RNA-dependent sterol aspartylation in fungi.

Diverting aminoacyl-transfer RNAs (tRNAs) from protein synthesis is a well-known process used by a wide range of bacteria to aminoacylate membrane constituents. By tRNA-dependently adding amino acids to glycerolipids, bacteria change their cell surface properties, which intensifies antimicrobial drug resistance, pathogenicity, and virulence. No equivalent aminoacylated lipids have been uncovered in any eukaryotic species thus far, suggesting that tRNA-dependent lipid remodeling is a process restricted to prokaryotes. We report here the discovery of ergosteryl-3ß-O-l-aspartate (Erg-Asp), a conjugated sterol that is produced by the tRNA-dependent addition of aspartate to the 3ß-OH group of ergosterol, the major sterol found in fungal membranes. In fact, Erg-Asp exists in the majority of "higher" fungi, including species of biotechnological interest, and, more importantly, in human pathogens like Aspergillus fumigatus We show that a bifunctional enzyme, ergosteryl-3ß-O-l-aspartate synthase (ErdS), is responsible for Erg-Asp synthesis. ErdS corresponds to a unique fusion of an aspartyl-tRNA synthetase-that produces aspartyl-tRNAAsp (Asp-tRNAAsp)-and of a Domain of Unknown Function 2156, which actually transfers aspartate from Asp-tRNAAsp onto ergosterol. We also uncovered that removal of the Asp modifier from Erg-Asp is catalyzed by a second enzyme, ErdH, that is a genuine Erg-Asp hydrolase participating in the turnover of the conjugated sterol in vivo. Phylogenomics highlights that the entire Erg-Asp synthesis/degradation pathway is conserved across "higher" fungi. Given the central roles of sterols and conjugated sterols in fungi, we propose that this tRNA-dependent ergosterol modification and homeostasis system might have broader implications in membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity.

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.

tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum.

Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Alanyl-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, whereas formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells.

Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides.

Six serotypes (Ia, Ib, II, III, IV, and V) cause nearly all group B streptococcal (GBS) disease globally. Capsular polysaccharide (CPS) conjugate vaccines aim to prevent GBS disease, however, licensure of a vaccine would depend on a standardized serological assay for measuring anti-CPS IgG responses. A multiplex direct Luminex-based immunoassay (dLIA) has been developed to simultaneously measure the concentration of serum IgG specific for the six prevalent GBS CPS serotypes. Assay validation was performed using serum samples obtained from human subjects vaccinated with an investigational 6-valent GBS CPS conjugate vaccine. Results for the assay are expressed as IgG concentrations (µg/mL) using a human serum reference standard composed of pooled sera from vaccinated subjects. The lower limits of quantitation (LLOQ) for all serotypes covered in the 6-plex GBS IgG dLIA fell within the range of 0.002-0.022 µg/mL IgG. Taken together, the 6-plex GBS IgG dLIA platform is specific for the six GBS serotypes included in Pfizer's investigational vaccine, has a wide dilution adjusted assay range, and is precise (<18.5% relative standard deviation) for all serotypes, and, therefore, is suitable for quantitatively measuring vaccine-induced or naturally acquired serotype-specific anti-CPS IgG responses against GBS.

Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides.

Measurement of IgG antibodies against group B streptococcus (GBS) capsular polysaccharide (CPS) by use of a standardized and internationally accepted multiplex immunoassay is important for the evaluation of candidate maternal GBS vaccines in order to compare results across studies. A standardized assay is also required if serocorrelates of protection against invasive GBS disease are to be established in infant sera for the six predominant GBS serotypes since it would permit the comparison of results across the six serotypes. We undertook an interlaboratory study across five laboratories that used standardized assay reagents and protocols with a panel of 44 human sera to measure IgG antibodies against GBS CPS serotypes Ia, Ib, II, III, IV, and V. The within-laboratory intermediate precision, which included factors like the lot of coated beads, laboratory analyst, and day, was generally below 20% relative standard deviation (RSD) for all six serotypes, across all five laboratories. The cross-laboratory reproducibility was < 25% RSD for all six serotypes, which demonstrated the consistency of results across the different laboratories. Additionally, anti-CPS IgG concentrations for the 44-member human serum panel were established. The results of this study showed assay robustness and that the resultant anti-CPS IgG concentrations were reproducible across laboratories for the six GBS CPS serotypes when the standardized assay was used.

