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1.
Angew Chem Int Ed Engl ; 62(25): e202301522, 2023 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-37099323

RESUMO

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.


Assuntos
Proteínas de Bactérias , Peptidoglicano Glicosiltransferase , Proteínas de Bactérias/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Peptidoglicano/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Membrana/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Parede Celular/metabolismo
2.
Braz J Microbiol ; 54(2): 609-618, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36973582

RESUMO

Shigellosis remains a worldwide health problem due to the lack of vaccines and the emergence of antibiotic-resistant strains. Shigella (S.) dysenteriae has rigid peptidoglycan (PG), and its tight regulation of biosynthesis and remodeling is essential for bacterial integrity. Lytic transglycosylases are highly conserved PG autolysins in bacteria that play essential roles in bacterial growth. However, their precise functions are obscure. We aimed to identify, clone, and express MltC, a unique autolysin in Escherichia (E.) coli C41 strain. The purification of recombinant MltC protein was performed using affinity chromatography and size-exclusion chromatography methods. The PG enzymatic activity of MltC was investigated using Zymogram and Fluorescein isothiocyanate (FITC)-labeled PG assays. Also, we aimed to detect its localization in bacterial fractions (cytoplasm and membrane) by western blot using specific polyclonal anti-MltC antibodies and its probable partners using immunoprecipitation and mass spectrometry applications. Purified MltC showed autolysin activity. Native MltC showed various locations in S. dysenteriae cells during different growth phases. In the Lag and early stationary phases, MltC was not found in cytoplasm and membrane fractions. However, it was detected in cytoplasm and membrane fractions during the exponential phase. In the late stationary phase, MltC was expressed in the membrane fraction only. Different candidate protein partners of MltC were identified that could be essential for bacterial growth and pathogenicity. This is the first study to suggest that MltC is indeed autolysin and could be a new drug target for the treatment of shigellosis by understanding its biological functions.


Assuntos
Disenteria Bacilar , Peptidoglicano Glicosiltransferase , Humanos , Peptidoglicano Glicosiltransferase/metabolismo , Shigella dysenteriae/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo
3.
J Bacteriol ; 204(12): e0023922, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36317921

RESUMO

The class A penicillin-binding proteins (aPBPs), PBP1A and PBP1B, are major peptidoglycan synthases that synthesize more than half of the peptidoglycan per generation in Escherichia coli. Whereas aPBPs have distinct roles in peptidoglycan biosynthesis during growth (i.e., elongation and division), they are semiredundant; disruption of either is rescued by the other to maintain envelope homeostasis and promote proper growth. Acinetobacter baumannii is a nosocomial pathogen that has a high propensity to overcome antimicrobial treatment. A. baumannii contains both PBP1A and PBP1B (encoded by mrcA and mrcB, respectively), but only mrcA deletion decreased fitness and contributed to colistin resistance through inactivation of lipooligosaccharide biosynthesis, indicating that PBP1B was not functionally redundant with the PBP1A activity. While previous studies suggested a distinct role for PBP1A in division, it was unknown whether its role in septal peptidoglycan biosynthesis was direct. Here, we show that A. baumannii PBP1A has a direct role in division through interactions with divisome components. PBP1A localizes to septal sites during growth, where it interacts with the transpeptidase PBP3, an essential division component that regulates daughter cell formation. PBP3 overexpression was sufficient to rescue the division defect in ΔmrcA A. baumannii; however, PBP1A overexpression was not sufficient to rescue the septal defect when PBP3 was inhibited, suggesting that their activity is not redundant. Overexpression of a major dd-carboxypeptidase, PBP5, also restored the canonical A. baumannii coccobacilli morphology in ΔmrcA cells. Together, these data support a direct role for PBP1A in A. baumannii division and highlights its role as a septal peptidoglycan synthase. IMPORTANCE Peptidoglycan biosynthesis is a validated target of ß-lactam antibiotics, and it is critical that we understand essential processes in multidrug-resistant pathogens such as Acinetobacter baumannii. While model systems such as Escherichia coli have shown that PBP1A is associated with side wall peptidoglycan synthesis, we show herein that A. baumannii PBP1A directly interacts with the divisome component PBP3 to promote division, suggesting a unique role for the enzyme in this highly drug-resistant nosocomial pathogen. A. baumannii demonstrated unanticipated resistance and tolerance to envelope-targeting antibiotics, which may be driven by rewired peptidoglycan machinery and may underlie therapeutic failure during antibiotic treatment.


