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Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-D-alanyl-D-alanine

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... No ornithine, which would be indicative of the presence of glutaminyl residues, was detected. (v) The involvement of the y-carboxyl group of the glutamic acid in the peptide bond was shown by Edman degradation (168,169). The first cycle removed the N-terminal alanine, and the N-terminal glutamic acid appeared. ...
... After the second cycle of the degradation, ammonia was liberated and N-terminal amino acids were no longer detectable, again demonstrating that NH3 was a substituent of the a-carboxyl group of glutamic acid. The Edman degradation was initially carried out on a carbohydrate free polypeptide fraction (169). It provided the first proof for the occurrence of an isoglutaminyl residue in the wall peptide moiety, which observation was in agreement with the previous demonstration (57) of the involvement of the y-linkage of D-GIu to the next amino acid in the nucleotide precursor of the wall. ...
... Consequently, some of the peptide subunits must be involved in a peptide-peptide link rather than an N-acetylmuramic acid-peptide link in order to accommodate the presence of unsubstituted N-acetylmuramic acid residues. (iii) The further observation that virtually all of the glycine residues in native walls were C terminal (168,169) dismissed the possibility that Gly was the cross-linking bridge. A comprehensive structure for the M. lysodeikticus peptidoglycan finally emerged and its establishment rested upon the following important contributions. ...
... The mode of action of β-lactams was identified about forty years after the discovery of penicillin (Park & Strominger, 1957;Tipper & Strominger, 1965). β-lactams are suicide inhibitors of the Penicillin-Binding Proteins (PBPs), whose inactivation leads to cell death. ...
... PBPs are efficaciously inactivated by βlactams through the formation of a stable acylenzyme between the catalytic serine of PBPs and the carbonyl moiety of the β-lactam ring. This reaction is thought to occur because of the analogy between the structure of the β-lactam ring and that of the D-Ala 4 -D-Ala 5 end of the peptidoglycan precursor (Tipper & Strominger, 1965). The similarity between β-lactams and the natural substrates of PBPs resides in the distribution of electrons in the β-lactam ring, which is proposed to mimic the distribution of three electrostatic-negative wells at the terminal D-Ala 4 -D-Ala 5 of the natural substrate of PBPs ...
... Penicillin-Binding Proteins (PBPs) are thus called because they were identified by their ability to covalently bind penicillin, the first member of the β-lactam family of antibiotics (Suginaka et al., 1972;Tipper & Strominger, 1965;Wise & Park, 1965). After the reaction of membrane extracts with radioactive penicillin, PBPs are separated by SDS-PAGE and numbered according to their relative mobility in descending order of size (Spratt, 1975). ...
Thesis
Antibiotic resistance is a growing and global threat to human health that has led to an acute need for the development of new antibiotics. Elucidating the mechanism of inhibition of antibiotic targets is crucial for the development of more potent drugs. The essentiality of peptidoglycan and more than seventy years of successful use of β-lactams have made polymerization of this major cell wall component an attractive and validated target for drug development. Active-site serine Penicillin-Binding Proteins (PBPs) have long been considered as the only enzymes catalyzing the essential cross-linking step of peptidoglycan polymerization. The thesis explores inhibition of a distinct family of enzymes, the active-site cysteine L,D-transpeptidases (LDTs), that have a preponderant role in peptidoglycan synthesis in Mycobacterium tuberculosis. We show that the efficacy of LDT inhibition by β-lactams is primarily governed by the reactivity of the four-membered ring. We propose that acylation of LDTs by β-lactams proceeds through formation of an amine anion intermediate, followed by a subsequent irreversible step that is essential for the antibacterial activity of the drugs. A fluorescence spectroscopy approach enabling kinetic analyses of the acylation steps was developed to explore inactivation mechanisms and to evaluate the efficacy of new synthetic drugs. We also identify diazabicyclooctanes (DBOs) as new pharmacophores that inactivate LDTs by formation of a thio-carbamoyl-enzyme. We discuss several mechanism-based strategies for rational optimization of LDT inhibitors belonging to the β-lactam and DBO families.
... 3,4 β-Lactams inhibit PBPs, initially by competing with the D-Ala-D-Ala terminus of the stem peptide substrate to give a non-covalent complex, which then reacts with the activesite catalytic serine [in Pseudomonas aeruginosa PBP3 (PaPBP3): Ser294] to give an acyl−enzyme complex which is stable over a biologically relevant timescale. 5 The effectiveness of β-lactams to treat Gram-negative infections caused by Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, and P. aeruginosa is increasingly compromised by serine-and/or metallo-βlactamases (SBLs and MBLs, respectively), 6,7 with >1800 BL variants identified. 7 Ambler classes A, C, and D BLs are SBLs, while those employing a zinc ion-mediated mechanism are Ambler class B BLs (MBLs). ...
... 1-Hydroxy-N-[2-(methylamino)-2-oxo-ethyl]-3H-2,1-benzoxaborole-6-carboxamide (5). General Protocol 1 was followed using the following quantities of reagents: 3 (100 mg, 0.56 mmol, 1 equiv); N,N-DMF (2 mL); 1,1-carbonyldiimidazole (182 mg, 1.12 mmol, 2 equiv); 2-amino-N-methylacetamide HCl (104 mg, 0.84 mmol, 1.5 equiv). ...
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The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.
... This mechanism deviates from pervious described mechanism for cross binding [44]. The penicillin nucleus is isoster to the terminal D-Ala-D-Ala unit essential in the cross binding process (the Tipper-Strominger hypothesis) [59]. By interacting with and acylating the serine hydroxyl group in the penicillin-binding transpeptidases [60] cross binding and consequently formation of a stable cell wall in the bacteria is prevented [44,52]. ...
... The compound, however, never was approved as a drug by the FDA. The ureidopenicillins azlocillin (Figure 13, 57), mezlocillin (58) and piperazillin (59) were developed in the 1970s. They are efficient for treatment of infections with Pseudomonas aeruginosa, Klebsiella and some Enterobacter species. ...
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The appearance of antibiotic drugs revolutionized the possibilities for treatment of diseases with high mortality such as pneumonia, sepsis, plaque, diphtheria, tetanus, typhoid fever, and tuberculosis. Today fewer than 1% of mortalities in high income countries are caused by diseases caused by bacteria. However, it should be recalled that the antibiotics were introduced in parallel with sanitation including sewerage, piped drinking water, high standard of living and improved understanding of the connection between food and health. Development of salvarsan, sulfonamides, and β-lactams into efficient drugs is described. The effects on life expectancy and life quality of these new drugs are indicated.
... 66 It is thought that β-lactam antibiotics mimic the natural substrate of the transpeptidase domain, the terminal portion of the peptidyl chain (D-Ala-D-Ala) (Figure 1.5). [66][67] Both the natural peptidoglycan substrate and βlactam acylate a catalytic serine within the active site of the transpeptidase active site of the PBP. 66 The natural peptidoglycan-enzyme acyl species can react with a second peptidoglycan chain (terminal glylyl) forming the desired peptidoglycan cross-link ( Figure 1.5). ...
