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.     

Cardiolipin Molecular Species with Shorter Acyl Chains Accumulate in Saccharomyces cerevisiae Mutants Lacking the Acyl Coenzyme A-binding Protein Acb1p: NEW INSIGHTS INTO ACYL CHAIN REMODELING OF CARDIOLIPIN*

The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates.Cardiolipin (CL)⁵ is a unique anionic glycerophospholipid with dimeric structure containing four acyl chains, which is almost exclusively localized to the mitochondrial inner membrane in eukaryotic cells (1, 2). CL has been shown to co-isolate with, and to be required for optimal activity of a number of enzymes in the respiratory chain (3–5), and it has been implicated in the stability and assembly of protein (super)complexes (6–8). In the presence of divalent cations and dependent on the acyl chain composition, CL has a propensity for membrane negative curvature, a property that may be important in, e.g. membrane fusion and fission (9, 10). In addition, CL is thought to serve as a proton trap in oxidative phosphorylation (11). In recent years, CL has also been implicated in apoptosis (12, 13).CL is synthesized in the inner mitochondrial membrane by condensation of PG and CDP-DAG, catalyzed by the cardiolipin synthase Crd1p (see Fig. 1; reviewed in Ref. 4). Compared with the other phospholipid classes, CL is enriched in unsaturated acyl chains, and the molecular species of CL possess a high degree of molecular symmetry (14). The CL-specific acyl chain pattern originates from substrate preferences during biosynthesis and subsequent remodeling by acyl chain exchange (15). The finding of an aberrant CL species profile in patients suffering from Barth syndrome, which results from mutations in the tafazzin gene (16), revealed the importance of CL remodeling, and set the stage for the identification of tafazzin as the acyltransferase involved (17, 18). The Drosophila homologue of tafazzin was shown to be a CoA-independent phospholipid transacylase with substrate preference for CL and PC (19).Open in a separate windowFIGURE 1.The cardiolipin biosynthetic pathway in the context of phospholipid biosynthesis in yeast. The enzymes of the CL biosynthetic pathway identified at the gene level are indicated: Cds1p, CDP-DAG synthase; Pgs1p, phosphatidylglycerolphosphate synthase; Crd1p, CL synthase; Taz1p, Tafazzin; Cld1p, CL-specific deacylase.The biosynthesis and remodeling of CL have been extensively studied in the yeast Saccharomyces cerevisiae. After synthesis by Crd1p, CL is subject to deacylation and reacylation, which involves the yeast homologue of tafazzin encoded by the TAZ1 gene. The yeast taz1 mutant has defects similar to those found in Barth syndrome, including reduced CL content, an aberrant CL species profile, and an accumulation of monolyso-CL (20). The bioenergetic coupling of isolated mitochondria from a taz1 mutant is compromised (21), which may be accounted for by the impaired assembly of the III₂IV₂ supercomplex (22). Recently, the CL-specific phospholipase Cld1p was identified, which functions upstream of Taz1p (23).Because the acyl chain composition of CL is important for its function, we investigated how the molecular species profile of CL is attained by using depletion of the 10-kDa cytosolic acyl-CoA-binding protein Acb1p as a tool to modify the cellular acyl chain content. Deletion of the ACB1 gene increases the cellular levels of C14 and C16 fatty acids at the expense of C18, without having adverse effects on cell growth or on the rate of glycerophospholipid synthesis (24–26). The changes in fatty acid composition are reflected to varying extents in the molecular species profile of phospholipids in Acb1p-depleted cells as determined by electrospray ionization-mass spectrometry (ESI-MS) (27, 28). We first determined by mass spectrometry that in the absence of Acb1p acyl chains shorter than C16 accumulate in CL as in the other phospholipid classes despite the Cld1p-Taz1p remodeling system. Using appropriate mutants and analysis by mass spectrometry, we investigated two possible origins of the shorter acyl chains in CL: (i) remodeling by Taz1p and (ii) de novo synthesis of CL from PG and CDP-DAG.     

External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data

The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.     

