Deacylated tRNA Accumulation Is a Trigger for Bacterial Antibiotic Persistence Independent of the Stringent Response.

Bacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation (phenylalanyl-tRNA synthetase [PheRS]) were used to study the effects of quality control on antibiotic persistence. Upon amino acid limitation, elevated PheRS quality control led to significant decreases in aminoacylated tRNAPhe accumulation and increased antibiotic persistence. This increase in antibiotic persistence was most pronounced (65-fold higher) when the relA-encoded tRNA-dependent stringent response was inactivated. The increase in persistence with elevated quality control correlated with ∼2-fold increases in the levels of the RNase MazF and the NTPase MazG and a 3-fold reduction in cellular NTP pools. These data reveal a mechanism for persister formation independent of the stringent response where reduced translation capacity, as indicated by reduced levels of aminoacylated tRNA, is accompanied by active reduction of cellular NTP pools which in turn triggers antibiotic persistence. IMPORTANCE Bacterial antibiotic persistence is a transient physiological state wherein cells become dormant and thereby evade being killed by antibiotics. Once the antibiotic is removed, bacterial persisters are able to resuscitate and repopulate. It is thought that antibiotic bacterial persisters may cause reoccurring infections in the clinical setting. The molecular triggers and pathways that cause bacteria to enter into the persister state are not fully understood. Our results suggest that accumulation of deacylated tRNA is a trigger for antibiotic persistence independent of the RelA-dependent stringent response, a pathway thought to be required for persistence in many organisms. Overall, this provides a mechanism where changes in translation quality control in response to physiological cues can directly modulate bacterial persistence.

An aminoacylation ribozyme evolved from a natural tRNA-sensing T-box riboswitch.

When the primitive translation system first emerged in the hypothetical RNA world, ribozymes could have been responsible for aminoacylation. Given that naturally occurring T-box riboswitches selectively sense the aminoacylation status of cognate tRNAs, we introduced a domain of random sequence into a T-box-tRNA conjugate and isolated ribozymes that were self-aminoacylating on the 3'-terminal hydroxyl group. One of them, named Tx2.1, recognizes the anticodon and D-loop of tRNA via interaction with its stem I domain, similarly to the parental T-box, and selectively charges N-biotinyl-L-phenylalanine (Bio-lPhe) onto the 3' end of the cognate tRNA in trans. We also demonstrated the ribosomal synthesis of a Bio-lPhe-initiated peptide in a Tx2.1-coupled in vitro translation system, in which Tx2.1 catalyzed specific tRNA aminoacylation in situ. This suggests that such ribozymes could have coevolved with a primitive translation system in the RNA world.

Drugging tRNA aminoacylation.

Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNAs to produce the aminoacyl-tRNAs (aa-tRNAs) required by ribosomes for translation of the genetic message into proteins. To ensure the accuracy of tRNA aminoacylation, and consequently the fidelity of protein synthesis, some aaRSs exhibit a proofreading (editing) site, distinct from the aa-tRNA synthetic site. The aaRS editing site hydrolyzes misacylated products formed when a non-cognate amino acid is used during tRNA charging. Because aaRSs play a central role in protein biosynthesis and cellular life, these proteins represent longstanding targets for therapeutic drug development to combat infectious diseases. Most existing aaRS inhibitors target the synthetic site, and it is only recently that drugs targeting the proofreading site have been considered. In the present study, we developed a robust assay for the high-throughput screening of libraries of inhibitors targeting both the synthetic and the proofreading sites of up to four aaRSs simultaneously. Thus, this assay allows for screening of eight distinct enzyme active sites in a single experiment. aaRSs from several prominent human pathogens (i.e., Mycobacterium tuberculosis, Plasmodium falciparum, and Escherichia coli) were used for development of this assay.

Discovery and characterization of a novel class of pyrazolopyrimidinedione tRNA synthesis inhibitors.

A high-throughput phenotypic screen for novel antibacterial agents led to the discovery of a novel pyrazolopyrimidinedione, PPD-1, with preferential activity against methicillin-resistant Staphylococcus aureus (MRSA). Resistance mapping revealed the likely target of inhibition to be lysyl tRNA synthetase (LysRS). Preliminary structure-activity relationship (SAR) studies led to an analog, PPD-2, which gained Gram-negative antibacterial activity at the expense of MRSA activity and resistance to this compound mapped to prolyl tRNA synthetase (ProRS). These targets of inhibition were confirmed in vitro, with PPD-1 showing IC50s of 21.7 and 35 µM in purified LysRS and ProRS enzyme assays, and PPD-2, 151 and 0.04 µM, respectively. The highly attractive chemical properties of these compounds combined with intriguing preliminary SAR suggest that further exploration of this compelling novel series is warranted.