Article
Staphylococcus aureus is a Gram-positive human pathogen of significant clinical importance. The development of resistance to numerous classes of clinically important antibiotics has exacerbated the need for the development of novel therapeutic modalities for the treatment of serious, drug-resistant S. aureus infections. One approach for developing new therapies is the discovery of anti-virulence compounds. Virulence in S. aureus is mainly regulated by the agr quorum sensing system. The agr system is a master regulator of staphylococcal virulence and utilises the thiolactone macrocyclic peptide, auto-inducing peptide (AIP) as the signal molecule. The agr system is based around two divergent promoters, agrP2 and agrP3. The agrP2 promoter drives expression of the agr operon, which consists of 4 genes, agrB, agrD, agrC and agrA. AgrB and AgrD are responsible for the synthesis of AIP. AgrD is the pre-pro-peptide of AIP and is processed in part by the membrane bound cysteine protease AgrB, a process which ultimately forms the thiolactone macrocycle within the AIP molecule. Inhibitors of AgrC and AgrA have been studied extensively, however at present, there remains limited study of inhibitors of AgrB This thesis describes research towards the development of inhibitors of AgrB through the production of analogues of the natural product ambuic acid. This represents the first study of analogues of ambuic acid as inhibitors of staphylococcal virulence. These analogues include the synthesis of a (-)-enantiomer of a truncated ambuic acid analogue and the production of a range of ketal analogues. In addition, the attempted synthesis of a range of silyl ether analogues was also carried out, unfortunately, it was only possible to synthesise one analogue. In addition, the full chemical synthesis of a cyclopropyl isostere of the epoxide within a truncated ambuic acid analogue was established. This isostere was produced to determine whether the epoxide is required for inhibition of the putative target AgrB in an effort to advance the knowledge of how these compounds function. The anti-agr activity of ambuic acid has been fully characterised in vitro. Ambuic acid was found to be a potent inhibitor of staphylococcal agr activity utilising a bioluminescent S. aureus reporter. In addition, ambuic acid was found to cause a reduction in the level of expression of the staphylococcal exotoxin α-haemolysin. Furthermore, utilising a bioluminescent S. aureus reporter strain which is incapable of synthesising AIP, but capable of sensing exogenous AIP (designed for the characterisation of inhibitors of AIP sensing), ambuic acid was found to have no effect. This suggests that ambuic acid inhibits the synthesis of AIP rather than the sensing of AIP. The testing of the synthesised analogues has been carried out in vitro using the bioluminescent S. aureus agr-activity reporter and pharmacological paramaters such as IC50 were determined. The majority of compounds were found to be potent inhibitors of agr activity, with the majority of compounds found to have IC50 values of < 10 μM. Surprisingly, the cyclopropyl isosteric analogue was found to be a potent inhibitor of agr activity, suggesting that the epoxide is not required for agr-inhibitory activity. These data, taken together represent the first study of analogues of the natural fungal product ambuic acid as inhibitors of staphylococcal virulence. In addition, these data show that these analogues are promising inhibitors of agr-activity and the production of further analogues could be used for developing a structure-activity understanding of how this class of compounds function.
... Since the discovery of the penicillin, many theories behind its mechanism of action have been reported in the literature. Tipper and Strominger (1965) contemplated that penicillin has a highly reactive b-lactam ring, which acrylates the cell wall transpeptidase at its nucleophilic active site. Another study by Ghuysen et al. declared that penicillin could be an allosteric inhibitor of cell wall transpeptidase (Ghuysen et al., 1974). ...
Chapter
Cancer is the second leading cause of death worldwide, with approximately one in six deaths attributed to this disease. Due to its complex requirements in treatment and management, cancer remains a global social challenge, driving extensive scientific efforts into the development of new technological advances and materials, to provide novel cancer therapies with enhanced efficacy and safety. Monotherapy or the administration of a sole anticancer drug is still a clinical standard in cancer therapy; however, these treatment options are often deemed less effective than combinational therapy. Over the past few years, research efforts into the use of mesoporous silica nanoparticles (MSNs) as delivery vehicles for drug combinations have increased significantly. Owing to their distinctly ordered porous structures, MSNs provide high loading capacities for various types of guest materials. In this chapter, we will touch upon the origin and synthesis of these versatile nanoparticles, as well as the many parameters by which MSNs can be tuned to effectively deliver drug combinations to tumors. We will also review various studies in which MSNs have been employed as nanocarriers for combinational therapies in cancer treatment. Finally, the obstacles associated with the use of MSNs and the future outlook of these multifaceted nanovehicles will be discussed.
... β-Lactam antibiotics are mimetics of the acyl-D-Ala-D-Ala terminus of the peptide stem of the nascent peptidoglycan in their recognition within the active site, per the Tipper-Strominger hypothesis. [47][48][49][50] The same mimicry holds for recognition of the acyl-D-Ala-D-Ala terminus of the nascent peptide stem at the allosteric site. We documented by X-ray crystallography that β-lactam antibiotics indeed bind noncovalently to the allosteric site. ...
Article
ConspectusThe 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-β-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 β-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 β-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 β-lactam antibiotics. These discoveries of small-molecule classes of antibacterials or of adjuvants to antibacterials hold promise in strategies for treatment of bacterial infections.
... So protein synthesis is stopped due to the failure of incorporation of amino acids (Chopra et al., 2001). On the other hand penicillin binds to DD-transpeptidase enzyme (also called penicillin binding protein-PBP) through b-lactam ring that inhibits the cross-linking between bacterial peptidoglycan chain causing disruption of the cell wall and cell death (Georgopapadakou and Liu, 1980;Waxman, 1980;Tipper and Strominger, 1965). But extensive repeated exposure of bacteria to these drugs caused the evolution of resistant strains. ...
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Background: Bacillus anthracis is a gram positive, spore forming, rod shaped bacteria which is the etiologic agent of anthrax-cutaneous, pulmonary and gastrointestinal. A recent outbreak of anthrax in a tropical region uncovered natural and in vitro resistance against penicillin, ciprofloxacin, quinolone due to over exposure of the pathogen to these antibiotics. This fact combined with the ongoing threat of using B. anthracis as a biological weapon proves that the identification of new therapeutic targets is urgently needed. Methods: In this computational approach various databases and online based servers were used to detect essential proteins of B. anthracis A0248. Protein sequences of B. anthracis A0248 strain were retrieved from the NCBI database which was then run in CD-hit suite for clustering. NCBI BlastP against the human proteome and similarity search against DEG were done to find out essential human non-homologous proteins. Proteins involved in unique pathways were analyzed using KEGG genome database and PSORTb, CELLO v.2.5, ngLOC-these three tools were used to deduce putative cell surface proteins. Results: Successive analysis revealed 116 proteins to be essential human non-homologs among which 17 were involved in unique metabolic pathways and 28 were predicted as membrane associated proteins. Both types of proteins can be exploited as they are unlikely to have homologous counterparts in the human host. Conclusion: Being human non-homologous, these proteins can be targeted for potential therapeutic drug development in future. Targets on unique metabolic and membrane-bound proteins can block cell wall synthesis, bacterial replication and signal transduction respectively.
... The core structure of these antibiotics is 3-carbon and 1-nitrogen ring known as β-lactam ring which is highly reactive. PBP are the enzymes responsible for cross-linkage of peptidoglycan components present in the bacterial cell wall [90]. The mechanism behind the antibiotic effect possessed by β-lactams is that they mimic the structure of natural D-Ala-D-Ala substrate of PBP and inhibit its work. ...
