In vivo target exploration of apidaecin based on Acquired Resistance induced by Gene Overexpression (ARGO assay).
ABSTRACT: Identifying the target molecules of antimicrobial agents is essential for assessing their mode of action. Here, we propose Acquired Resistance induced by Gene Overexpression (ARGO) as a novel in vivo approach for exploring target proteins of antimicrobial agents. The principle of the method is based on the fact that overexpression of the expected target protein leads to reduced sensitivity to the antimicrobial agent. We applied this approach to identify target proteins of the antimicrobial peptide apidaecin, which is specifically effective against Gram-negative bacteria. To this end, a set of overexpression Escherichia coli clones was tested, and peptide chain release factor 1, which directs the termination of translation, was found as a candidate, suggesting that apidaecin inhibits the termination step of translation. This finding was confirmed in vivo and in vitro by evaluating the inhibitory activity of apidaecin towards lacZ reporter gene expression, which is tightly dependent on its stop codon. The results of this study demonstrate that apidaecin exerts its antimicrobial effects partly by inhibiting release factors.
Project description:Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.
Project description:During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome.
Project description:Many antibiotics stop bacterial growth by inhibiting different steps of protein synthesis. However, no specific inhibitors of translation termination are known. Proline-rich antimicrobial peptides, a component of the antibacterial defense system of multicellular organisms, interfere with bacterial growth by inhibiting translation. Here we show that Api137, a derivative of the insect-produced antimicrobial peptide apidaecin, arrests terminating ribosomes using a unique mechanism of action. Api137 binds to the Escherichia coli ribosome and traps release factor (RF) RF1 or RF2 subsequent to the release of the nascent polypeptide chain. A high-resolution cryo-EM structure of the ribosome complexed with RF1 and Api137 reveals the molecular interactions that lead to RF trapping. Api137-mediated depletion of the cellular pool of free release factors causes the majority of ribosomes to stall at stop codons before polypeptide release, thereby resulting in a global shutdown of translation termination.
Project description:The conjugation of the cationic antimicrobial peptide, apidaecin Ib, to the anionic photosensitizer, 5(4'-carboxyphenyl)-10,15,20-triphenylporphyrin (cTPP), afforded a new antibacterial agent effective, under light activation, against both Gram-positive and Gram-negative bacteria. At low concentrations (1.5-15 ?M) the conjugate was able to reduce the survival of Escherichia coli cells by 3-4 log10, and most notably, it resulted photoactive also against hard-to-treat Pseudomonas aeruginosa, although at higher concentration (60 ?M). Under similar conditions, the photosensitizer alone was only photoactive against Staphylococcus aureus while the unconjugated peptide was inactive against all the bacterial strains tested. This study shows the possibility of obtaining new broad-spectrum apidaecin-photosensitizer conjugates with potent antibacterial activity.
Project description:Drug resistance is a major problem in antibacterial chemotherapy. Apidaecins, which refer to a series of small, proline-rich antimicrobial peptides, are predominantly active against many drug-resistant bacteria. The apidaecins have special antibacterial mechanisms, and are non-toxic for human cells, a prerequisite for using them as novel antibiotic drugs. However, no efficient non-tagged apidaecin expression system has been reported, which is the limiting factor for their application. Here we successfully generated a Pichia pastoris transformant expressing and secreting apidaecin. However, expression was unstable and poor. Analysis of this revealed that the integration plasmid was frequently lost and that apidaecin expression resulted in cell death. Using N-methyl-N-nitro-N-nitroso-guanidine mutagenesis and selection, a mutant strain Apmu4 was derived, in which the rate of loss of the integration plasmid was much lower after induction, and which produced improved titres of apidaecin. Additionally, we discovered that using glucose as the sole carbon source to pre-culture the strain before induction could greatly enhance apidaecin production. A pilot-scale 10?L fermentation yielded 418?mg/L of recombinant apidaecin, which represents the highest reported yield of apidaecin. Consequently, this study reports the first super heterologous expression and secretion of apidaecin in yeast.