Effect of hydrogen peroxide on the biosynthesis of heme and proteins: potential implications for the partitioning of Glu-tRNA(Glu) between these pathways.

Glutamyl-tRNA (Glu-tRNA(Glu)) is the common substrate for both protein translation and heme biosynthesis via the C5 pathway. Under normal conditions, an adequate supply of this aminoacyl-tRNA is available to both pathways. However, under certain circumstances, Glu-tRNA(Glu) can become scarce, resulting in competition between the two pathways for this aminoacyl-tRNA. In Acidithiobacillus ferrooxidans, glutamyl-tRNA synthetase 1 (GluRS1) is the main enzyme that synthesizes Glu-tRNA(Glu). Previous studies have shown that GluRS1 is inactivated in vitro by hydrogen peroxide (H2O2). This raises the question as to whether H2O2 negatively affects in vivo GluRS1 activity in A. ferrooxidans and whether Glu-tRNA(Glu) distribution between the heme and protein biosynthesis processes may be affected by these conditions. To address this issue, we measured GluRS1 activity. We determined that GluRS1 is inactivated when cells are exposed to H2O2, with a concomitant reduction in intracellular heme level. The effects of H2O2 on the activity of purified glutamyl-tRNA reductase (GluTR), the key enzyme for heme biosynthesis, and on the elongation factor Tu (EF-Tu) were also measured. While exposing purified GluTR, the first enzyme of heme biosynthesis, to H2O2 resulted in its inactivation, the binding of glutamyl-tRNA to EF-Tu was not affected. Taken together, these data suggest that in A. ferrooxidans, the flow of glutamyl-tRNA is diverted from heme biosynthesis towards protein synthesis under oxidative stress conditions.

Innate immune and chemically triggered oxidative stress modifies translational fidelity.

Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

High levels of tRNA abundance and alteration of tRNA charging by bortezomib in multiple myeloma.

In multiple myeloma (MM), malignant plasma cells produce large amounts of antibodies and have highly active protein translational machinery. It is not known whether regulation of the abundance and aminoacylation (charging) of transfer RNA (tRNA) takes place in myeloma cells to accommodate for the increased amount of protein translation. Using tRNA-specific microarrays, we demonstrate that tRNA levels are significantly elevated in MM cell lines compared to normal bone marrow cells. We furthermore show that the addition of the proteasome inhibitor, bortezomib (Velcade, PS-341) results in decreased charging levels of tRNAs, in particular those coding for hydrophobic amino acids. These results suggest that tRNA properties are altered in MM to accommodate for its increased need for protein translation, and that proteasome inhibition directly impacts protein synthesis in MM through effects on tRNA charging.

Crystallographic and mutational studies of seryl-tRNA synthetase from the archaeon Pyrococcus horikoshii.

Seryl-tRNA synthetase (SerRS) catalyzes the ligation of serine to the 3'-end of serine tRNA (tRNA(Ser)), which is typical of the type-2 tRNAs characterized by a long extra arm. The SerRSs are divided into two types, the archaeal/eukaryal and bacterial types. In this study, we solved the crystal structures of the SerRS from the archaeon Pyrococcus horikoshii bound with 5'-O-[N-(L-seryl)-sulfamoyl]-adenosine at 2.6 A and with ATP at 2.8 A, as well as in the apo form at 3.0 A. P. horikoshii SerRS recognizes the seryl and adenylate moieties in a manner similar to those of the bacterial and mitochondrial SerRSs from Thermus thermophilus and Bos taurus, respectively, but different from that of the unusual SerRS from the methanogenic archaeon Methanosarcina barkeri. P. horikoshii SerRS efficiently aminoacylated not only P. horikoshii tRNA(Ser) but also bacterial tRNA(Ser)s from T. thermophilus and Escherichia coli. Models of P. horikoshii SerRS bound with the T. thermophilus and P. horikoshii tRNA(Ser)s suggested that the helical domain of P. horikoshii SerRS is involved in the extra arm binding. This region of P. horikoshii SerRS has additional basic residues as compared with T. thermophilus SerRS, and a Trp residue specific to the archaeal/eukaryal SerRSs. Mutational analyses revealed that the basic and Trp residues are important for tRNA aminoacylation. P. horikoshii SerRS has the archaea-specific insertion, which collaborates with the core domain to form a basic channel leading to the active site. Two sulfate ions are bound to the channel, suggesting that the tRNA 3' region might bind to the channel.

SPARK: a new peptidyl transferase activity assay.

