes, the Mur ligases (MurC, D, E and F). Among them, MurD is responsible for the ATP-dependent addition of d-glutamic acid to UDP-MurNAc-l-Ala, a reaction which involves acyl-phosphate and tetrahedral intermediates. Like most enzymes of peptidoglycan biosynthesis, MurD constitutes an attractive target for the design and synthesis of new antibacterial agents. Escherichia coli MurD has been the first Mur ligase for which the tridimensional (3D) structure was solved. Thereafter, several co-crystal structures with different ligands or inhibitors were released. In the present review, we will deal with work performed on substrate specificity, reaction mechanism and 3D structure of E. coli MurD. Then, a part of the review will be devoted to recent work on MurD orthologs from species other than E. coli and to cellular organization of Mur ligases and in vivo regulation of the MurD activity. Finally, we will review the different classes of MurD inhibitors that have been designed and assayed to date with the hope of obtaining new antibacterial compounds.

Background: MurE controls stereochemical incorporation of lysine or diaminopimelate into peptidoglycan stem peptides. Results: The structure of S. aureus MurE reveals an unexpected lack of specificity for lysine within the active site. Conclusion: Incorporation of lysine is supported by the comparatively high concentration of cytoplasmic lysine, not enzyme specificity. Significance: This study provides new perspectives in targeting Gram-positive peptidoglycan assembly for antimicrobial discovery., Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

International audience; Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials.

vanGCd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanGCd cluster to confer resistance. We showed that expression of vanGCd is inducible by vancomycin and that VanGCd , VanXYCd and VanTCd are functional, exhibiting D-Ala : D-Ser ligase, D,D-dipeptidase and D-Ser racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans-complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sublethal concentration of vancomycin led to production of UDP-MurNAc-pentapeptide[D-Ser], suggesting that the vanGCd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP-MurNAc-tetrapeptide, UDP-MurNAc-pentapeptide[D-Ala] and UDP-MurNAc-pentapeptide[D-Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra-Tetra is amidated on the meso-diaminopimelic acid. Taken together, our results suggest that the vanGCd gene cluster is functional and is prevented from promoting vancomycin resistance in C. difficile.