Assuntos
Acinetobacter baumannii , Infecção Hospitalar , Proteínas de Escherichia coli , Peptidoglicano Glicosiltransferase , Humanos , Acinetobacter baumannii/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Escherichia coli , Antibacterianos/farmacologia , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/química , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano Glicosiltransferase/metabolismo
4.
Bioorg Med Chem ; 67: 116819, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35635930

RESUMO

A series of salicylanilide compounds was previously identified as antibacterial agents that inhibit the peptidoglycan formation. To find the exact binding mode, we synthesized a benzophenone-containing salicylanilide compound (1) and used it as a photoaffinity probe to label Acinetobacter baumannii penicillin-binding protein (PBP1b). After incubation and photo-irradiation, the labeled protein was subjected to trypsin digestion, dialysis enrichment, LC-ESI-MS/MS analysis, and Mascot search to reveal an octadecapeptide sequence 364RQLRTEYQESDLTNQGLR381 that was labeled at E372. Our molecular docking experiments suggest a hydrophobic pocket surrounded by R367 and E372 is the binding site of salicylanilide 1. The pocket lies in between the transglycosylase and transpeptidase domains, thus binding of salicylanilide 1 can block the propagation pathway to disrupt the growth of peptidoglycan chain.


Assuntos
Peptidoglicano Glicosiltransferase , Benzofenonas/farmacologia , Escherichia coli/metabolismo , Simulação de Acoplamento Molecular , Peptidoglicano , Peptidoglicano Glicosiltransferase/química , Peptidoglicano Glicosiltransferase/metabolismo , Marcadores de Fotoafinidade , Salicilanilidas , Espectrometria de Massas em Tandem
5.
Commun Biol ; 5(1): 107, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35115684

RESUMO

The peptidoglycan (PG) cell wall provides shape and structure to most bacteria. There are two systems to build PG in rod shaped organisms: the elongasome and divisome, which are made up of many proteins including the essential MreB and PBP2, or FtsZ and PBP3, respectively. The elongasome is responsible for PG insertion during cell elongation, while the divisome is responsible for septal PG insertion during division. We found that the main elongasome proteins, MreB and PBP2, can be inhibited without affecting growth rate in a quorum sensing-independent density-dependent manner. Before cells reach a particular cell density, inhibition of the elongasome results in different physiological responses, including intracellular vesicle formation and an increase in cell size. This inhibition of MreB or PBP2 can be compensated for by the presence of the class A penicillin binding protein, PBP1B. Furthermore, we found this density-dependent growth resistance to be specific for elongasome inhibition and was consistent across multiple Gram-negative rods, providing new areas of research into antibiotic treatment.


Assuntos
Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Contagem de Células , Cefalexina/farmacologia , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Tioureia/administração & dosagem , Tioureia/análogos & derivados , Tioureia/farmacologia
6.
Proc Natl Acad Sci U S A ; 118(35)2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34429361

RESUMO

A cell wall made of the heteropolymer peptidoglycan (PG) surrounds most bacterial cells. This essential surface layer is required to prevent lysis from internal osmotic pressure. The class A penicillin-binding proteins (aPBPs) play key roles in building the PG network. These bifunctional enzymes possess both PG glycosyltransferase (PGT) and transpeptidase (TP) activity to polymerize the wall glycans and cross-link them, respectively. In Escherichia coli and other gram-negative bacteria, aPBP function is dependent on outer membrane lipoproteins. The lipoprotein LpoA activates PBP1a and LpoB promotes PBP1b activity. In a purified system, the major effect of LpoA on PBP1a is TP stimulation. However, the relevance of this activation to the cellular function of LpoA has remained unclear. To better understand why PBP1a requires LpoA for its activity in cells, we identified variants of PBP1a from E. coli and Pseudomonas aeruginosa that function in the absence of the lipoprotein. The changes resulting in LpoA bypass map to the PGT domain and the linker region between the two catalytic domains. Purification of the E. coli variants showed that they are hyperactivated for PGT but not TP activity. Furthermore, in vivo analysis found that LpoA is necessary for the glycan synthesis activity of PBP1a in cells. Thus, our results reveal that LpoA exerts a much greater control over the cellular activity of PBP1a than previously appreciated. It not only modulates PG cross-linking but is also required for its cognate synthase to make PG glycans in the first place.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Parede Celular/enzimologia , Reagentes de Ligações Cruzadas/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Lipoproteínas/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética
7.
Nat Commun ; 12(1): 2775, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986273

