Microcin C: biosynthesis, mode of action, and potential as a lead in antibiotics development

The natural compound Microcin C (McC) is a Trojan horse inhibitor of aspartyl tRNA synthetases endowed with strong antibacterial properties, in which a heptapeptide moiety is responsible for active transport of the inhibitory metabolite part into the bacterial cell. The intracellularly formed aspartyl AMP analogue carries a chemically more stable phosphoramidate linkage, in comparison to the labile aspartyl-adenylate, and in addition is esterified with a 3-aminopropyl moiety. Therefore, this compound can target aspartyl-tRNA synthetase. The biochemical production and secretion of McC, and the possibilities to develop new classes of antibiotics using the McC Trojan horse concept in combination with sulfamoylated adenosine analogues will be discussed briefly.     

细菌持留与抗生素表型耐药机制

持留菌是细菌群体中一小部分具有表型耐药的细菌。自1944年被发现后,近几十年来因其在慢性持续性感染和生物膜感染中的重要作用而得到越来越多的重视。已有的研究结果表明,细菌持留的机理复杂,涉及的相关信号通路有毒素-抗毒素系统、细胞能量代谢及蛋白核酸合成等生理状态的降低、DNA保护修复系统、蛋白酶系统、反式翻译、外排泵系统等。虽然不同细菌的持留机理有一定的相似性和保守性,但不同细菌的持留机制也存在差异,如毒素-抗毒素系统在大肠埃希菌(Escherichia coli)中的过度激活可导致持留菌增加,但在金黄色葡萄球菌(Staphylococcus aureus)中却并无相同作用。本文从持留菌的研究历史出发,综述了当前对革兰氏阴性菌和阳性菌的持留机制方面的研究进展,同时探讨了在持留菌相关感染疾病方面的治疗策略,以期为更好地解决持留菌带来的问题,缩短治疗时间提供新的思路。     

Antimicrobial peptide resistance mechanisms of human bacterial pathogens

The critical role played by antimicrobial peptides (AMPs) in mammalian innate immunity is increasingly recognized. Bacteria differ in their intrinsic susceptibility to AMPs, and the relative resistance of some important human pathogens to these defense molecules is now appreciated as an important virulence phenotype. Experimental analysis has identified diverse mechanisms of bacterial AMP resistance including altered cell surface charge, active efflux, production of proteases or trapping proteins, and modification of host cellular processes. The contribution of these resistance mechanisms to pathogenesis is confirmed through direct comparison of wild-type bacteria and AMP-sensitive mutants using in vivo infection models. Knowledge of the molecular basis of bacterial AMP resistance may provide new targets for antimicrobial therapy of human infectious diseases.     

细菌耐受噬菌体感染的分子机制研究进展

噬菌体是能感染细菌的病毒。为了抵抗噬菌体的感染,细菌进化出多种抵抗噬菌体感染的机制,这些机制的阐析极大地促进了基因编辑领域的发展,同时也为噬菌体治疗的开展奠定了基础。本文就细菌针对噬菌体感染的各个环节所进行的抵抗及其分子机制进行了简要综述,同时讨论了这些防御系统的存在对细菌自身的影响,分析了当前细菌耐受噬菌体机制研究存在的局限性,并对未来研究进行了展望。     

Structures,mechanisms and inhibitors of undecaprenyl diphosphate synthase: A cis-prenyltransferase for bacterial peptidoglycan biosynthesis

Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.     