Calibration of a serum reference standard for Group B streptococcal polysaccharide conjugate vaccine development using surface plasmon resonance.

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality worldwide. Development of a maternal vaccine to protect newborns through placentally transferred antibody is considered feasible based on the well-established relationship between anti-GBS capsular polysaccharide (CPS) IgG levels at birth and reduced risk of neonatal invasive GBS. An accurately calibrated serum reference standard that can be used to measure anti-CPS concentrations is critical for estimation of protective antibody levels across serotypes and potential vaccine performance. For this, precise weight-based measurement of anti-CPS IgG in sera is required. Here, we report an improved approach for determining serum anti-CPS IgG levels using surface plasmon resonance with monoclonal antibody standards, coupled with a direct Luminex-based immunoassay. This technique was used to quantify serotype-specific anti-CPS IgG levels in a human serum reference pool derived from subjects immunized with an investigational six-valent GBS glycoconjugate vaccine.

Development of a continuous assay for high throughput screening to identify inhibitors of the purine salvage pathway in Plasmodium falciparum.

Malaria, an infectious disease caused by protozoan parasites from the genus Plasmodium, represents a serious global health threat. The continued emergence of drug resistant strains has severely decreased current antimalarial drug efficacy and led to a perpetual race for drug discovery. Most protozoan parasites, including Plasmodium spp., are unable to synthesize purines de novo and instead rely on an essential purine salvage pathway for acquisition of purines from the infected host. Because purines are essential for Plasmodium growth and survival, the enzymes of the purine salvage pathway represent promising targets for drug discovery. Target-based high-throughput screening (HTS) assays traditionally focus on a single target, which severely limits the screening power of this type of approach. To circumvent this limitation, we have reconstituted the purine salvage pathway from Plasmodium falciparum in an assay combining four drug targets. This assay was developed for HTS and optimized to detect partial inhibition of any of the four enzymes in the pathway. Inhibitors of several enzymes in the pathway were identified in a pilot screen, with several compounds exhibiting effective inhibition when provided in micromolar amounts.

A Quantitative Spectrophotometric Assay to Monitor the tRNA-Dependent Pathway for Lipid Aminoacylation In Vitro.

The transfer RNA (tRNA)-dependent pathway for lipid aminoacylation is a two-step pathway composed of (1) a tRNA aminoacylation step catalyzed by an aminoacyl-tRNA synthetase, forming a specific aa-tRNA, and (2) a tRNA-dependent transfer step in which the amino acid acylating the tRNA is transferred to an acceptor lipid. The latter step is catalyzed by a transferase located within the cytoplasmic membrane of certain bacteria. Lipid aminoacylation modifies the biochemical properties of the membrane and enhances resistance of some pathogens to various classes of antimicrobial agents and components of the innate immune response. Lipid aminoacylation has also been linked to increased virulence of various pathogenic bacteria. Inhibition of this mechanism would render pathogens more susceptible to existing drugs or to natural defenses of a host organism. Because lipid aminoacylation is widespread in many bacterial genera and absent from eukaryotes, and because the tRNA aminoacylation step of this pathway is also used in protein biosynthesis (a process essential for bacterial life), this pathway represents an attractive target for drug design. We have reconstituted the lipid aminoacylation pathway in vitro and optimized it for high-throughput screening of libraries of compounds to simultaneously identify inhibitors targeting each step of the pathway in a single assay.