RESUMO

The pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of ß-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.


Assuntos
Antibacterianos/farmacologia , Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , beta-Lactamas/farmacologia , Acetilglucosamina/química , Aldeídos/química , Microscopia Crioeletrônica , Ácidos Murâmicos/química , Oligossacarídeos/farmacologia , Peptidoglicano/biossíntese , Conformação Proteica , Domínios Proteicos/fisiologia
8.
PLoS Genet ; 17(4): e1009366, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33857142

RESUMO

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try to understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.


Assuntos
Proteínas de Bactérias/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Proteínas de Ligação às Penicilinas/ultraestrutura , Peptidoglicano Glicosiltransferase/ultraestrutura , Conformação Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/química , Peptidoglicano/genética , Peptidoglicano/ultraestrutura , Peptidoglicano Glicosiltransferase/química , Peptidoglicano Glicosiltransferase/genética , Peptidil Transferases/química , Peptidil Transferases/genética , Peptidil Transferases/ultraestrutura
9.
Mol Microbiol ; 116(1): 41-52, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33709487

RESUMO

Until recently, class A penicillin-binding proteins (aPBPs) were the only enzymes known to catalyze glycan chain polymerization from lipid II in bacteria. Hence, the discovery of two novel lipid II polymerases, FtsW and RodA, raises new questions and has consequently received a lot of attention from the research community. FtsW and RodA are essential and highly conserved members of the divisome and elongasome, respectively, and work in conjunction with their cognate class B PBPs (bPBPs) to synthesize the division septum and insert new peptidoglycan into the lateral cell wall. The identification of FtsW and RodA as peptidoglycan glycosyltransferases has raised questions regarding the role of aPBPs in peptidoglycan synthesis and fundamentally changed our understanding of the process. Despite their dethronement, aPBPs are essential in most bacteria. So, what is their function? In this review, we discuss recent progress in answering this question and present our own views on the topic.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/biossíntese , Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Staphylococcus aureus/metabolismo , Streptococcus pneumoniae/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
10.
Mol Microbiol ; 116(1): 329-342, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33660879

RESUMO

The integrity of the cell envelope of E. coli relies on the concerted activity of multi-protein machineries that synthesize the peptidoglycan (PG) and the outer membrane (OM). Our previous work found that the depletion of lipopolysaccharide (LPS) export to the OM induces an essential PG remodeling process involving LD-transpeptidases (LDTs), the glycosyltransferase function of PBP1B and the carboxypeptidase PBP6a. Consequently, cells with defective OM biogenesis lyse if they lack any of these PG enzymes. Here we report that the morphological defects, and lysis associated with a ldtF mutant with impaired LPS transport, are alleviated by the loss of the predicted OM-anchored lipoprotein ActS (formerly YgeR). We show that ActS is an inactive member of LytM-type peptidoglycan endopeptidases due to a degenerated catalytic domain. ActS is capable of activating all three main periplasmic peptidoglycan amidases, AmiA, AmiB, and AmiC, which were previously reported to be activated only by EnvC and/or NlpD. Our data also suggest that in vivo ActS preferentially activates AmiC and that its function is linked to cell envelope stress.