Synthetic Microcin C Analogs Targeting Different Aminoacyl-tRNA Synthetases

Microcin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the α-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNA^Asp by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.Microcins are small (<10-kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (17). Three microcins, B, C, and J, form a subgroup of posttranslationally modified microcins. Members of this subgroup have highly unusual structures and inhibit cellular enzymes that are validated targets for antibacterial drug development (25). Posttranslationally modified microcins are attractive as drug candidates because of their strong antibacterial action and because virtually limitless numbers of their derivatives can be generated by means of mutation, chemical synthesis, or both. Microcin B (McB), a 43-residue peptide with thiazole and indole rings (13), inhibits DNA gyrase (21). Microcin J, a 21-amino-acid peptide, assumes an unusual threaded lasso structure (2, 23, 27) and inhibits bacterial RNA polymerase (1, 18). The structure of the subject of this study, McC (compound 1) is shown in Fig. ​Fig.1a.1a. McC is a heptapeptide with a formylated N-terminal methionine and a C-terminal aspartate whose α-carboxyl group is covalently linked to adenosine through an N-acyl phosphoramide bond (10, 14). The phosphoramidate of McC is additionally modified by an O-propylamine group (9).Open in a separate windowFIG. 1.Structures and synthesis of McC analogs. (a) Structures of microcin C (compound 1) and its processing product (compound 2). (b) Structures of synthetic McC analogs 7 to 9 and their expected processing products, compounds 4 to 6, which are established inhibitors of AspRS, GluRS, and LeuRS, respectively. (c) Structure of Asp-AMP (compound 3), the natural reaction intermediate of AspRS. Compounds 2 and 4 are nonhydrolyzable analogs of this compound. (d) Synthesis of compounds 7 to 9, which starts from compounds 4 to 6. Hereto the hexapeptide was coupled to the sulfamoyl precursors 4-6 via the coupling agent DIC, followed by removal of the Fmoc protecting group: (i) Fmoc-MRTGNA-OH, HOBt, DIC, DIPEA; (ii) Et₃N/DMF (1:1 [vol/vol]).The passage of McC through the inner layer of the Escherichia coli cell wall is carried out by the YejABEF transporter (19). Once inside the cell, McC is specifically processed by one of the several broad-specificity E. coli cytoplasmic aminopeptidases (12). The product of processing, modified aspartyl-adenylate (compound 2) (15), closely resembles Asp-AMP (compound 3) (Fig. ​(Fig.1c),1c), the natural reaction intermediate of the tRNA^Asp aminoacylation reaction catalyzed by AspRS. However, because the bond between the α-carboxyl of C-terminal aspartate and the phosphoramidate nitrogen is nonhydrolyzable, compound 2 inhibits AspRS. Unprocessed McC has no effect on tRNA^Asp aminoacylation, while processed McC has no effect on McC-sensitive cells at concentrations at which intact McC strongly inhibits cell growth. Thus, McC is a Trojan horse inhibitor (22): the peptide part allows McC to enter sensitive cells, where it gets processed, liberating the inhibitory part of the drug.Aminoacyl-tRNA synthetases (aaRSs) carry out the condensation of genetically encoded amino acids with cognate tRNAs. When 1 of the 20 aaRSs present in the cell is inhibited, the corresponding tRNA is not charged. This leads to protein synthesis inhibition and cell growth arrest. In principle, variation of the last amino acid of the McC peptide, the product of the mccA gene, should allow investigators to obtain McC derivatives targeting aaRSs other than AspRS. Unfortunately, the results of systematic structure-activity analyses of the McC peptide revealed that substitutions in the seventh codon of mccA invariably prevented McC production, presumably by interfering with posttranslational modifications of the MccA peptide by the McC maturation enzymes (11). Indeed, in vitro analysis showed that the C-terminal asparagine of MccA is required for the addition of the adenosine moiety by the MccB protein (24).Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of their corresponding aaRSs (5, 20, 26). However, these compounds show low in vivo activities due to limited membrane permeability and the absence of a transporter for these compounds. Here, we show that through chemical attachment of aminoacyl-sulfamoyl adenosines to the first 6 amino acids of the MccA peptide, potent antibacterial agents can be generated. The new compounds share the Trojan horse mechanism of action with McC but target aaRSs specified by the last amino acid of the peptide moiety.     