Article
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Background Penicillin G amidase/acylases from microbial sources is a unique enzyme that belongs to the N-terminal nucleophilic hydrolase structural superfamily. It catalyzes the selective hydrolysis of side chain amide/acyl bond of penicillins and cephalosporins whereas the labile amide/acyl bond in the β-lactam ring remains intact. Main body of abstract This review summarizes the production aspects of PGA from various microbial sources at optimized conditions. The minimal yield from wild strains has been extensively improved using varying strain improvement techniques like recombination and mutagenesis; further applied for the subsequent synthesis of 6-aminopenicillanic acid, which is an intermediate molecule for synthesis of a wide range of novel β-lactam antibiotics. Immobilization of PGA has also been attempted to enhance the durability of enzyme for the industrial purposes. Short conclusion The present review provides an emphasis on exploitation of E. coli to enhance the microbial production of PGA. The latest achievements in the production of recombinant enzymes have also been discussed. Besides E. coli , other potent microbial strains with PGA activity must be explored to enhance the yields. Graphical abstract
... Some antibiotics, such as aminoglycosides and macrolides (spiromycin and clindamycin), are not only used to tackle bacterial infections but also against the parasite Toxoplasma gondii 24 , and clindamycin also against malaria if used together with other compounds in combinatorial therapeutics. This is thought to be due to the similarity of the translation machinery as seen for other protozoans 25 -lactam antibiotics are analogues of D-alanyl-D-alanine, the last two residues of the pentapeptide chain, and possess a remarkably similar conformation 27 . When present, penicillin competes for the PBP, acylating a critical catalytic serine residue in the active site of the PBP, which is then unable to perform the transpeptidase reaction 28 . ...
Thesis
Bacteria display several intrinsic mechanisms which confers them the ability to cope with disadvantageous situations, such as nutrient deprivation, environmental inter/intra-species competition, managing adaptation to detrimental conditions, and handling effects of antibacterial compounds.In a global context of antibiotic resistance accelerated by anthropogenic activities, gram negative bacteria display intrinsic resistance mechanisms. The complex and dynamic multilayered envelope, coated with lipopolysaccharides (LPS), confers these bacteria increased survivability. Biosynthesis of these complex glycolipids is initiated in the cytoplasm, and its transport proceeds along the inner membrane, periplasm, until reaching the outer membrane, with a dedicated biosynthetic pathway and transport machinery.The Lipopolysaccharide Transport (Lpt) machinery comprises 7 fundamental proteins (LptA to LptG) that span the entire envelope. More specifically, at the inner membrane, LptB2FG ABC transporter couples ATP hydrolysis with LPS extraction. LptB2 cycles ATP while LptF/G interact with LPS and carry it towards LptC and LptA in the periplasm.This machinery uses a conserved architecture with dedicated jellyroll domains present on LptF, LptG, LptC and LptA that assemble into a bridge that allow LPS flow to the outer membrane.Molecules that would disrupt protein-protein interactions between the different jellyroll domains of the Lpt system could become potent cell wall inhibitors. Thanatin, a natural occurring antimicrobial peptide, has been described as targeting the jellyroll domains of the machinery. We screened its effect in the disruption of LptC-LptA complex. Thanatin binds to LptA but not LptC and inhibits the assembly of the complex at low nM concentrations, showing the potential of targeting Lpt Jellyroll-jellyroll interactions.The network of interactions between the Inner membrane complex, LptB2FG and periplasmic LptC and LptA is not fully understood. LptB2FG was produced in detergent micelles and within nanodisc particles, to probe interactions with LptC and LptA at an atomic scale, using Nuclear Magnetic Resonance (NMR) and biophysical techniques.In the assembly of the LptB2FGCA bridge, LptC and LptF interact mostly through the jellyroll domains. A mutation in the LptF jellyroll (R212 residue) rendered LptC presence facultative in vivo.Biophysical and biochemical characterization showed unaltered interaction of mutant LptB2FG with LptC and LptA, whereas ATPase activity showed lack of regulation by presence of its partners. This led us to propose that R212 is a checkpoint in the LptF jellyroll, acting as a hub for LptB2FG to sense proper assembly of the machinery.When LptB2FGCA complex is assembled in vitro, LptB2 was found capable of catalyzing phosphotransfer between ADP molecules, generating ATP and AMP, a novel activity (Adenylate Kinase) previously undescribed for this protein. Being a topic of very recent interest in the literature, the role of dual-function transporters is not understood. To characterize the balance between ATPase and AK, we mutated LptB2 on key ABC motifs to probe possible location for AK activity. LptB2FG studied in nanodisc particles, suggests that balance between activities depends on the dynamic assembly of LptB2FGCA, with regulatory mechanisms possibly not being shared between both activities. Structural characterization of LptB2 in apo and nucleotide bound-state was initiated .This project, focused on the essential Lpt system, sheds light on the importance of protein-protein interactions as targets for designing future antimicrobial compounds. It could also be worth evaluating if dual-function transporters, involved in cell wall synthesis and drug export, are valid targets for future drug screenings.
... The results revealed the roles of the indole NH and the C2 carboxylate in potent inhibition; N-methylation or replacement of the C2 carboxylate reduced activity by ~1,000 fold (3-7; Supplementary Tables 1 and 2). C3 modification was found to be useful, as shown by studies with C3 aryl InCs (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)Supplementary Table 3). In general, the C4, C5 and C6 positions were less amenable to diverse derivatization (27-37; Supplementary Tables 4 and 5). ...
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Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure–activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL–carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.
... In addition to the 4 types of PBPs (PBP1, PBP2, PBP3, and PBP4), MRSA has the potential to express PBP2a, which is resistant to the action of methicillin and some other β-lactams. 3 Among the structural domains, the transpeptidase (TPase) domain plays a crucial role in bacterial life. 4,5 PBP2a is a product of the resistance gene mecA, which functions as a surrogate transpeptidase when other PBPs are inhibited. ...
... The assignment of proteins detected at altered levels in the presence of imipenem to functional categories provides important clues about the mechanisms employed by our E. roggenkampii ST232 study isolate for dealing with the detrimental effects of this antibiotic. In the first place, imipenem is a potent inhibitor of cell wall biogenesis, blocking either the transpeptidation or carboxylation reactions catalyzed by PBPs [33]. As a consequence, the peptidoglycan layer of the growing bacteria will be weakened, which may lead to cell death by lysis, especially in hypotonic environments [4]. ...
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Gram-negative bacteria belonging to the Enterobacter cloacae complex are increasingly implicated in difficult-to-treat nosocomial infections, as exemplified by a recently characterized highly carbapenem-resistant clinical Enterobacter roggenkampii isolate with sequence type (ST) 232. While mechanisms of carbapenem resistance are well-understood, little is known about the responses of highly drug-resistant bacteria to these antibiotics. Our present study was therefore aimed at charting the responses of the E. roggenkampii ST232 isolate to the carbapenem imipenem, using a ‘stable isotope labeling of amino acids in cell culture’ approach for quantitative mass spectrometry. This unveiled diverse responses of E. roggenkampii ST232 to imipenem, especially altered levels of proteins for cell wall biogenesis, central carbon metabolism, respiration, iron–sulfur cluster synthesis, and metal homeostasis. These observations suggest a scenario where imipenem-challenged bacteria reduce metabolic activity to save resources otherwise used for cell wall biogenesis, and to limit formation of detrimental reactive oxygen species at the cytoplasmic membrane due to respiration and Fenton chemistry. We consider these observations important, because knowing the adaptive responses of a highly resistant bacterium of the E. cloacae complex to last-resort antibiotics, such as imipenem, provides a ‘sneak preview’ into the future development of antibiotic resistance in this emerging group of pathogens.
... The transpeptidases link NAG and NAM, whereas endopeptidase and carboxypeptidase control the extent of the cross-linking of PG (10). In the presence of b-lactam antibiotics, cell wall transpeptidases are inhibited, leading to the accumulation of non-cross-linked strands of PG, and the cell is not able to process all of the anhMPs for recycling to the periplasmic space for their integration in the PG (15,16). This inability of the cell to process the enhanced levels of anhMPs leads to anhMP accumulation in the cytoplasm. ...