Assuntos
Membrana Externa Bacteriana/fisiologia , Carboxipeptidases/metabolismo , Endopeptidases/metabolismo , Escherichia coli/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Carboxipeptidases/genética , Membrana Celular/fisiologia , Parede Celular/metabolismo , Endopeptidases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Lipopolissacarídeos/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Plasmídeos/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Estresse Fisiológico/fisiologia
11.
J Bacteriol ; 203(10)2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33649146

RESUMO

Lytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan must be unfailingly stable to preserve cell integrity, but must also be dynamically remodeled for the cell to grow, divide, and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG, and flagellar insertion is aided by localized activity of a dedicated PG lyase in Gram-negative bacteria. To date, there is no known dedicated lyase in Gram-positive bacteria for the insertion of flagella. Here, we take a reverse-genetic candidate-gene approach and find that cells mutated for the lytic transglycosylase CwlQ exhibit a severe defect in flagellum-dependent swarming motility. We further show that CwlQ is expressed by the motility sigma factor SigD and is secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells with mutations of CwlQ remain proficient for flagellar biosynthesis even when mutated in combination with four other lyases related to motility (LytC, LytD, LytF, and CwlO). The PG lyase (or lyases) essential for flagellar synthesis in B. subtilis, if any, remains unknown.IMPORTANCE Bacteria are surrounded by a wall of peptidoglycan and early work in Bacillus subtilis was the first to suggest that bacteria needed to enzymatically remodel the wall to permit insertion of the flagellum. No PG remodeling enzyme alone or in combination, however, has been found to be essential for flagellar assembly in B. subtilis Here, we take a reverse-genetic candidate-gene approach and find that the PG lytic transglycosylase CwlQ is required for swarming motility. Subsequent characterization determined that while CwlQ was coexpressed with motility genes and is secreted by the flagellar secretion apparatus, it was not required for flagellar synthesis. The PG lyase needed for flagellar assembly in B. subtilis remains unknown.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/fisiologia , Flagelos/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Movimento , Mutação , Peptidoglicano Glicosiltransferase/genética , Fator sigma/metabolismo , Sistemas de Secreção Tipo III/metabolismo
12.
Elife ; 102021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33625355

RESUMO

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as ß-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin-binding proteins (PBPs) are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here, we developed a novel Förster resonance energy transfer-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high-throughput screening for new antimicrobials.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano/biossíntese , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo
13.
Acc Chem Res ; 54(4): 917-929, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33512995

RESUMO

The need for new classes of antibacterials is genuine in light of the dearth of clinical options for the treatment of bacterial infections. The prodigious discoveries of antibiotics during the 1940s to 1970s, a period wistfully referred to as the Golden Age of Antibiotics, have not kept up in the face of emergence of resistant bacteria in the past few decades. There has been a renewed interest in old drugs, the repurposing of the existing antibiotics and pairing of synergistic antibiotics or of an antibiotic with an adjuvant. Notwithstanding, discoveries of novel classes of these life-saving drugs have become increasingly difficult, calling for new paradigms. We describe, herein, three strategies from our laboratories toward discoveries of new antibacterials and adjuvants using computational and multidisciplinary experimental methods. One approach targets penicillin-binding proteins (PBPs), biosynthetic enzymes of cell-wall peptidoglycan, for discoveries of non-ß-lactam inhibitors. Oxadiazoles and quinazolinones emerged as two structural classes out of these efforts. Several hundred analogs of these two classes of antibiotics have been synthesized and fully characterized in our laboratories. A second approach ventures into inhibition of allosteric regulation of cell-wall biosynthesis. The mechanistic details of allosteric regulation of PBP2a of Staphylococcus aureus, discovered in our laboratories, is outlined. The allosteric site in this protein is at 60 Å distance to the active site, whereby ligand binding at the former makes access to the latter by the substrate possible. We have documented that both quinazolinones and ceftaroline, a fifth-generation cephalosporin, bind to the allosteric site in manifestation of the antibacterial activity. Attempts at inhibition of the regulatory phosphorylation events identified three classes of antibacterial adjuvants and one class of antibacterials, the picolinamides. The chemical structures for these hits went through diversification by synthesis of hundreds of analogs. These analogs were characterized in various assays for identification of leads with adjuvant and antibacterial activities. Furthermore, we revisited the mechanism of bulgecins, a class of adjuvants discovered and abandoned in the 1980s. These compounds potentiate the activities of ß-lactam antibiotics by the formation of bulges at the sites of septum formation during bacterial replication, which are points of structural weakness in the envelope. These bulges experience rupture, which leads to bacterial death. Bulgecin A inhibits the lytic transglycosylase Slt of Pseudomonas aeruginosa as a likely transition-state mimetic for its turnover of the cell-wall peptidoglycan. Once damage to cell wall is inflicted by a ß-lactam antibiotic, the function of Slt is to repair the damage. When Slt is inhibited by bulgecin A, the organism cannot cope with it and would undergo rapid lysis. Bulgecin A is an effective adjuvant of ß-lactam antibiotics. These discoveries of small-molecule classes of antibacterials or of adjuvants to antibacterials hold promise in strategies for treatment of bacterial infections.


