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MBio Jun 2022β-Lactam antibiotics exploit the essentiality of the bacterial cell envelope by perturbing the peptidoglycan layer, typically resulting in rapid lysis and death. Many...
β-Lactam antibiotics exploit the essentiality of the bacterial cell envelope by perturbing the peptidoglycan layer, typically resulting in rapid lysis and death. Many Gram-negative bacteria do not lyse but instead exhibit "tolerance," the ability to sustain viability in the presence of bactericidal antibiotics for extended periods. Antibiotic tolerance has been implicated in treatment failure and is a stepping-stone in the acquisition of true resistance, and the molecular factors that promote intrinsic tolerance are not well understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. Carbapenem β-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections, but treatment failure is increasingly prevalent. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate tolerance to high-level meropenem treatment, form spheroplasts upon exposure to the antibiotic, and revert to normal growth after antibiotic removal. Using transcriptomics and genetic screens, we show that several genes associated with outer membrane integrity maintenance and efflux promote tolerance, likely by limiting entry into the periplasm. Genes associated with peptidoglycan homeostasis in the periplasm and cytoplasm also answered our screen, and their disruption compromised cell envelope barrier function. Finally, we defined the enzymatic activity of the tolerance determinants penicillin-binding protein 7 (PBP7) and ElsL (a cytoplasmic ld-carboxypeptidase). These data show that outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to A. baumannii meropenem tolerance. Carbapenem treatment failure associated with "superbug" infections has rapidly increased in prevalence, highlighting the urgent need to develop new therapeutic strategies. Antibiotic tolerance can directly lead to treatment failure but has also been shown to promote the acquisition of true resistance within a population. While some studies have addressed mechanisms that promote tolerance, factors that underlie Gram-negative bacterial survival during carbapenem treatment are not well understood. Here, we characterized the role of peptidoglycan recycling in outer membrane integrity maintenance and meropenem tolerance in A. baumannii. These studies suggest that the pathogen limits antibiotic concentrations in the periplasm and highlight physiological processes that could be targeted to improve antimicrobial treatment.
Topics: Acinetobacter baumannii; Anti-Bacterial Agents; Carbapenems; Gram-Negative Bacteria; Meropenem; Microbial Sensitivity Tests; Peptidoglycan
PubMed: 35638738
DOI: 10.1128/mbio.01001-22 -
The FEBS Journal Aug 2022The peptidoglycan (PG) cell wall is an essential polymer for the shape and viability of bacteria. Its protective role is in great part provided by its mesh-like... (Review)
Review
The peptidoglycan (PG) cell wall is an essential polymer for the shape and viability of bacteria. Its protective role is in great part provided by its mesh-like character. Therefore, PG-cross-linking enzymes like the penicillin-binding proteins (PBPs) are among the best targets for antibiotics. However, while PBPs have been in the spotlight for more than 50 years, another class of PG-cross-linking enzymes called LD-transpeptidases (LDTs) seemed to contribute less to PG synthesis and, thus, has kept an aura of mystery. In the last years, a number of studies have associated LDTs with cell wall adaptation to stress including β-lactam antibiotics, outer membrane stability, and toxin delivery, which has shed light onto the biological meaning of these proteins. Furthermore, as some species display a great abundance of LD-cross-links in their cell wall, it has been hypothesized that LDTs could also be the main synthetic PG-transpeptidases in some bacteria. In this review, we introduce these enzymes and their role in PG biosynthesis and we highlight the most recent advances in understanding their biological role in diverse species.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Cell Wall; Penicillin-Binding Proteins; Peptidoglycan; Peptidyl Transferases
PubMed: 34109739
DOI: 10.1111/febs.16066 -
ACS Chemical Biology Sep 2022, the major fungal pathogen in humans, is under the strong influence of bacterial peptidoglycan fragments to undergo the yeast-to-hyphae transition, a key virulent step...
, the major fungal pathogen in humans, is under the strong influence of bacterial peptidoglycan fragments to undergo the yeast-to-hyphae transition, a key virulent step in pathogenesis and infections. However, due to the synthetic difficulties of obtaining peptidoglycan fragments for biological studies, mechanistic details of how recognizes and uptakes these peptidoglycan fragments have not been well elucidated. Notably, previous works have solely focused on the synthetic peptidoglycan ligand, muramyl dipeptide (MDP), despite its poor hyphal-inducing activity in . In this work, we isolated and purified natural peptidoglycan fragments via enzymatic degradation of bacteria cell wall sacculi and chemoenzymatically installed a series of functional d-amino acids into the natural muropeptide, creating peptidoglycan probes that bear photoaffinity, bio-orthogonal, or fluorescent functionality. Using these chemoenzymatic peptidoglycan probes, we established that natural peptidoglycan fragments, which are potent hyphal-inducers, interact with the Cyr1 sensor protein in the in-gel fluorescence assay as well as in pulldown studies. Moreover, we established that bacterial peptidoglycan probes enter cells via an energy-dependent endocytic process.