Article
Antimicrobial resistance is a global health problem that requires a better understanding of the mechanisms that bacteria use to resist antibiotics. Bacteria inhabiting the plant rhizosphere are a potential source of antibiotic resistance, but their mechanisms controlling antibiotic resistance are poorly understood. A. baldaniorum Sp245 is a rhizobacterium that is known for its characteristic resistance to ampicillin.
... Penicillin binding protein (PBP) was a membrane protein located on the cell membrane and involved in maintaining the shape and normal function of bacteria (Chen and Williams 1987). Penicillin played a bactericidal effect by inhibiting bacterial cell wall biosynthesis and causing bacterial cell death (Tipper and Strominger 1965). In this study, the HBA/HP-β-CD inclusion compound increased the expression of penicillin binding protein (A0AJZ7) and penicillin binding protein 1A (A0A468CGA7) by 1.27 times and 1.28 times. ...
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Food safety affected by food-borne pathogen has received increasing attention by researchers. Listeriamonocytogenes (L. monocytogenes), widespread in the environment, could easily cause some diseases. The aim of this study was to investigate how L. monocytogenes ATCC 19,115 regulated and shaped its proteome in response to hexahydro-β-acids (HBA) formed inclusion complex with hydroxypropyl-β-cyclodextrin (HP-β-CD), compared to untreated cells growing under optimal conditions. HP-β-CD enhanced the solubility of HBA to 0.589 g/100 mL. The minimum inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of HBA/HP-β-CD to L. monocytogenes were 25 μg/mL and 100 μg/mL, respectively. Scanning electron microscope (SEM) images demonstrated that HBA could destroy the cell membrane of L. monocytogenes. The proteomic analysis revealed that 2882 proteins were initially identified, among which 153 and 201 proteins were differentially upregulated and downregulated respectively. Changes of L. monocytogenes proteome in response to treatments were mainly related to carbohydrate metabolism, protein synthesis, ribosome composition proteins, cell wall composition proteins, and cell division anomalies process. This research is conducive to understanding the molecular mechanisms underlying the inhibiting effects of HBA/HP-β-CD against L. monocytogenes, providing novel insights for further development of HBA/HP-β-CD antimicrobial agents. Key points • MIC and MBC of HBA/HP-β-CD to L. monocytogeneswere 25 μg/mL and 100 μg/mL. • HBA/HP-β-CD cause significant changes in bacterial proteome. • The process of ribosome composition and carbohydrate metabolism was inhibited. Graphical abstract
... PRODUCTION OF f8-LACTAM ANTIBIOTICS ,8-Lactam antibiotics are distinct in that a filactam antibiotic, penicillin, was the first antibiotic discovered (58) and in that ,8-lactam antibiotics have been widely used for chemotherapy of infectious diseases in the 40 years since penicillin's rediscovery. This widespread use of ,B-lactam antibiotics stems from their intrinsic antibacterial activity together with a highly specific inhibitory effect on the biosynthesis of peptidoglycan (212,227,228), a unique structure of the bacterial cell wall, and from their quite low toxicity to animals, including humans. Furthermore, the discovery of 6-aminopenicillanic acid and 7-aminocephalosporanic acid (95,115,194) and their preparation by enzymic (penicillin amidase, EC 3.5.1.11) ...
... This conclusion was initially based on the pioneering work of Tipper and Strominger reported in 1965, who noticed that one of the possible conformations of the terminal D-Ala 4 -D-Ala 5 dipeptide of stem pentapeptide is similar to that of penicillin ( Figure 1A). [20] By analogy, we present a carbapenem with a conformation that simulates the D-Ala 4 -D-Ala 5 ( Figure 1A) or L-Lys 3 -D-Ala 4 ( Figure 1B) extremity of the pentapeptide or tetrapeptide stem peptides, which are used as acyl donor by transpeptidases of the D,D and L,D specificities, respectively. Several groups have investigated the possibility of modifying the structure of the substituents linked to the bicyclic core of βlactams to improve drug binding and antibacterial activity. ...
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The carbapenem class of β‐lactams has been optimized against Gram‐negative bacteria producing extended‐spectrum β‐lactamases by introducing substituents at position C 2 . Carbapenems are currently investigated for the treatment of tuberculosis since these drugs are potent covalent inhibitors of L,D‐transpeptidases involved in mycobacterial cell wall assembly. We sought to optimize carbapenems for inactivation of these unusual targets by exploiting the nucleophilicity of the C 8 hydroxyl group to introduce chemical diversity. Since β‐lactams are structure analogues of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido‐carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, due to the positive impact of a phenethylthio substituent introduced at position C 2 but the peptidomimetics added at position C 8 did not further improve activity. Thus, position C 8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems.
... In infections, disrupting constitutive mechanisms that ensure pathogen survival and/or replication often cures the disease. For example, penicillin inhibits the enzyme transpeptidase, which is found in Gram positive bacteria (Tipper & Strominger, 1965;Wise & Park, 1965). Transpeptidase catalyzes the crosslinking of molecules of the glycoprotein peptidoglycan, which strengthens the bacterial call wall, allowing the cell wall to provide the bacteria with the structural rigidity needed for survival. ...
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... 13 All of these antibiotics contain a four-membered β-lactam ring, which ensures antibiotic binding to penicillin-binding proteins and, consequently, inhibition of bacterial cell wall biosynthesis. 14,15 Clinically used β-lactam compounds can be divided into four different groups: penicillins, cephalosporins, carbapenems, and monobactams, of which carbapenems play a critical role as potent antibiotics reserved for the most serious Gram-negative infections where alternatives are limited. 16 Emerging resistance against β-lactams is evident, and especially in Gram-negative bacteria, β-lactamase enzymes are the main resistance mechanism against these drugs. ...
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This volume brings current knowledge of proteomics technologies and related developments with special reference to diseases caused by microbes. The editor has compiled chapters written by expert academicians which distill the information about useful methods in microbial proteomics for the benefit of readers. Chapters cover several methods used to investigate the microbial proteome and special topics such as antimicrobial drug resistance mechanisms, biomarker developments, post-translational modifications. Key Features: -Overview of several biochemical methods in proteomics -Full-color, high-quality images of the most frequent technologies and applications -Concise, well organized, and didactic format -Updates in basic applied information -Bibliographic references -Information on proteomics for tuberculosis treatment This reference work is intended for researchers seeking information on laboratory techniques applied in proteomics research and microbiology.
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The biological diversity of the unicellular bacteria-whether assessed by shape, food, metabolism, or ecological niche-surely rivals (if not exceeds) that of the multicellular eukaryotes. The relationship between bacteria whose ecological niche is the eukaryote, and the eukaryote, is often symbiosis or stasis. Some bacteria, however, seek advantage in this relationship. One of the most successful-to the disadvantage of the eukaryote-is the small (less than 1 μm diameter) and nearly spherical Staphylococcus aureus bacterium. For decades, successful clinical control of its infection has been accomplished using β-lactam antibiotics such as the penicillins and the cephalosporins. Over these same decades S. aureus has perfected resistance mechanisms against these antibiotics, which are then countered by new generations of β-lactam structure. This review addresses the current breadth of biochemical and microbiological efforts to preserve the future of the β-lactam antibiotics through a better understanding of how S. aureus protects the enzyme targets of the β-lactams, the penicillin-binding proteins. The penicillin-binding proteins are essential enzyme catalysts for the biosynthesis of the cell wall, and understanding how this cell wall is integrated into the protective cell envelope of the bacterium may identify new antibacterials and new adjuvants that preserve the efficacy of the β-lactams.