Recognition and clinical significance of mechanisms of bacterial resistance to beta-lactams

Resistance to beta-lactams may be difficult to recognize. This is due to the difficulty in detecting these resistances, when the routine tests performed in diagnostic laboratories are interpreted in the usual manner. Since failure to recognize this type of resistance may have serious consequences for the patient, it is essential that it be detected when present. For the detection of methicillin resistance ofStaphylococcus aureus a standardized method using either a medium containing 5% NaCl or a low incubation temperature is advocated. Methicillin resistance ofS. epidermidis can only be recognized reliably by means of a quantitative test and incubation for 42–48 h. Resistance ofHaemophilus influenzae to ampicillin may be intrinsic or it may be caused by a TEM beta-lactamase; a beta-lactamase test should be used to detect the latter type of resistance. Inducible cephalosporinase may be responsible for the rapid development of resistance of some bacterial species to cefamandole, even during therapy. If a stable beta-lactamase production is attained by mutation, resistance to other beta-lactams will usually be present as well. Routine induction tests should be performed for all isolates of species ofEnterobacter, Serratia, Citrobacter andProteus, indole-positive. The same type of ‘hidden’ resistance may be present inPseudomonas aeruginosa, with regard to cefotaxime and other third-generation cephalosporins. Beta-lactamase-positiveNeisseria gonorrhoeae can easily be recognized by a beta-lactamase test. In addition, the results of diffusion tests allow one to distinguish between beta-lactamase-positive and beta-lactamase-negative strains. Recognition of those strains ofN. gonorrhoeae having a decreased susceptibility to penicillin is only possible when well-standardized quantitative tests are used.     

Microcin 7: purification and properties

Microcin 7 is an antibiotic peptide, produced and excreted to the culture medium by E. coli strains harboring the plasmid pRYC7. This peptide was extracted from the culture media by adsorbing it on octadecyl silica. It was purified by gel filtration on Sephadex G-25 and reverse phase high performance liquid chromatography. Its amino acid composition is the following: Ala (0.8), Arg (1.9), Asx (1.9), Gly (1.5), Met (0.8) and Thr (0.9). The purified peptide dose not react with ninhydrin and it is resistant to carboxypeptidase degradation, indicating that the molecule may be a cyclic or end-blocked oligopeptide.     

Molecular mechanisms involved in bacterial speck disease resistance of tomato

An important recent advance in the field of plant-microbe interactions has been the cloning of genes that confer resistance to specific viruses, bacteria, fungi or nematodes. Disease resistance (R) genes encode proteins with predicted structural motifs consistent with them having roles in signal recognition and transduction. The future challenge is to understand how R gene products specifically perceive defence-eliciting signals from the pathogen and transduce those signals to pathways that lead to the activation of plant defence responses. In tomatoes, the Pto kinase (product of the Pto R gene) confers resistance to strains of the bacterial speck pathogen, Pseudomonas syringae pv. tomato, that carry the corresponding avirulence gene avrPto. Resistance to bacterial speck disease is initiated by a mechanism involving the physical interaction of the Pto kinase and the AvrPto protein. This recognition event initiates signalling events that lead to defence responses including an oxidative burst, the hypersensitive response and expression of pathogenesis-related genes. Pto-interacting (Pti) proteins have been identified that appear to act downstream of the Pto kinase and our current studies are directed at elucidating the roles of these components.     

Combatting antimicrobial resistance via the cysteine biosynthesis pathway in bacterial pathogens

Antibiotics are the cornerstone of modern medicine and agriculture, and rising antibiotic resistance is one the biggest threats to global health and food security. Identifying new and different druggable targets for the development of new antibiotics is absolutely crucial to overcome resistance. Adjuvant strategies that either enhance the activity of existing antibiotics or improve clearance by the host immune system provide another mechanism to combat antibiotic resistance. Targeting a combination of essential and non-essential enzymes that play key roles in bacterial metabolism is a promising strategy to develop new antimicrobials and adjuvants, respectively. The enzymatic synthesis of L-cysteine is one such strategy. Cysteine plays a key role in proteins and is crucial for the synthesis of many biomolecules important for defense against the host immune system. Cysteine synthesis is a two-step process, catalyzed by two enzymes. Serine acetyltransferase (CysE) catalyzes the first step to synthesize the pathway intermediate O-acetylserine, and O-acetylserine sulfhydrylase (CysK/CysM) catalyzes the second step using sulfide or thiosulfate to produce cysteine. Disruption of the cysteine biosynthesis pathway results in dysregulated sulfur metabolism, altering the redox state of the cell leading to decreased fitness, enhanced susceptibility to oxidative stress and increased sensitivity to antibiotics. In this review, we summarize the structure and mechanism of characterized CysE and CysK/CysM enzymes from a variety of bacterial pathogens, and the evidence that support targeting these enzymes for the development of new antimicrobials or antibiotic adjuvants. In addition, we explore and compare compounds identified thus far that target these enzymes.     

