Todd A. Black

Lesions in Teichoic Acid Biosynthesis in Staphylococcus aureus Lead to a Lethal Gain of Function in the Otherwise Dispensable Pathway

An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus.

Genetic strategies for antibacterial drug discovery.

The availability of genome sequences is revolutionizing the field of microbiology. Genetic methods are being modified to facilitate rapid analysis at a genome-wide level and are blossoming for human pathogens that were previously considered intractable. This revolution coincided with a growing concern about the emergence of microbial drug resistance, compelling the pharmaceutical industry to search for new antimicrobial agents. The availability of the new technologies, combined with many genetic strategies, has changed the way that researchers approach antibacterial drug discovery.

Impact of Antifungal Prophylaxis on Colonization and Azole Susceptibility of Candida Species

ABSTRACT Two large studies compared posaconazole and fluconazole or itraconazole for prophylaxis in subjects undergoing allogeneic hematopoietic stem cell transplantation or subjects with acute myelogenous leukemia. To assess the impact of prophylaxis on colonization and the development of resistance in Saccharomyces yeasts, identification and susceptibility testing were performed with yeasts cultured at regular intervals from mouth, throat, and stool samples. Prior to therapy, 34 to 50% of the subjects were colonized with yeasts. For all three drugs, the number of positive Candida albicans cultures decreased during drug therapy. In contrast, the proportion of subjects with positive C. glabrata cultures increased by two- and fourfold in the posaconazole and itraconazole arms, respectively. Likewise, in the fluconazole arm the proportion of subjects with positive C. krusei cultures increased twofold. C. glabrata was the species that most frequently exhibited decreases in susceptibility, and this trend did not differ significantly between the prophylactic regimens. For the subset of subjects from whom colonizing C. glabrata isolates were recovered at the baseline and the end of treatment, approximately 40% of the isolates exhibited more than fourfold increases in MICs during therapy. Molecular typing of the C. albicans and C. glabrata isolates confirmed that the majority of the baseline and end-of-treatment isolates were closely related, suggesting that they were persistent colonizers and not newly acquired. Overall breakthrough infections by Candida species were very rare (∼1%), and C. glabrata was the colonizing species that was the most frequently associated with breakthrough infections.

Evernimicin Binds Exclusively to the 50S Ribosomal Subunit and Inhibits Translation in Cell-Free Systems Derived from both Gram-Positive and Gram-Negative Bacteria

ABSTRACT Evernimicin (SCH 27899) is a new antibiotic with activity against a wide spectrum of gram-positive bacteria and activity against some gram-negative bacteria. Previous metabolic labeling studies indicated that evernimicin specifically inhibited protein synthesis inStaphylococcus aureus. Using a susceptibleEscherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli. In cell-free translation assays with extracts from either E. coli orS. aureus, evernimicin had a 50% inhibitory concentration of approximately 125 nM. In contrast, cell-free systems derived from wheat germ and rabbit reticulocytes were inhibited only by very high levels of evernimicin. Evernimicin did not promote transcript misreading. [14C]evernimicin specifically bound to the 50S subunit from E. coli. Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli andS. aureus and 50S subunits from E. colireturned dissociation constants of 84, 86, and 160 nM, respectively. In binding experiments, performed in the presence of excess quantities of a selection of antibiotics known to bind to the 50S subunit, only the structurally similar drug avilamycin blocked binding of [14C]evernimicin to ribosomes.

Temporal Appearance of Plasmid-Mediated Quinolone Resistance Genes

ABSTRACT One hundred fifty AAC(6′)-Ib-positive gram-negative isolates collected between 1981 and 1991 were examined by PCR for the presence of the aac(6′)-Ib-cr variant and other plasmid-mediated quinolone resistance (PMQR) genes. None had the aac(6′)-Ib-cr variant, qnrA, qnrS, qnrC, or qepA, but two strains collected in 1988 had qnrB alleles, making these the earliest known PMQR genes.