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Chapter
Proteome deals with complete set of proteins, expressed by a genome of a cell present inside of an organism at a specific time period. It includes expression study, information of modifications in proteins, interactions with other protein biomolecules, etc. The goal of proteomics is to know new protein information, whether a protein is over expressed or under expressed in specific situation and overall gives information of its effect on an organism. Thousands of proteins can be examined at a time as compared to other methodologies. Many advanced technologies have been evolved to investigate proteome in depth and generate a huge amount of data. The most common high throughput techniques such as polyacrylamide & agarose gel electrophoresis and their advanced versions in combination with mass spectrometry are being used in modern proteomics. The study of microbial proteome data helps us to know how bacteria get resistant to particular drug and which biomolecules are involved in that process. Database also gives the opportunity to develop better drugs that target new places on bacterial surface or new drugs on the same target. The advancement of proteomics technique and their applications in microbial research has granted a new hope to explore disease biomarkers and the development of diagnostic assays. Microbial protein's benefits are extensively used in the agricultural sector. Proteomics profiling has a key role in disease identification in humans and animals. Thanks to protein information as new targets are identified and more safer and effective drugs are produced.
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A particulate enzyme system from strains of Escherichia coli is described which catalyzes the utilization of the uridine nucleotides, UDP-N-acetylmuramyl-l-Ala d-Glu-α, ε-diaminopimelyl-d-Ala-d-Ala and UDP-N-acetylglucosamine, for peptidoglycan synthesis. Unlike the systems previously studied in gram-positive cocci, this particulate enzyme catalyzes the terminal cross-linking reaction in cell wall synthesis, a transpeptidation in which a d-alanine residue is lost from the end of one of the N-acetylmuramyl-pentapeptide residues incorporated into the product. The d-alanine residue is also lost from the end of the second unit involved in the cross-linking, apparently through the action of a d-alanine carboxypeptidase. Both the transpeptidase and the d-alanine carboxypeptidase are inhibited by various penicillins and cephalosporins.
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Many nascent proteins that are destined to be anchored to plasma membranes by a phosphatidylinositol glycan (PI-G) are in the range of 50-70 kDa so that changes of 2-3 kDa between precursors and products during processing are not easily detected. Furthermore, PI-G-anchored proteins are generally glycosylated so that changes between the nascent (prepro) proteins and the mature products are not due simply to the loss of signal peptides. These problems have made it difficult to monitor the processing of the prepro form of wild type human placental alkaline phosphatase (PLAP) in a cell-free system. We have designed a smaller and simpler substrate of PI-G “transamidase” derived by deletion of approximately 60% of the internal sequence of preproPLAP 513. This engineered protein, preprominiPLAP 208, retains the NH2⁻ and COOH-terminal signal peptides of PLAP as well as all the epitopes for site-directed antibodies of the latter, but is devoid of glycosylation sites, the active site, and most of the cysteine residues. With preprominiPLAP, it has been possible to demonstrate, in a cell-free system, step by step conversion to the pro form and then to the mature form, with the concomitant loss of the appropriate signal peptides. These changes were shown to be time- and enzyme concentration-dependent. Studies with Asp-179 site-directed mutants of preprominiPLAP showed the same specificity for amino acids with a monosubstituted β carbon at the cleavage/attachment site that were found previously with wild type PLAP.
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A particulate d-alanine carboxypeptidase present in Bacillus subtilis has been studied. This enzyme has a pH optimum near 5, and is activated by Zn⁺² cations. The enzyme is inactivated by penicillin G and activity cannot be restored by washing or by treating it with penicillinase. ¹⁴C-Penicillin G is also bound to this particulate enzyme. Both the binding of penicillin G and the inactivation of the carboxypeptidase by penicillin G are reversed by treatment with neutral hydroxylamine, and the kinetics of these two processes is identical. d-Alanine carboxypeptidase activity is also inhibited by sulfhydryl reagents such as iodoacetate in a manner similar to their inhibition of penicillin binding. The possibility that the particulate d-alanine carboxypeptidase may be an uncoupled transpeptidase is discussed.
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The lipid intermediates in peptidoglycan synthesis in Staphylococcus aureus, N-acetylmuramyl(-pentapeptide)-P-P-lipid and N-acetylglucosaminyl-N-acetylmuramyl(-pentapeptide)-P-P-lipid, act as acceptors of ammonia in an ATP-dependent reaction in which the α-carboxyl group of glutamic acid is amidated. Procedures have been developed for the separation of the amide and the amide-free forms of the lipid intermediates, and it has been shown that the amide-free lipid intermediate can substitute for the uridine nucleotide substrates in the amidation reaction. Some properties of the reaction are reported.
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An uncross-linked monomer (nascent peptidoglycan unit) accumulates in cells of Staphylococcus aureus treated with low concentrations of penicillin G or with ampicillin, methicillin, or cephalothin. The uncross-linked monomer has been isolated, and analyses indicate that it represents a prefabricated subunit of the wall bearing both of the d-alanine residues of its pentapeptide precursor as well as an open pentaglycine chain. Pulse labeling experiments indicate that this uncross-linked unit is a direct precursor of the cross-linked peptidoglycan and that its cross-linking is inhibited by penicillin G. At high concentrations of penicillin G, wall synthesis ceases abruptly and no accumulation of the nascent peptidoglycan units is observed. These data have been obtained in support of the hypothesis that penicillins are substrate analogues of the d-alanyl-d-alanine end of the nascent peptidoglycan units and that they acylate the transpeptidase which catalyzes the cross-linking reaction. The data may also provide an explanation of the paradoxical observation that the killing rate in S. aureus by penicillin G is higher at low concentrations than at high concentrations of the antibiotic. In the presence of low concentrations a weakened wall may be formed, thus rendering the organisms more susceptible to lysis than at high concentrations of penicillin where wall synthesis abruptly ceases.
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d-Alanine carboxypeptidase I, which removes the terminal d-alanine residue of the uridine nucleotide, UDP-N-acetylmuramyl pentapeptide, and several related compounds occurs in both the particulate and soluble fractions of Escherichia coli and several other gram-negative bacteria. The soluble enzyme has been purified 120-fold and some of its properties are reported. The enzyme is competitively inhibited by penicillins and cephalosporins at very low concentrations. d-Alanine carboxypeptidase II, which catalyzes the hydrolysis of the penultimate d-alanine residue of the uridine nucleotide substrate, has also been purified and separated from d-alanine carboxypeptidase I. This enzyme is not inhibited by penicillins and cephalosporins. The possible physiological substrate and function of these enzymes are discussed.
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Peptidoglycan: Structure, Synthesis, and Regulation, Page 1 of 2 Abstract Peptidoglycan is a defining feature of the bacterial cell wall. Initially identified as a target of the revolutionary beta-lactam antibiotics, peptidoglycan has become a subject of much interest for its biology, its potential for the discovery of novel antibiotic targets, and its role in infection. Peptidoglycan is a large polymer that forms a mesh-like scaffold around the bacterial cytoplasmic membrane. Peptidoglycan synthesis is vital at several stages of the bacterial cell cycle: for expansion of the scaffold during cell elongation and for formation of a septum during cell division. It is a complex multifactorial process that includes formation of monomeric precursors in the cytoplasm, their transport to the periplasm, and polymerization to form a functional peptidoglycan sacculus. These processes require spatio-temporal regulation for successful assembly of a robust sacculus to protect the cell from turgor and determine cell shape. A century of research has uncovered the fundamentals of peptidoglycan biology, and recent studies employing advanced technologies have shed new light on the molecular interactions that govern peptidoglycan synthesis. Here, we describe the peptidoglycan structure, synthesis, and regulation in rod-shaped bacteria, particularly Escherichia coli, with a few examples from Salmonella and other diverse organisms. We focus on the pathway of peptidoglycan sacculus elongation, with special emphasis on discoveries of the past decade that have shaped our understanding of peptidoglycan biology.