细菌对利奈唑胺的耐药机制及检测方法研究进展

利奈唑胺是一种重要的噁唑烷酮类药物, 对临床上具重要意义的革兰阳性菌, 包括多重耐药葡萄球菌、耐万古霉素肠球菌等有抗菌作用。目前虽然细菌耐利奈唑胺的情况并不严重, 但临床耐药株的出现值得关注。本文综述了细菌对利奈唑胺的耐药机制及检测方法。其耐药机制主要由细菌单个碱基突变和甲基转移酶的产生所介导, 还有一些耐药机制尚不明了。检测方法主要有微生物学和分子生物学方法, 部分方法尚需进一步完善。     

Extended targeting potential and improved synthesis of Microcin C analogs as antibacterials

Microcin C (McC) (1) is a potent antibacterial compound produced by some Escherichia coli strains. McC functions through a Trojan-Horse mechanism: it is actively taken up inside a sensitive cell through the function of the YejABEF-transporter and then processed by cellular aminopeptidases. Processed McC (2) is a non-hydrolysable aspartyl-adenylate analog that inhibits aspartyl-tRNA synthetase (AspRS). A new synthesis is described that allows for the production of a wide variety of McC analogs in acceptable amounts. Using this synthesis a number of diverse compounds was synthesized with altered target specificity. Further characteristics of the YejABEF transporters were determined using these compounds.     

The RimL Transacetylase Provides Resistance to Translation Inhibitor Microcin C

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.     

Aspartyl-tRNA synthetase is the target of peptide nucleotide antibiotic Microcin C

Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.     

The mechanisms of hepatitis C virus resistance to interferon

The aim of this review is to describe the role of hepatitis C proteins, non-structural protein 5A and envelope protein E2, in resistance to interferon alpha. These proteins contain interferon induced-protein kinase R binding domains. The binding renders the kinase inactive; therefore the phosphorylation of translation factor eIF2 is inhibited. The studies indicate that phosphorylation of eIF4E is also inhibited. As a result, with the sufficient pool of active eIF2 in infected cell, synthesis of viral proteins proceeds while cap- and cap binding factors-, among them eIF4E, -dependent synthesis of host proteins is diminished. It seems this process is one of the molecular mechanisms responsible for the resistance of hepatitis C virus to interferon, persistence in infected cell and the resultant difficulties in treatment of infected individuals.     

Exposure to host resistance mechanisms drives evolution of bacterial virulence in plants

Bacterial pathogenicity to plants and animals has evolved through an arms race of attack and defense. Key players are bacterial effector proteins, which are delivered through the type III secretion system and suppress basal defenses . In plants, varietal resistance to disease is based on recognition of effectors by the products of resistance (R) genes . When recognized, the effector or in this scenario, avirulence (Avr) protein triggers the hypersensitive resistance reaction (HR), which generates antimicrobial conditions . Unfortunately, such gene-for-gene-based resistance commonly fails because of the emergence of virulent strains of the pathogen that no longer trigger the HR . We have followed the emergence of a new virulent pathotype of the halo-blight pathogen Pseudomonas syringae pv. phaseolicola within leaves of a resistant variety of bean. Exposure to the HR led to the selection of strains lacking the avirulence (effector) gene avrPphB (or hopAR1), which triggers defense in varieties with the matching R3 resistance gene. Loss of avrPphB was through deletion of a 106 kb genomic island (PPHGI-1) that shares features with integrative and conjugative elements (ICElands) and also pathogenicity islands (PAIs) in diverse bacteria . We provide a molecular explanation of how exposure to resistance mechanisms in plants drives the evolution of new virulent forms of pathogens.