Assuntos
Adjuvantes Imunológicos/química , Antibacterianos/química , Sítio Alostérico , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Descoberta de Drogas , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Simulação de Dinâmica Molecular , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/antagonistas & inibidores , Peptidoglicano Glicosiltransferase/metabolismo , Pseudomonas aeruginosa/enzimologia , Quinazolinonas/química , Quinazolinonas/metabolismo , Staphylococcus aureus/metabolismo
14.
Nat Microbiol ; 6(5): 584-593, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33495624

RESUMO

Synthesis of septal peptidoglycan (sPG) is crucial for bacterial cell division. FtsW, an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a peptidoglycan glycosyltransferase (PGTase). Despite its importance, the septal PGTase activity of FtsW has not been demonstrated in vivo. How its activity is spatiotemporally regulated in vivo has also remained elusive. Here, we confirmed FtsW as an essential septum-specific PGTase in vivo using an N-acetylmuramic acid analogue incorporation assay. Next, using single-molecule tracking coupled with genetic manipulations, we identified two populations of processively moving FtsW molecules: a fast-moving population correlated with the treadmilling dynamics of the essential cytoskeletal FtsZ protein and a slow-moving population dependent on active sPG synthesis. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving population. Our results suggest a two-track model, in which inactive sPG synthases follow the 'Z-track' to be distributed along the septum and FtsN promotes their release from the Z-track to become active in sPG synthesis on the slow 'sPG-track'. This model provides a mechanistic framework for the spatiotemporal coordination of sPG synthesis in bacterial cell division.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Parede Celular/química , Parede Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Membrana/genética , Peptidoglicano/metabolismo , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano Glicosiltransferase/metabolismo , Imagem Individual de Molécula
15.
mBio ; 12(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402533

RESUMO

Despite dogma suggesting that lipopolysaccharide/lipooligosaccharide (LOS) was essential for viability of Gram-negative bacteria, several Acinetobacter baumannii clinical isolates produced LOS- colonies after colistin selection. Inactivation of the conserved class A penicillin-binding protein, PBP1A, was a compensatory mutation that supported isolation of LOS-A. baumannii, but the impact of PBP1A mutation was not characterized. Here, we show that the absence of PBP1A causes septation defects and that these, together with ld-transpeptidase activity, support isolation of LOS-A. baumannii PBP1A contributes to proper cell division in A. baumannii, and its absence induced cell chaining. Only isolates producing three or more septa supported selection of colistin-resistant LOS-A. baumannii PBP1A was enriched at the midcell, where the divisome complex facilitates daughter cell formation, and its localization was dependent on glycosyltransferase activity. Transposon mutagenesis showed that genes encoding two putative ld-transpeptidases (LdtJ and LdtK) became essential in the PBP1A mutant. Both LdtJ and LdtK were required for selection of LOS-A. baumannii, but each had distinct enzymatic activities in the cell. Together, these findings demonstrate that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity remodel the cell envelope to support selection of colistin-resistant LOS-A. baumanniiIMPORTANCE The increasing prevalence of antibiotic treatment failure associated with Gram-negative bacterial infections highlights an urgent need to develop new alternative therapeutic strategies. The last-line antimicrobial colistin (polymyxin E) targets the ubiquitous outer membrane lipopolysaccharide (LPS)/LOS membrane anchor, lipid A, which is essential for viability of most diderms. However, several LOS-Acinetobacter baumannii clinical isolates were recovered after colistin selection, suggesting a conserved resistance mechanism. Here, we characterized a role for penicillin-binding protein 1A in A. baumannii septation and intrinsic ß-lactam susceptibility. We also showed that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity support isolation of colistin-resistant LOS-A. baumannii.