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Penicillin and related antibiotics disrupt cell wall synthesis in bacteria causing the downstream misactivation of cell wall hydrolases called autolysins to induce cell lysis. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae (Sp), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked teichoic acids (LTAs). Because LytA binds to both species of teichoic acids, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation during exponential growth and premature lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active PG synthesis. Our results support a model in which the WTA tailoring activity of WhyD during exponential growth directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.
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β ‐lactams, the cornerstone of antibiotherapy, inhibit multiple and partially redundant targets referred to as transpeptidases or penicillin‐binding proteins. These enzymes catalyze the essential cross‐linking step of the polymerization of cell wall peptidoglycan. The understanding of the mechanisms of action of β ‐lactams and of resistance to these drugs requires the development of reliable methods to characterize their targets. Here, we describe an activity‐based purification method of β ‐lactam targets based on click and release chemistry. We synthesized alkyne‐carbapenems with suitable properties with respect to the kinetics of acylation of a model target, the Ldt fm L , D ‐transpeptidase, the stability of the resulting acylenzyme, and the reactivity of the alkyne for the cycloaddition of an azido probe containing a biotin moiety for affinity purification and a bioorthogonal cleavable linker. The probe provided access to the fluorescent target in a single click and release step.
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β-lactamases are a major cause of rapidly emerging and spreading antibiotic resistance. Currently β-lactamase inhibitors (BLIs) in clinical use act only on Ambler Class A, C and some class D lactamases. The urgent need to identify new BLIs recently lead to FDA approval of boron-based compounds BLIs, e.g. Vaborbactam. The boron-based proteasome inhibitors Bortezomib and Ixazomib are used in cancer therapy as multiple myeloma drugs but they also bind to Ser-/Thr- proteases. In this study we show the crystal structures of the β-lactamase CTX-M-14 with covalently bound Bortezomib and Ixazomib at high resolutions of 1.3 and 1.1 Å, respectively. Ixazomib is well defined in electron density whereas Bortezomib show some disorder which corresponds to weaker inhibition efficiency observed for Ixazomib. Both inhibitors mimic the deacylation transition state of β-lactam hydrolysis, because they replace the deacylating water molecule. We further investigate differences in binding of Bortezomib/Ixazomib to CTX-M-14 and its target proteases as well as known β-lactamase drugs. Our findings can help to use Bortezomib/Ixazomib as lead compounds for development of new BLIs.
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Amoxicillin (AMX), one of the micro-amount hazardous pollutants, was frequently detected in environments, and of great risks to environments and human health. Microbial degradation is a promising method to eliminate pollutants. In this study, an efficient AMX-degrading strain, Ads-6, was isolated and characterized. Strain Ads-6, belonging to the genus Bosea, was also able to grow on AMX as the sole carbon and nitrogen source, with a removal of ~60% TOC. Ads-6 exhibited strong AMX-degrading ability at initial concentrations of 0.5–2 mM and pH 6–8. Addition of yeast extract could significantly enhance its degrading ability. Many degradation intermediates were identified by HPLC-MS, including new ones such as two phosphorylated products which were firstly defined in AMX degradation. A new AMX degradation pathway was proposed accordingly. Moreover, the results of comparative transcriptomes and proteomes revealed that β-lactamase, L, D-transpeptidase or its homologous enzymes were responsible for the initial degradation of AMX. Protocatechuate branch of the beta-ketoadipate pathway was confirmed as the downstream degradation pathway. These results in the study suggested that Ads-6 is great potential in biodegradation of antibiotics as well as in the bioremediation of contaminated environments.
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Penicillin-binding proteins (PBPs) are integral to bacterial cell division as they mediate the final steps of cell wall maturation. Selective fluorescent probes are useful for understanding the role of individual PBPs, including their localization and activity during growth and division of bacteria. For the development of new selective probes for PBP imaging, several β-lactam antibiotics were screened, as they are known to covalently bind PBP in vivo. The PBP inhibition profiles of 16 commercially available β-lactam antibiotics were evaluated in an unencapsulated derivative of the D39 strain of Streptococcus pneumoniae, IU1945. These β-lactams have not previously been characterized for their PBP inhibition profiles in S. pneumoniae and these data augment those obtained from a library of 20 compounds that we previously reported. We investigated seven penicillins, three carbapenems, and six cephalosporins. Most of these β-lactams were found to be co-selective for PBP2x and PBP3, as was noted in our previous studies. Six out of 16 antibiotics were selective for PBP3 and one molecule was co-selective for PBP1a and PBP3. Overall, this work expands the chemical space available for development of future β-lactam-based probes for specific pneumococcal PBP labeling and these methods can be used for the development of probes for PBP labelling in other bacterial species.
Preprint
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Penicillin and related antibiotics disrupt cell wall synthesis in bacteria and induce lysis by misactivating cell wall hydrolases called autolysins. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae ( Sp ), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked lipoteichoic acids. Because LytA binds to these polymers, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active PG synthesis. Our results support a model in which the WTA tailoring activity of WhyD directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.
Chapter
Daptomycin is a lipopeptide antibacterial drug with broad-spectrum activity against Gram-positive pathogens. Daptomycin has a unique mechanism of action which is not entirely understood. The first license for use by the United States Food and Drug Administration was in September 2003 for the treatment of infections caused by Gram-positive bacteria using a once-daily dosing regimen. The current licensed usages of daptomycin are complicated skin and skin structure infections in adult and pediatric patients, Staphylococcus aureus bloodstream infections in adult patients including those with right-sided infective endocarditis and S. aureus bloodstream infections in pediatric patients. The Clinical and Laboratory Standards Institute recommends that antimicrobial susceptibility testing (AST) is undertaken using the broth dilution method with cation-adjusted Mueller-Hinton broth (CAMHB) and the addition of 50 μg mL− 1 of calcium. Antibacterial breakpoints are available for Staphylococcus spp., β-haemolytic Streptococcus spp., viridans group Streptococcus spp. and Enterococcus spp. Acquired daptomycin resistance remains rare. There now exists a wealth of data on the use of daptomycin for non-licensed indications, including bone and joint infections, left-sided endocarditis and for the treatment of multidrug-resistant Gram positive organisms such as methicillin-resistant S. aureus and vancomycin-resistant enterococci.
Chapter
The β‐lactam class of antibacterials is a cornerstone of human health. For nearly eight decades, their unparalleled clinical efficacy and clinical safety have made the β‐lactam class preeminent in the treatment of bacterial infection. The relatively brief period in human history during which the β‐lactams have exerted this benefit is a period characterized by continuous medicinal chemistry innovation, seen visibly in the progression from the penicillins to the complex ensemble of β‐lactams (now including also cephalosporins, monobactams, and carbapenems) used in the clinic. The key force behind this innovation is the progressive evolution by bacteria of resistance mechanisms. Today, highly resistant bacteria challenge the way medicinal chemists contemplate the creative alteration of β‐lactam structures, the way the pharmaceutical industry develops β‐lactams (and other antibacterial) structures, and the way the medical community uses antibacterials. This article gives a concise summary of the history of the β‐lactams. Its emphasis is recent structural innovation with respect to the β‐lactams, and with respect to structurally related classes that act to preserve the clinical activity of the β‐lactams through inhibition of bacterial β‐lactam‐hydrolyzing, and thus β‐lactam‐deactivating, enzymes. We integrate these chemistry advances with new biological discoveries with respect to the bactericidal mechanism of the β‐lactams and with respect to bacterial resistance mechanisms. The combination of these perspectives is a foundational perspective to guide the medicinal chemistry future of the β‐lactams.