Topics: Acetylmuramyl-Alanyl-Isoglutamine; Amino Acids; Bacteria; Candida albicans; Cell Wall; Humans; Ligands; Peptidoglycan
PubMed: 35968762
DOI: 10.1021/acschembio.2c00468 -
Chemistry (Weinheim An Der Bergstrasse,... Aug 2022The biosynthesis, breakdown, and modification of peptidoglycan (PG) play vital roles in both bacterial viability and in the response of human physiology to bacterial... (Review)
Review
The biosynthesis, breakdown, and modification of peptidoglycan (PG) play vital roles in both bacterial viability and in the response of human physiology to bacterial infection. Studies on PG biochemistry are hampered by the fact that PG is an inhomogeneous insoluble macromolecule. Chemical synthesis is therefore an important means to obtain PG fragments that may serve as enzyme substrates and elicitors of the human immune response. This review outlines the recent advances in the synthesis and biochemical studies of PG fragments, PG biosynthetic intermediates (such as Park's nucleotides and PG lipids), and PG breakdown products (such as muramyl dipeptides and anhydro-muramic acid-containing fragments). A rich variety of synthetic approaches has been applied to preparing such compounds since carbohydrate, peptide, and phospholipid chemical methodologies must all be applied.
Topics: Cell Wall; Humans; Macromolecular Substances; Muramic Acids; Peptidoglycan
PubMed: 35560956
DOI: 10.1002/chem.202200788 -
International Journal of Molecular... Mar 2022The synthesis of a peptidoglycan septum is a fundamental part of bacterial fission and is driven by a multiprotein dynamic complex called the divisome. FtsW and FtsI are... (Review)
Review
The synthesis of a peptidoglycan septum is a fundamental part of bacterial fission and is driven by a multiprotein dynamic complex called the divisome. FtsW and FtsI are essential proteins that synthesize the peptidoglycan septum and are controlled by the regulatory FtsBLQ subcomplex and the activator FtsN. However, their mode of regulation has not yet been uncovered in detail. Understanding this process in detail may enable the development of new compounds to combat the rise in antibiotic resistance. In this review, recent data on the regulation of septal peptidoglycan synthesis is summarized and discussed. Based on structural models and the collected data, multiple putative interactions within FtsWI and with regulators are uncovered. This elaborates on and supports an earlier proposed model that describes active and inactive conformations of the septal peptidoglycan synthesis complex that are stabilized by these interactions. Furthermore, a new model on the spatial organization of the newly synthesized peptidoglycan and the synthesis complex is presented. Overall, the updated model proposes a balance between several allosteric interactions that determine the state of septal peptidoglycan synthesis.
Topics: Bacterial Proteins; Cell Wall; Membrane Proteins; Peptidoglycan
PubMed: 35408901
DOI: 10.3390/ijms23073537 -
Current Opinion in Cell Biology Feb 2021Bacterial cell division is orchestrated by the divisome, a protein complex centered on the tubulin homolog FtsZ. FtsZ polymerizes into a dynamic ring that defines the... (Review)
Review
Bacterial cell division is orchestrated by the divisome, a protein complex centered on the tubulin homolog FtsZ. FtsZ polymerizes into a dynamic ring that defines the division site, recruits downstream proteins, and directs peptidoglycan synthesis to drive constriction. Recent studies have documented treadmilling of FtsZ polymer clusters both in cells and in vitro. Emerging evidence suggests that FtsZ dynamics are regulated largely by intrinsic properties of FtsZ itself and by the membrane anchoring protein FtsA. Although FtsZ dynamics are broadly required for Z-ring assembly, their role(s) during constriction may vary among bacterial species. These recent advances set the stage for future studies to investigate how FtsZ dynamics are physically and/or functionally coupled to peptidoglycan metabolic enzymes to direct efficient division.
Topics: Bacteria; Bacterial Proteins; Cell Division; Cell Wall; Cytoskeletal Proteins; Peptidoglycan
PubMed: 33220539
DOI: 10.1016/j.ceb.2020.10.013 -
Proceedings of the National Academy of... Jun 2023AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of...
AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX-EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX-EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC.