The formation of peptide bonds is the central chemical reaction during protein synthesis and is catalyzed by the peptidyl transferase center residing in the large ribosomal subunit. This active site is composed of universally conserved rRNA nucleosides. The peptidyl transferase center is by far the most frequently used target site of natural antibiotics in the cell. Here we describe a novel, simple, and convenient method to assess peptide bond formation which we named SPARK. The basic principle of SPARK is the use of two reaction substrates that closely resemble the natural tRNA substrates (one is biotinylated and the other carries a tritium label) that become covalently connected during transpeptidation. Formation of this peptide bond then allows capture and direct quantification of the radiolabled product, now joined to the biotin group, using the scintillation proximity assay technology. Binding of the tritiated radioligand to streptavidin-coated beads causes the excitation of the bead-embedded scintillant, thus resulting in the detection of radioactivity. Since no product purification step is required, SPARK is amenable to simple automation, which makes it useful in high-throughput screens of natural or synthetic compound libraries in the search for novel antibiotics.

Methods for identifying compounds that specifically target translation.

This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and aminoacylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model "universal" mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a specific target of an antibiotic within the translational pathway of prokaryotic cells.

Spotlight on targeting aminoacyl-tRNA synthetases for the treatment of fungal infections.

This month's Spotlight on... focuses on targeting aminoacyl-tRNA synthetases for the treatment of fungal infections. The emergence of drug-resistant strains of bacterial and fungal pathogens has led to the development of new antibiotic strategies. Aminoacyl-tRNA synthetases, crucial for protein synthesis, represent a new target for the treatment of different infectious diseases.

Differential activation of system A and betaine/GABA transport in MDCK cell membranes by hypertonic stress.

Accumulation of osmolytes by renal cells is due in part to increased uptake via specific transporters. These include amino acid transport system A and the betaine/GABA transporter (BGT1). Transport changes have been characterized using intact cells which makes the intracellular mechanisms difficult to determine. In this study the hypertonic upregulation of system A and BGT1 was studied directly at the membrane level in Madin-Darby canine kidney (MDCK) cells. Both system A and BGT1 transport systems were detected in an isolated membrane fraction containing plasma membranes. System A transport was increased in membranes prepared from cells after 6 h hypertonic stress (449 mosmol/kg) but BGT1 activity was minimal and not different from isotonic controls. The increase in system A was blocked by inhibitors of RNA and protein synthesis. BGT1 transport was induced in membranes prepared after 24 h hypertonicity. At this time system A activity in the membrane fraction remained increased, unlike the downregulation observed in intact MDCK cells. We conclude that differential upregulation of system A and BGT1 by hypertonic stress is due to intrinsic changes in these transporters at the membrane level. In contrast, the downregulation of system A in intact cells when hypertonicity is prolonged for 24 h is likely due to the action of an intracellular repressor that is not present in the isolated membranes.

The effectiveness of the bonding of amino acid by tRNA of livers of rats experimentally exposed to cadmium and barium.

The process of amino acid activation takes place in the presence of specific aminoacyl-tRNA synthetases. These enzymes, together with ATP, catalyse the bonding of tRNA and amino acids (5). The effectiveness of binding amino acid by tRNA has a direct influence on the intensity of protein biosynthesis. This effectiveness varies in various metabolic states of the cell (16). Heavy metals influence cellular metabolism. They usually reduce enzyme activity, block chemical reactions, bind with proteins, interact with other elements and even break certain bonds (1, 8, 9, 11, 14). Experiments in the present work were conducted in order to determine changes in the effectiveness of amino acid binding by tRNA of the livers of rats receiving heavy metals such as cadmium and barium in their feed.

Alteration in the conformational stability of collagen caused by the incorporation of the lysine analogue S-2-aminoethylcysteine.

We studied the effects of the lysine analogue S-2-aminoethylcysteine on the activation of lysyl tRNA and on the secretion and conformational stability of newly synthesized type I collagen in embryonic chick tendon fibroblasts. The analogue competed efficiently with lysine for activation onto tRNA without affecting significantly the activation of other amino acids (Km for lysine: 1.6 microM; Ki for S-2-aminoethylcysteine: 1.4 microM). The analogue also profoundly inhibited the synthesis and secretion of [14C]procollagen but did not affect the synthesis or secretion of non-collagenous proteins. Although the [14C]proline-labeled procollagen synthesized in the presence of S-2-aminoethylcysteine contained normal levels of hydroxyproline, it was susceptible to digestion with pepsin at 25 degrees C, indicating that incorporation of the analogue altered the conformational stability of the collagen triple helix. This analogue should be a powerful tool to further study the role of lysine on collagen structure and to determine how altered collagen structure affects its synthesis and secretion. Furthermore, this analogue may be a potent and selective inhibitor of collagen accumulation in pathologic conditions accompanied by tissue fibrosis.

Antidegenerative effects of Mg(2+)-valproate in cultured cerebellar neurons.