Chapter
Infectious diseases are the world’s second most significant cause of human death. Staphylococcus aureus is perhaps the human’s greatest concern because of its inherent virulence and its ability to cause a wide variety of life-threatening infections and its capability to adapt under various conditions. The drug resistance of S. aureus has gradually increased due to the adaptation of bacteria and the excessive use of antibiotics. There are many anti-staphylococcus drugs; however, they quickly lose their therapeutic value due to the resistance mechanisms developed by the bacteria. The major fundamental mechanisms of antimicrobial resistance are enzymatic degradation of antibacterial drugs, alteration of bacterial proteins that are antimicrobial targets and changes in membrane permeability to antibiotics. S. aureus develops resistance to beta-lactamase through the acquisition of a genomic island called staphylococcus cassette chromosome (SCC mec) which carries methicillin resistance determinant called mecA. Biofilm formation and quorum sensing of S. aureus have shown resistance to different antibiotics. Therefore, understanding the drug resistance of MRSA (methicillin-resistant Staphylococcus aureus) correctly and elucidating its drug resistance mechanism at the molecular level is of great importance for the treatment of S. aureus infections.
Chapter
Multidrug-resistant bacteria are currently considered as an appearing global disease and a major public health problem. We have recently witnessed an impressive increase in the proportion and absolute number of pathogen bacteria resistant to multiple antimicrobial agents. Bacteria develop resistance to antimicrobials through a large variety of mechanisms; however, the overuse and/or misuse of antibiotics are the key driver of emerging resistance. Despite the fact that antimicrobial resistance is inevitable, human activity is effective in detecting the borders of resistance. Globalization and travel make a significant contribution on the spread of antimicrobial resistance. Alternative therapies like phage therapy may attenuate the selective pressure of antimicrobial chemotherapy. Studies on alternative therapies including, immune stimulation, vaccination, topical agents are still under investigation.
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In the current study, the polyurethane acrylate (PUA) polymer was synthesized by the addition reaction between an isophorone diisocyanate (IPDI) and 2-hydroxyethyl acrylate and cured by polyol. Different properties of the synthesized PUA were determined through diverse analysis methods. The polyurethane acrylate (PUA)/natural filler-based composite (rhizome water extract of Costus speciosus) was prepared as an antifouling agent. The results revealed that the lowest weight loss percentages were detected at 2 wt% PUA/natural filler composite loadings with Escherichia coli (ATCC 23,282) and Pseudomonas aeruginosa (ATCC 10,145). The decreased weight loss percentage may be attributed to the well dispersed natural composite resulting in a slippery surface that can prevent fouling adhesion. It was concluded that the PUA/natural filler composite might be considered an eco-friendly and economical solution to the biofouling problem. Key points • A novel strategy for anti-biofouling. • A new composite reduced Gram-negative bacteria.
Article
The β-lactams are the most widely used antibacterial agents worldwide. These antibiotics, a group that includes the penicillins and cephalosporins, are covalent inhibitors that target bacterial penicillin-binding proteins and disrupt peptidoglycan synthesis. Bacteria can achieve resistance to β-lactams in several ways, including the production of serine β-lactamase enzymes. While β-lactams also covalently interact with serine β-lactamases, these enzymes are capable of deacylating this complex, treating the antibiotic as a substrate. In this tutorial-style review, we provide an overview of the β-lactam antibiotics, focusing on their covalent interactions with their target proteins and resistance mechanisms. We begin by describing the structurally diverse range of β-lactam antibiotics and β-lactamase inhibitors that are currently used as therapeutics. Then, we introduce the penicillin-binding proteins, describing their functions and structures, and highlighting their interactions with β-lactam antibiotics. We next describe the classes of serine β-lactamases, exploring some of the mechanisms by which they achieve the ability to degrade β-lactams. Finally, we introduce thel,d-transpeptidases, a group of bacterial enzymes involved in peptidoglycan synthesis which are also targeted by β-lactam antibiotics. Although resistance mechanisms are now prevalent for all antibiotics in this class, past successes in antibiotic development have at least delayed this onset of resistance. The β-lactams continue to be an essential tool for the treatment of infectious disease, and recent advances (e.g., β-lactamase inhibitor development) will continue to support their future use.
Article
OMVs are spherical buds derived from the outer membrane of Gram-negative bacteria, containing lipopolysaccharide (LPS) and phospholipids, proteins, nucleic acids, peptidoglycan, and other cellular molecules. Some of those molecules, such as LPS and peptidoglycan associated lipoprotein (Pal), have been shown to be toxic and to contribute to sepsis-related inflammation. While OMVs are released constitutively from Gram-negative bacteria, environmental factors like temperature changes, stress, and antibiotics can enhance OMV release. We proposed that some antibiotics, such as beta lactams that target the peptidoglycan layer of the cell, enhance OMV release more than other types of antibiotics. We used ultracentrifugation, immunoblotting, and nanoparticle tracking analysis to purify, detect, and roughly quantify OMV release from Escherichia coli in the presence of different antibiotics. Preliminary results support our hypothesis, although data collection is ongoing. Finally, we propose a novel use of released OMVs as molecular biomarkers for bacterial sepsis.
Preprint
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A lack of direct single-cell readouts for bacterial kinase activity remains a major barrier to our understanding of most signaling systems. At the single-cell-level, protein kinase activity is typically inferred by the activity of downstream transcriptional reporters. Complicating this approach in vivo, promoters are often co-regulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we have designed and constructed new, direct and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor that responds to phosphorylation. We demonstrate the utility of these reporters in measuring kinase activity in population-based and single-cell assays during various growth phases and antibiotic treatments. These sensors respond to a highly conserved bacterial Ser/Thr kinase, PrkC that has no known dedicated transcription factor and whose regulon is known to be convolved with an essential signaling system. We used these new sensors to measure PrkC activity in colonies, bulk culture, and single cells. Together these new sensors provide evidence for considerable heterogeneity in PrkC activity in actively growing populations. We further demonstrate that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). This is consistent with a model where PrkC senses and responds to blocks in the extracellular steps in cell wall synthesis. As the design of these phosphorylation sensors is modular, we anticipate that this work may have broad applications to other bacterial signaling systems in the future.
Chapter
Infectious diseases are spreading at a very fast pace in the world, and around one-fifth of the population is affected every year. Though antibiotics are the first choice of drugs for the treatment of these diseases, but their use is limited because of the rapid increase in development of antibiotic resistance. This becomes a major challenge in the management of infections. New antibiotics that can combat the problem of resistance and provide effective treatment for curing the infectious diseases are the need of the day; however, the number of new antibiotics getting approvals is declining over the years. To overcome these problems, one of the strategies is the use of phytotherapeutics. Owing to their multimode of actions and various properties such as antibacterial, antioxidant, and antiinflammatory, phytocompounds, especially in combinations, offer an alternative over antibiotics. This chapter discusses the activity of phytocompounds, their synergistic behavior, mechanism of action with other drugs, and their nanocarrier-based formulations.