Topics: Escherichia coli Proteins; Peptidoglycan; N-Acetylmuramoyl-L-alanine Amidase; Escherichia coli; Amidohydrolases; Bacterial Proteins
PubMed: 37276423
DOI: 10.1073/pnas.2302580120 -
International Journal of Medical... Nov 2019The ability to recover components of their own cell wall is a common feature of bacteria. This was initially recognized in the Gram-negative bacterium Escherichia coli,... (Review)
Review
The ability to recover components of their own cell wall is a common feature of bacteria. This was initially recognized in the Gram-negative bacterium Escherichia coli, which recycles about half of the peptidoglycan of its cell wall during one cell doubling. Moreover, E. coli was shown to grow on peptidoglycan components provided as nutrients. A distinguished recycling enzyme of E. coli required for both, recovery of the cell wall sugar N-acetylmuramic acid (MurNAc) of the own cell wall and for growth on external MurNAc, is the MurNAc 6-phosphate (MurNAc 6P) lactyl ether hydrolase MurQ. We revealed however, that most Gram-negative bacteria lack a murQ ortholog and instead harbor a pathway, absent in E. coli, that channels MurNAc directly to peptidoglycan biosynthesis. This "anabolic recycling pathway" bypasses the initial steps of peptidoglycan de novo synthesis, including the target of the antibiotic fosfomycin, thus providing intrinsic resistance to the antibiotic. The Gram-negative oral pathogen Tannerella forsythia is auxotrophic for MurNAc and apparently depends on the anabolic recycling pathway to synthesize its own cell wall by scavenging cell wall debris of other bacteria. In contrast, Gram-positive bacteria lack the anabolic recycling genes, but mostly contain one or two murQ orthologs. Quantification of MurNAc 6P accumulation in murQ mutant cells by mass spectrometry allowed us to demonstrate for the first time that Gram-positive bacteria do recycle their own peptidoglycan. This had been questioned earlier, since peptidoglycan turnover products accumulate in the spent media of Gram-positives. We showed, that these fragments are recovered during nutrient limitation, which prolongs starvation survival of Bacillus subtilis and Staphylococcus aureus. Peptidoglycan recycling in these bacteria however differs, as the cell wall is either cleaved exhaustively and monosaccharide building blocks are taken up (B. subtilis) or disaccharides are released and recycled involving a novel phosphomuramidase (MupG; S.aureus). In B. subtilis also the teichoic acids, covalently bound to the peptidoglycan (wall teichoic acids; WTAs), are recycled. During phosphate limitation, the sn-glycerol-3-phosphate phosphodiesterase GlpQ specifically degrades WTAs of B. subtilis. In S. aureus, in contrast, GlpQ is used to scavenge external teichoic acid sources. Thus, although bacteria generally recover their own cell wall, they apparently apply distinct strategies for breakdown and reutilization of cell wall fragments. This review summarizes our work on this topic funded between 2011 and 2019 by the DFG within the collaborative research center SFB766.
Topics: Anti-Bacterial Agents; Bacteria; Cell Wall; Glycoside Hydrolases; Metabolic Networks and Pathways; Muramic Acids; Peptidoglycan; Phosphoric Diester Hydrolases; Species Specificity; Teichoic Acids
PubMed: 31296364
DOI: 10.1016/j.ijmm.2019.06.006 -
Nature Reviews. Microbiology Jul 2019The Gram-negative envelope is a complex structure that consists of the inner membrane, the periplasm, peptidoglycan and the outer membrane, and protects the bacterial... (Review)
Review
The Gram-negative envelope is a complex structure that consists of the inner membrane, the periplasm, peptidoglycan and the outer membrane, and protects the bacterial cell from the environment. Changing environmental conditions can cause damage, which triggers the envelope stress responses to maintain cellular homeostasis. In this Review, we explore the causes, both environmental and intrinsic, of envelope stress, as well as the cellular stress response pathways that counter these stresses. Furthermore, we discuss the damage to the cell that occurs when these pathways are aberrantly activated either in the absence of stress or to an excessive degree. Finally, we review the mechanisms whereby the σ response constantly acts to prevent cell death caused by highly toxic unfolded outer membrane proteins. Together, the recent work that we discuss has provided insights that emphasize the necessity for proper levels of stress response activation and the detrimental consequences that can occur in the absence of proper regulation.
Topics: Bacterial Outer Membrane; Bacterial Proteins; Cell Membrane; Cell Wall; Gene Expression Regulation, Bacterial; Gram-Negative Bacteria; Lipopolysaccharides; Microbial Viability; Peptidoglycan; Sigma Factor; Stress, Physiological
PubMed: 31150012
DOI: 10.1038/s41579-019-0199-0 -
The ISME Journal Jul 2023Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic...
Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.
Topics: Phylogeny; Peptidoglycan; Bacteria; Chloroflexi; Phenotype
PubMed: 37041326
DOI: 10.1038/s41396-023-01405-0