Assuntos
Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Lipopolissacarídeos/deficiência , Proteínas de Ligação às Penicilinas/metabolismo , Peptidil Transferases/metabolismo , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Lipídeo A/metabolismo , Lipopolissacarídeos/genética , Testes de Sensibilidade Microbiana , Peptidoglicano Glicosiltransferase
16.
Mol Microbiol ; 115(6): 1170-1180, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33278861

RESUMO

Bacterial cells are surrounded by a peptidoglycan (PG) cell wall. This structure is essential for cell integrity and its biogenesis pathway is a key antibiotic target. Most bacteria utilize two types of synthases that polymerize glycan strands and crosslink them: class A penicillin-binding proteins (aPBPs) and complexes of SEDS proteins and class B PBPs (bPBPs). Although the enzymatic steps of PG synthesis are well characterized, the steps involved in terminating PG glycan polymerization remain poorly understood. A few years ago, the conserved lytic transglycosylase MltG was identified as a potential terminase for PG synthesis in Escherichia coli. However, characterization of the in vivo function of MltG was hampered by the lack of a growth or morphological phenotype in ΔmltG cells. Here, we report the isolation of MltG-defective mutants as suppressors of lethal deficits in either aPBP or SEDS/bPBP PG synthase activity. We used this phenotype to perform a domain-function analysis for MltG, which revealed that access to the inner membrane is important for its in vivo activity. Overall, our results support a model in which MltG functions as a terminase for both classes of PG synthases by cleaving PG glycans as they are being actively synthesized.


Assuntos
Parede Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano/biossíntese , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peptidoglicano Glicosiltransferase/genética
17.
Biochem J ; 478(1): 41-59, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33196080

RESUMO

Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-step Escherichia coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB, encoding a peptidoglycan-synthesizing enzyme and cpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, the growth of ΔmrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.


Assuntos
Membrana Celular/metabolismo , Parede Celular/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Proteínas Quinases/metabolismo , Tolerância ao Sal/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Citosol/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Floculação , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Mutação Puntual , Proteínas Quinases/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética
18.
mBio ; 11(6)2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144379

RESUMO

Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.IMPORTANCEEscherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division.


Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Ligação às Penicilinas/metabolismo , Antibacterianos/farmacologia , Cefsulodina/farmacologia , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Proteínas de Ligação às Penicilinas/química , Peptidoglicano/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Ligação Proteica , Conformação Proteica
19.
J Biol Chem ; 295(52): 18256-18265, 2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33109614

RESUMO

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75-Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.


Assuntos
Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Ligação Proteica , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética
20.
Elife ; 92020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32897856

RESUMO

Bacteria surround themselves with peptidoglycan, an adaptable enclosure that contributes to cell shape and stability. Peptidoglycan assembly relies on penicillin-binding proteins (PBPs) acting in concert with SEDS-family transglycosylases RodA and FtsW, which support cell elongation and division respectively. In Bacillus subtilis, cells lacking all four PBPs with transglycosylase activity (aPBPs) are viable. Here, we show that the alternative sigma factor σI is essential in the absence of aPBPs. Defects in aPBP-dependent wall synthesis are compensated by σI-dependent upregulation of an MreB homolog, MreBH, which localizes the LytE autolysin to the RodA-containing elongasome complex. Suppressor analysis reveals that cells unable to activate this σI stress response acquire gain-of-function mutations in the essential histidine kinase WalK, which also elevates expression of sigI, mreBH and lytE. These results reveal compensatory mechanisms that balance the directional peptidoglycan synthesis arising from the elongasome complex with the more diffusive action of aPBPs.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/biossíntese , Fator sigma/genética , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/genética , Peptidoglicano Glicosiltransferase/antagonistas & inibidores , Peptidoglicano Glicosiltransferase/metabolismo , Fator sigma/metabolismo , Regulação para Cima
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