Article
By catalyzing a 3-3 cross-link in peptidoglycan, l,d-transpeptidases (Ldts) can cause resistance to β-lactams in some pathogens in vitro. However, the prevalence of Ldt and Ldt-mediated responses to different β-lactams in vivo have never been explored. Here, we apply an in vivo metabolic labeling strategy to study their biodistributions and Ldt-induced bacterial responses to β-lactams in the mouse gut microbiota. A tetrapeptide-based fluorescent probe that functions as a substrate for Ldts in Gram-positive bacteria efficiently labels ∼18% of total gut bacteria after gavage, suggesting Ldts' high prevalence in gut microbiota. The cellular distributions of 3-3 cross-links on three gut bacterial species were then identified with the aid of fluorescence in situ hybridization to identify the bacterial taxa. After oral administration of two β-lactams, ampicillin and meropenem, only the latter efficiently inhibits the tetrapeptide labeling, suggesting that Ldts may be able to cause resistance to some β-lactams in the mammalian gut.
Article
Reaction of walls with anhydrous hydrazine gave fairly reproducible results enabling the identification and quantitative determination of “C-terminal” groups. Liberation of “C-terminal” residues by anhydrous hydrazine was maximal on heating for 8 h at 100°. A variety of groups have been found by hydrazinolysis suggesting that individual wall peptides may differ in detailed structure. Free amino groups have been identified and determined for a variety of walls and changes in both free amino groups and C-terminal groups after removal of the teichoic acids have been established.Results of hydrazinolysis, reaction with fluorodinitrobenzene and digestion with Ghuysen's Streptomyces F2b enzyme suggest that alanine is the principal amino acid linked to muramic acid in various walls and that lysine is the site of some unusual structures of the wall mucopeptide. It is suggested that the N-terminal amino groups (alanine, aspartic acid, glutamic acid or glycine) may be located as single amino acid substituents on the ε-NH2 group of lysine in certain walls. Streptomyces F2b enzyme appears to contain an enzyme splitting ε-lysyl-glycine, or ε-lysyl-glycyl sequences in certain cell-wall mucopeptides.Partial hydrazinolysis does not seem to give any marked enrichment of the amino sugar backbone of the wall.A method of preparing bis-dinitrophenyl α,ε-diaminopimelic acid as a reference compound for these studies has been described.
Article
T2-phage enzyme or lysozyme remove from the complex cell wall of Escherichia coli B an integral component of one of its layers. The material is released in the form of several chemically closely related split products which have been characterized as mucopeptides. Two of these fragments, quantitatively predominant, were isolated in pure and crystalline form. One of them, designated C5, is made up from one residue each of N-acetyl-glucosamine, N-acetyl-muramic acid, glutamic acid, α,ϵ-diaminopimelic acid, and alanine. The molecule of the other fragment, C6, comprises one additional alanine residue. Ultracentrifugal determination of molecular weights definitely established the size of the fragments C5 and C6 to be as indicated.The arrangement of amino sugars and amino acids within each of the two fragments was cleared up in part. A short peptide chain, comprising either one (C5) or two (C6) alanines, one glutamic acid and one α,ϵ-diaminopimelic acid must be linked by its amino endgroup to the carboxyl group of N-acetylmuramic acid which carries the N-acetylglucosaminyl-residue attached by a β-glucosidic link.
Article
An unidentified species of the fungus, Chalaropsis, produced extracellular enzymes that lysed Staphylococcus aureus and several other gram-positive bacteria.The lytic enzymes have been purified by ion-exchange chromatography and separated into two fractions which have been designated Chalaropsis A and B enzymes. The B enzyme constitutes over 90% of the total lytic activity of crude filtrates and has been obtained as a relatively pure protein. The A enzyme constitutes less than 10% of the initial activity and has not been extensively purified. Both enzymes are N-acetylhexosaminidases that act on the cell wall of S. aureus. The combined effect of the enzymes on the lysis of S. aureus is additive.The B enzyme is a low molecular weight protein with an isoelectric point slightly above pH 7. It is not a chitinaee.
Article
1.1. The rupture of [35S]penicillin treated Bacillus cereus in a pressure cell solubilized the majority of the specifically bound radioactivity. Upon conversion of [35S]penicillin treated cells to protoplasts approx. 95% of the label was found in the lysozyme digest supernatant and not associated with the protoplasts. In either case the bound label was still associated with a large molecular weight component.2.2. The bound label was not found to be associated with the ribosomes, messenger RNA, purified cell wall, or purified cytoplasmic membrane. Localization of the bound penicillin to a component closely associated with the cytoplasmic membrane-cell wall area is strengthened by the finding that a combination of lipase (EC 3.1.1.3) and trypsin (EC 3.4.4.4) caused a major portion of the bound radioactivity to become dialyzable. This latter result indicates that the cell structure to which penicillin binds is a lipoprotein.3.3. The release of the penicillin-receptor complex during protoplast formation explains the results that protoplasts do not fix [35S]penicillin similar to whole cells and that protoplasts cannot be induced to a higher rate of penicillinase (penicillin amidohydrolase, EC 3.5.2.6) formation by addition of penicillin. A 30 to 60 min induction of the inducible strain with penicillin is required for the subsequently formed protoplasts to synthesize penicillinase at comparable rates to induced whole cells.
Article
The staphylolytic enzyme recently isolated from cultures of a Chalaropsis species by Hash is shown to be an acetylmurainidase that cleaves all the glycosidic linkages of N-acetylmuramic acid and N,O-diacetylmuramic acid in the cell wall of Staphylococcus aureus strain Copenhagen. It is similar in specificity to the "32 enzyme" from Streptomyces albus but it differs from egg-white lysozyme whose activity is inhibited by the presence of O-acetyl groups.
Article
Continuous, cell-shaped mucopolymer layers were isolated from cell walls of some unstable and stable L-forms of Proteus mirabilis. The chemical compositions of these non-rigid mucopolymers and of normal rigid mucopolymer basal-structures of rod-shaped Proteus bacteria were very similar. Electron microscopy showed that the protein and mucopolymer of the complex “rigid layers” were organized differently in cell walls of rods and L-form cells. The results suggest that the rigid-layer protein plays a role in the morphogenesis of the mucopolymer.
Article
Ensign, J. C. (University of Illinois, Urbana), and R. S. Wolfe. Lysis of bacterial cell walls by an enzyme isolated from a myxobacter. J. Bacteriol. 90 395–402. 1965.—An exoenzyme which lyses intact cells, heat-killed cells, and cell walls of Arthrobacter crystallopoietes was purified 60-fold from the growth liquor obtained from a myxobacter (strain AL-1). The lytic enzyme was produced during growth of the organism in a number of complex media, the maximal amount of enzyme being produced in yeast extract broth. The purified enzyme lysed at different rates a number of gram-positive bacteria. With the exception of Rhodospirillum rubrum, Spirillum itersonii, and S. serpens, the gram-negative bacteria tested were not attacked.
These have been described in detail in the previous paper. All experiments were performed on 5-month-old Strong A female mice on the
  • Materials Methods
Methods and Materials.-These have been described in detail in the previous paper. All experiments were performed on 5-month-old Strong A female mice on the 6th day after an intraperitoneal injection of Ehrlich ascites cells.
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Autoradiography: Smears of Ehrlich ascites cells were fixed in methanol and autoradiographs
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BY RENATO BASERGAI RICHARD D. ESTENSEN4 AND ROBERT 0. PETERSEN DEPARTMENT OF PATHOLOGY, NORTHWESTERN UNIVERSITY MEDICAL SCHOOL, CHICAGO Communicated by Shields Warren, June 24, 1965