Project description:Proline-rich antimicrobial peptide apidaecin (Api) inhibits bacterial protein synthesis in a distinctive way. In vitro biochemical and structural studies showed that Api binds in the nascent peptide exit tunnel of the ribosome that has completed translation of the gene and has released the newly-synthesized protein. Once in the tunnel, Api traps the release factors on the post-termination ribosome leading to cessation of translation. The mode of Api action in the bacterial cell, however, had remained unknow. By analyzing distribution of ribosomes on mRNAs in Api-treated cells using Ribo-seq approach we uncovered a range of effects that stem from the unique mechanism of Api action. Exposure of bacterial cells to Api results in arrest of translation at stop codons, likely in a post-release state, due to the immediate Api action, as well as in a pre-release state due to the depletion of available release factors. In addition, arrest of translation at the end of the open reading frame (ORF) leads to a pronounced queuing of the translating ribosomes behind the one arrested at the stop codon. One of the major consequences of the Api action is a dramatically increased stop codon bypass by the ribosomes paused in a pre-release state, leading to accumulation of proteins with C-terminal extensions, as confirmed by whole-cell proteomics analysis. Stop codon bypass, that occurs either in 0-frame, by misincorporation of a near-cognate aminoacyl-tRNA at the stop codon, or via frameshifting allows for a fraction of the translating ribosomes to reach the 3’ ends of mRNA transcripts. The pervasive stalling of pre-release ribosomes at the stop codons and at the RNA transcripts ends triggers activation of the cellular ribosome rescue systems which, likely due to Api action as well, remain ineffective. While major Api effects are directed towards the ribosome at the end of the ORFs, cells exposed to Api show a somewhat increased ribosome occupancy of the start codons. Understanding the unique mode of Api to inhibit translation termination in the cell can help envisioning the development of treatments for genetic diseases and research tools for genome exploration.
Project description:Apidaecins are a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. They are predominantly active against the gram-negative bacteria. Previous studies mainly focused on the identification of their internal macromolecular targets, few addressed on the action of apidaecins on the molecules, especially proteins, of bacterial cell membrane. In this study, iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered membrane proteins of Escherichia coli cells incubated with one isoform of apidaecins--apidaecin IB. Cell division protease ftsH, an essential regulator in maintenance of membrane lipid homeostasis, was found to be overproduced in cells incubated with apidaecin IB. Its over-expression intensified the degradation of cytoplasmic protein UDP-3-O-acyl-N- acetylglucosamine deacetylase, which catalyzes the first committed step in the biosynthesis of the lipid A moiety of LPS, and thus leaded to the further unbalanced biosynthesis of LPS and phospholipids. Our findings suggested a new antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.
Project description:ArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export of l-arginine (Arg) in Escherichia coli and l-lysine (Lys) and Arg in Corynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane of E. coli, we have employed a combination of cysteine accessibility in situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Cout configuration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO function in vivo and that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO function in vivo and their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomer in vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.The orthologous proteins LysE of C. glutamicum and ArgO of E. coli function as exporters of the basic amino acids l-arginine and l-lysine and the basic amino acid l-arginine, respectively, and LysE can functionally substitute for ArgO when expressed in E. coli Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic and in silico studies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.
Project description:Proline-rich antimicrobial peptides (PrAMPs) represent promising alternative therapeutic options for the treatment of multidrug-resistant bacterial infections. PrAMPs are predominantly active against Gram-negative bacteria by inhibiting protein expression via at least two different modes of action, i.e., blocking the ribosomal exit tunnel of 70S ribosomes (oncocin-type binding) or inhibiting the assembly of the 50S ribosomal subunit (apidaecin-type binding). The in vivo efficacy and favorable biodistribution of oncocins confirmed the therapeutic potential of short PrAMPs for the first time, whereas the in vivo evaluation of apidaecins is still limited despite the promising efficacy of apidaecin-analog Api88 in an intraperitoneal murine infection model. Here, the in vivo efficacy of apidaecin-analog Api137 was studied, which rescued all NMRI mice from a lethal intraperitoneal infection with E. coli ATCC 25922 when administered three times intraperitoneal at doses of 0.6 mg/kg starting 1 h after infection. When Api88 and Api137 were administered intravenous or intraperitoneal at doses of 5 and 20 mg/kg, their plasma levels were similarly low (<3 ?g/mL) and four-fold lower than for oncocin-analog Onc72. This contradicted earlier expectation based on the very low serum stability of Api88 with a half-life time of only ~5 min compared to ~6 and ~3 h for Api137 and Onc72, respectively. Pharmacokinetic data relying on a sensitive mass spectrometry method utilizing multiple reaction monitoring and isotope-labeled peptides revealed that Api88 and Api137 were present in blood, urine, and kidney, and liver homogenates at similar levels accompanied by the same major metabolites comprising residues 1-16 and 1-17. The pretended discrepancy was solved, when all peptides were incubated in peritoneal lavage. Api137 was rapidly degraded at the C-terminus, while Api88 was rather stable despite releasing the same degradation products. Onc72 was very stable explaining its higher plasma levels compared to Api88 and Api137 after intraperitoneal administration illuminating its good in vivo efficacy. The data indicate that the degradation of therapeutic peptides should be studied in serum and further body fluids. Moreover, the high efficacy in murine infection models and the fast clearance of Api88 and Api137 within ~60 min after intravenous and ~90 min after intraperitoneal injections indicate that their in vivo efficacy relates to the maximal peptide concentration achieved in blood.