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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 -
Biological Chemistry Apr 2015Backbone cyclization has a profound impact on the biological activity and thermal and proteolytic stability of proteins and peptides. Chemical methods for cyclization... (Review)
Review
Backbone cyclization has a profound impact on the biological activity and thermal and proteolytic stability of proteins and peptides. Chemical methods for cyclization are not always feasible, especially for large peptides or proteins. Recombinant Staphylococcus aureus sortase A shows potential as a new tool for the cyclization of both proteins and peptides. In this review, the scope and background of the sortase-mediated cyclization are discussed. High efficiency, versatility, and easy access make sortase A a promising cyclization tool, both for recombinant and chemo-enzymatic production methods.
Topics: Amino Acid Sequence; Aminoacyltransferases; Bacterial Proteins; Carbohydrate Sequence; Cyclization; Cysteine Endopeptidases; Models, Molecular; Molecular Sequence Data; Peptides; Peptides, Cyclic; Peptidoglycan; Protein Conformation; Protein Engineering; Proteins; Recombinant Proteins; Staphylococcus aureus
PubMed: 25581753
DOI: 10.1515/hsz-2014-0260 -
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 -
Seminars in Cell & Developmental Biology Apr 2019Chemokines are a family of small proteins best known for their ability to orchestrate immune cell trafficking and recruitment to sites of infection. Their role in... (Review)
Review
Chemokines are a family of small proteins best known for their ability to orchestrate immune cell trafficking and recruitment to sites of infection. Their role in promoting host defense is multiplied by a number of additional receptor-dependent biological activities, and most, but not all, chemokines have been found to mediate direct antimicrobial effects against a broad range of microorganisms. The molecular mechanism(s) by which antimicrobial chemokines kill bacteria remains unknown; however, recent observations have expanded our fundamental understanding of chemokine-mediated bactericidal activity to reveal increasingly diverse and complex actions. In the current review, we present and consider mechanistic insights of chemokine-mediated antimicrobial activity against bacteria. We also discuss how contemporary advances are reshaping traditional paradigms and opening up new and innovative avenues of research with translational implications. Towards this end, we highlight a developing framework for leveraging chemokine-mediated bactericidal and immunomodulatory effects to advance pioneering therapeutic approaches for treating bacterial infections, including those caused by multidrug-resistant pathogens.
Topics: Animals; Anti-Infective Agents; Bacteria; Bacterial Infections; Cell Membrane; Cell Wall; Chemokines; Drug Resistance, Bacterial; Humans; Peptidoglycan; Protein Structure, Secondary; Structure-Activity Relationship
PubMed: 29432954
DOI: 10.1016/j.semcdb.2018.02.003 -
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 -
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 -
ACS Infectious Diseases Jul 2020This review highlights recent efforts to detect bacteria using engineered small molecules that are processed and incorporated similarly to their natural counterparts.... (Review)
Review
This review highlights recent efforts to detect bacteria using engineered small molecules that are processed and incorporated similarly to their natural counterparts. There are both scientific and clinical justifications for these endeavors. The use of detectable, cell-wall targeted chemical probes has elucidated microbial behavior, with several fluorescent labeling methods in widespread laboratory use. Furthermore, many existing efforts including ours, focus on developing new imaging tools to study infection in clinical practice. The bacterial cell wall, a remarkably rich and complex structure, is an outstanding target for bacteria-specific detection. Several cell wall components are found in bacteria but not mammals, especially peptidoglycan, lipopolysaccharide, and teichoic acids. As this review highlights, the development of laboratory tools for fluorescence microscopy has vastly outstripped related positron emission tomography (PET) or single photon emission computed tomography (SPECT) radiotracer development. However, there is great synergy between these chemical strategies, which both employ mimicry of endogenous substrates to incorporate detectable structures. As the field of bacteria-specific imaging grows, it will be important to understand the mechanisms involved in microbial incorporation of radionuclides. Additionally, we will highlight the clinical challenges motivating this imaging effort.
Topics: Bacteria; Cell Wall; Peptidoglycan; Positron-Emission Tomography; Teichoic Acids
PubMed: 32433879
DOI: 10.1021/acsinfecdis.9b00515 -
Current Biology : CB Apr 2018To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods...
To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially-that is, parallel to the cell long axis-throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog.
Topics: Alphaproteobacteria; Animals; Bacterial Proteins; Cell Wall; Nematoda; Peptidoglycan; Symbiosis
PubMed: 29576473
DOI: 10.1016/j.cub.2018.02.028