Microbial genetics: Genetic strategies for antibacterial drug discovery

The availability of genome sequences is revolutionizing the field of microbiology. Genetic methods are being modified to facilitate rapid analysis at a genome-wide level and are blossoming for human pathogens that were previously considered intractable. This revolution coincided with a growing concern about the emergence of microbial drug resistance, compelling the pharmaceutical industry to search for new antimicrobial agents. The availability of the new technologies, combined with many genetic strategies, has changed the way that researchers approach antibacterial drug discovery.

EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high‐level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell‐free translation assays and direct‐binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid‐borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin‐sensitive strain 30‐fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin‐binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

Evernimicin (SCH27899) Inhibits a Novel Ribosome Target Site: Analysis of 23S Ribosomal DNA Mutants

ABSTRACT Spontaneous mutants of susceptible clinical and laboratory isolates of Streptococcus pneumoniae exhibiting reduced susceptibility to evernimicin (SCH27899; MIC, 0.5 to 4.0 mg/liter) were selected on plates containing evernimicin. Four isolates that did not harbor mutations in rplP (which encodes ribosomal protein L16) were further analyzed. Whole chromosomal DNA or PCR products of the 23S ribosomal DNA (rDNA) operons from these mutants could be used to transform the susceptible S. pneumoniae strain R6 to resistance at frequencies of 10−5 and 10−4, respectively, rates 10- to 100-fold lower than that for a single-allele chromosomal marker. The transformants appeared slowly (48 to 72 h) on selective medium, and primary transformants passaged on nonselective medium produced single colonies that displayed heterogeneous susceptibilities to evernimicin. A single passage on selective medium of colonies derived from a single primary transformant homogenized the resistance phenotype. Sequence analysis of the 23S rDNA and rRNA from the resistant mutants revealed single, unique mutations in each isolate at the equivalent Escherichia coli positions 2469 (A → C), 2480 (C → T), 2535 (G → A), and 2536 (G → C). The mutations map within two different stems of the peptidyltransferase region of domain V. Because multiple copies of rDNA are present in the chromosome, gene conversion between mutant and wild-type 23S rDNA alleles may be necessary for stable resistance. Additionally, none of the characterized mutants showed cross-resistance to any of a spectrum of protein synthesis inhibitors, suggesting that the target site of evernimicin may be unique.

Mutations in Ribosomal Protein L16 Conferring Reduced Susceptibility to Evernimicin (SCH27899): Implications for Mechanism of Action

ABSTRACT A clinical isolate of Streptococcus pneumoniae (SP#5) that showed decreased susceptibility to evernimicin (MIC, 1.5 μg/ml) was investigated. A 4,255-bp EcoRI fragment cloned from SP#5 was identified by its ability to transform evernimicin-susceptibleS. pneumoniae R6 (MIC, 0.03 μg/ml) such that the evernimicin MIC was 1.5 μg/ml. Nucleotide sequence analysis of this fragment revealed that it contained portions of the S10-spc ribosomal protein operons. The nucleotide sequences of resistant and susceptible isolates were compared, and a point mutation (thymine to guanine) that causes an Ile52-Ser substitution in ribosomal protein L16 was identified. The role of this mutation in decreasing susceptibility to evernimicin was confirmed by direct transformation of the altered L16 gene. The presence of the L16 mutation in the resistant strain suggests that evernimicin is an inhibitor of protein synthesis. This was confirmed by inhibition studies using radiolabeled substrates, which showed that the addition of evernimicin at sub-MIC levels resulted in a rapid decrease in the incorporation of radiolabeled isoleucine in a susceptible isolate (SP#3) but was much less effective against SP#5. The incorporation of isoleucine showed a linear response to the dose level of evernimicin. The incorporation of other classes of labeled substrates was unaffected or much delayed, indicating that these were secondary effects.

Screening for Antibacterial Inhibitors of the UDP-3-O-(R-3-Hydroxymyristoyl)-N-Acetylglucosamine Deacetylase (LpxC) Using a High-Throughput Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)- Nacetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire™ mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 µg/mL). The results show that mass spectrometry—based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.