We have investigated in the present study the effect of Mg(2+)-valproate on necrotic degeneration induced by an excitotoxic insult in primary culture of cerebellar neurons, that is an homogeneous population of glutamatergic neurons. Mg(2+)-valproate protected cultures against glutamate-induced neurotoxicity, acting as an indirect N-methyl-D-aspartate (NMDA) receptor antagonist, thus reducing free radical formation and affecting the biochemical parameters (i.e. 45Ca(2+)-influx, cyclic GMP formation, inositol phospholipid hydrolysis and protein kinase C translocation) that undergo modifications following NMDA receptor activation in cerebellar granule cells.

Characterization of versilin-sensitive sites in self-sensitive producer and sensitive non-producer or unrelated organism.

Studies on self-sensitivity of producer mutant vs. sensitivity of non-producer parent and unrelated organism showed that versilin inhibited spore germination and sporulation in the self-sensitive producer mutant, non-producer parent Aspergillus versicolor N5 and the unrelated sensitive Trichophyton rubrum. Sporulation appeared to be more sensitive than spore germination. The inhibition of in vivo synthesis of protein was very marked, but inhibition of RNA and DNA was slight and moderate, respectively. Thus versilin was not specific in its action, but the principal sensitive site was protein synthesis, as further suggested by inhibition of polyU-directed in vitro synthesis of polyphenylalanine. The activation of leucine was unaffected, but the formation of leucyl-tRNA was severely inhibited in all three strains. The differences in sensitivities between the strains were the same, whether as whole cells or as cell-free extracts. Thus the nature of the sensitive site appeared to be identical in the self-sensitive producer and sensitive non-producer or unrelated organism.

Inactivation of human arginine-143, lysine-143, and isoleucine-143 Cu,Zn superoxide dismutases by hydrogen peroxide: multiple mechanisms for inactivation.

Site-specific mutants of human Cu,Zn superoxide dismutase (Cu,ZnSOD) have been prepared in which the active-site arginine at position 143 (i.e., SODR143) has been replaced by either lysine (SODK143) or isoleucine (SODI143). As reported previously (W.F. Beyer, Jr., et al. (1987) J. Biol. Chem. 262, 11182-11187), SODK143 and SODI143 have 43 and 11%, respectively, of the catalytic activity of SODR143. H2O2, at low concentrations, acts as an affinity reagent for the inactivation of SODR143. At pH 9.0 and 25 degrees C, the process is characterized by a half-saturation constant for H2O2, K50, of 5.1 mM and a maximum pseudo-first-order rate constant for inactivation, Kmax, of 0.53 min-1. At pH 11.5, the corresponding values are 0.63 mM and 1.23 min-1. The active species in the inactivation is likely HO2-, as previously found with yeast and bovine Cu,ZnSODs (see C.L. Borders, Jr., and I. Fridovich (1985) Arch. Biochem. Biophys. 241, 472-476). SODK143 is also inactivated by HO2- by an affinity mechanism, i.e., one where reversible binding of H2O2 (HO2-) is a prerequisite for inactivation. At pH values of 9.0 and 11.5, the kmax values are 0.92 and 1.08 min-1, respectively; however, the corresponding K50 values increase to 42.5 and 15.8 mM, respectively. SODI143 is also inactivated by H2O2, but no evidence for an affinity mechanism was found; instead, a second-order kinetic mechanism was observed. Inactivation of each of the three enzymes is accompanied by the loss of one histidine per subunit. At elevated concentrations of H2O2, a second nonaffinity mechanism of inactivation of both SODR143 and SODK143 was found, in which a second equivalent of H2O2 reacts with the Cu,ZnSOD.HO2- complex to give a competing second-order inactivation. It appears that the positive charge of arginine-143 plays a role in the binding of HO2- at the active site of human Cu,ZnSOD, and that replacement of the arginine by lysine gives an enzyme with a similar affinity mechanism of inactivation, but with a greatly reduced affinity for HO2-. However, replacement with isoleucine causes an entirely different mechanism of inactivation; this raises the possibility that the mechanism of enzyme catalysis of superoxide dismutation by SODI143 is also different.

The initiator tRNA acceptance assay as a short-term test for carcinogens. 4. Results with 20 mycotoxins.

The activity of 20 mycotoxins was tested by the recently developed initiator tRNA acceptance assay for carcinogens. With the exception of citrinin, all compounds carcinogenic for rodents stimulated the charging of tRNA with L-methionine. In three out of four non-carcinogenic mycotoxins the test was negative. Five carcinogenic mycotoxins were negative in mutagenicity tests but positive in the acceptance assay indicating that also non-mutagenic carcinogens may be detected by the latter procedure.