Gross deletions/duplications in <Emphasis Type="Italic">PROS1</Emphasis> are relatively common in point mutation-negative hereditary protein S deficiency

Hereditary protein S (PS) deficiency is an autosomal disorder caused by mutations in the PS gene (PROS1). Conventional PCR-based mutation detection identifies PROS1 point mutations in approximately 50% of the cases. To verify if gross copy number variations (CNVs) are often present in point mutation-negative hereditary PS deficiency we used multiplex ligation-dependent probe amplification (MLPA) as a detection tool in samples from individuals with a high probability of having true PS deficiency. To this end, DNA samples from nine PS deficient probands with family members (seven type I and two type III) and nine isolated probands (three type I and six type III), in whom PROS1 mutations were not found by DNA sequencing, were evaluated. An independent quantitative PCR (qPCR) was performed to confirm the findings of the MLPA assay. Family members were also tested when DNA was available. Gross abnormalities of PROS1 were found in six out of eighteen probands. In three probands complete deletion of the gene was detected. Two probands had a partial deletion involving different parts of the gene (one from exon 4 through 9 and another from exon 9 through 11). One family showed a duplication of part of PROS1. qPCR analysis was in accordance with these results. In conclusion, this study substantiates that gross gene abnormalities in PROS1 are relatively common in hereditary PS deficient patients and that MLPA is a useful tool for direct screening of CNVs in PROS1 point mutation-negative individuals.     

O-glycosylation of protein subpopulations in alcohol-extracted rice proteins

Mucin-type O-glycosylation has been well characterized in mammalian systems but not in plants. In this study, the purified alcohol-soluble, non-reduced protein (prolamin) fraction from rice seed was investigated for the occurrence of O-linked oligosaccharides. As storage prolamins are unlikely to be O-glycosylated, any O-glycosylation found was likely to belong to co-extracted proteins, whether because of association with the protein body or solubility. SDS-PAGE and MS analyses revealed 14 and 16kDa protein families in fractions that bound to the lectins peanut agglutinin (PNA), Vicia villosa lectin (VVL) and Jacalin, indicative of the presence of O-linked saccharides. Enzymatic cleavage, fluorescent labeling and high-performance liquid chromatography (HPLC) analysis demonstrated a peak consistent with Gal-beta-(1-->3)-GalNAc, with similar MS/MS fragmentation. Additionally, upon chemical analysis, a GlcNAc-containing O-linked carbohydrate moiety was discovered. Protein blotting with anti-O-GlcNAc antibody (clone CTD110.6) was positive in a subpopulation of the 14kDa alcohol-soluble protein fraction, but a hot capping experiment was negative. Therefore, the GlcNAc residue in this case is unlikely to be terminal. Additionally, a positive reaction with CTD110.6mAb cannot be taken as absolute proof of O-GlcNAc modification and further confirmatory experiments should be employed. We hypothesize that O-glycosylation may contribute to protein functionality or regulation. Further investigation is required to identify the specific proteins with these modifications. This 'reverse' approach could lead to the identification of proteins involved in mRNA targeting, signaling, translation, anchoring or maintenance of translational quiescence and may be applied to germinating rice seed extracts for further elucidation of protein function and regulation.     

Priority effects in experimental populations of the cyanobacterium Microcystis

The arrival order of colonists in developing populations can have a lasting influence on community and population structure, a phenomenon referred to as priority effects. To explore whether such priority effects are important in determining strain composition of populations of the cyanobacterium Microcystis , four Microcystis strains, isolated from a single lake and differing in functional traits, were grown during 4 weeks in the laboratory in all possible pairwise combinations, with the two strains either inoculated at the same time or with a time lag of 1 week, in the presence or absence of grazing Daphnia magna . The relative abundance of strains in the mixtures was assessed using denaturing gradient gel electrophoresis, and the growth rate of each strain in the mixtures was determined for the last 2 weeks of the experiment. We observed strong effects of inoculation order on the final population structure, and these effects were influenced by grazing Daphnia . The priority effects were strain-specific and occurred in two directions: some of the strains grew slower while others grew faster when inoculated second compared with when inoculated first. Our results indicate that priority effects may have a profound impact on strain composition of Microcystis populations.     

Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

Background  

Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols.     

The role of thin filament cooperativity in cardiac length-dependent calcium activation

Length-dependent activation (LDA) is a prominent feature of cardiac muscle characterized by decreases in the Ca²⁺ levels required to generate force (i.e., increases in Ca²⁺ sensitivity) when muscle is stretched. Previous studies have concluded that LDA originates from the increased ability of (strong) cross-bridges to attach when muscle is lengthened, which in turn enhances Ca²⁺ binding to the troponin C (TnC) subunit of the troponin complex. However, our results demonstrate that inhibition of strong cross-bridge attachment with blebbistatin had no effect on the length-dependent modulation of Ca²⁺ sensitivity (i.e., EC₅₀) or Ca²⁺ cooperativity, suggesting that LDA originates upstream of cross-bridge attachment. To test whether LDA arises from length dependence of thin-filament activation, we replaced native cTnC with a mutant cTnC (DM-TnC) that is incapable of binding Ca²⁺. Although progressive replacement of native cTnC with DM-TnC caused an expected monotonic decrease in the maximal force (F_max), DM-TnC incorporation induced much larger increases in EC₅₀ and decreases in Ca²⁺ cooperativity at short lengths than at long lengths. These findings support the conclusion that LDA arises primarily from the influence of length on the modulation of the Ca²⁺ cooperativity arising from interaction between adjacent troponin-tropomyosin complexes on the thin filament.     

Association between Type 2 Diabetes Loci and Measures of Fatness

Background

Type 2 diabetes (T2D) is a metabolic disorder characterized by disturbances of carbohydrate, fat and protein metabolism and insulin resistance. The majority of T2D patients are obese and obesity by itself may be a cause of insulin resistance. Our aim was to evaluate whether the recently identified T2D risk alleles are associated with human measures of fatness as characterized with Dual Energy X-ray Absorptiometry (DEXA).

Methodology/Principal Findings

Genotypes and phenotypes of approximately 3,000 participants from cross-sectional ERF study were analyzed. Nine single nucleotide polymorphisms (SNPs) in CDKN2AB, CDKAL1, FTO, HHEX, IGF2BP2, KCNJ11, PPARG, SLC30A8 and TCF7L2 were genotyped. We used linear regression to study association between individual SNPs and the combined allelic risk score with body mass index (BMI), fat mass index (FMI), fat percentage (FAT), waist circumference (WC) and waist to hip ratio (WHR). Significant association was observed between rs8050136 (FTO) and BMI (p = 0.003), FMI (p = 0.007) and WC (p = 0.03); fat percentage was borderline significant (p = 0.053). No other SNPs alone or combined in a risk score demonstrated significant association to the measures of fatness.

Conclusions/Significance

From the recently identified T2D risk variants only the risk variant of the FTO gene (rs8050136) showed statistically significant association with BMI, FMI, and WC.     

Plasma Concentrations of Oseltamivir and Oseltamivir Carboxylate in Critically Ill Children on Extracorporeal Membrane Oxygenation Support

Introduction

To evaluate the effect of extracorporeal membrane oxygenation (ECMO) support on pharmacokinetics of oseltamivir and oseltamivir carboxylate (OC) in children.

Methodology

Steady state 0–12 hour pharmacokinetic sampling was performed in new influenza A (H1N1) infected children treated with oseltamivir while on ECMO support. Cmax, Cmin and AUC_{0–12 h} were calculated. The age-specific oseltamivir dosage was doubled to counter expected decreased plasma drug concentrations due to increased volume of distribution on ECMO support.

Principal Findings

Three patients were enrolled aged 15, 6 and 14 years in this pharmacokinetic case series. For two children the OC plasma concentrations were higher than those found in children and adults not on ECMO. These increased plasma concentrations related to the increased oseltamivir dosage and decreased kidney function. In one patient suboptimal plasma concentrations coincided with a decreased gastric motility.

Conclusion

Oseltamivir pharmacokinetics do not appear to be significantly influenced by ECMO support. Caution is required in case of nasogastric administration and decreased gastric motility. Due to the limited number of (paediatric) patients available further multicenter studies are warranted.