Hein-Peter Kroll

New Class of Bacterial Phenylalanyl-tRNA Synthetase Inhibitors with High Potency and Broad-Spectrum Activity

ABSTRACT Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNAPhe), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 μg/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNAPhe charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA+ parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy.

Interaction of human complement proteins with serum-sensitive and serum-resistant strains of Escherichia coli

Exposure of serum-susceptible Escherichia coli strains to lethal concns of lysozyme-free human serum resulted in stable binding of complement components to the outer membrane (OM), but not to the cytoplasmic membrane (CM). The short prekilling phase of the reaction was accompanied by binding of C3b; loss of viability was immediately preceeded by stable deposition onto the OM of the component proteins of the membrane attack complex. During the early stages of the active killing phase, bound monomeric C9 could be resolved into two distinct bands on SDS-polyacrylamide gels. Serum exposure lead to a progressive loss of CM recoverability, which appeared to result from partial degradation of CM phospholipids. In contrast, exposure of a resistant E, coli strain to human serum resulted in little change in the membrane profile and very little stable deposition of terminal complement components onto the OM.

Straightforward Syntheses of Furanomycin Derivatives and their Biological Evaluation

Several types of furanomycin analogues were synthesized and investigated with respect to their antibacterial activity. Two different synthetic pathways were developed, based on aldol reactions/ring closing metathesis and an ester enolate Claisen rearrangement. Only the natural product and its desmethyl derivative showed antibacterial activity, pointing towards a narrow structure-activity relationship.

Serological response to type 1-like somatic fimbriae in diarrheal infection due to classical enteropathogenic Escherichia coli

Fimbriae from enteropathogenic Escherichia coli strain E2349/69 (0127:H6) and its plasmid-minus derivative, MAR20, were purified and characterized as type 1-like by their physicochemical and hemagglutination patterns. Sera from adult volunteers challenged with the diarrheagenic parent strain and the attenuated plasmid-minus derivative were examined to detect an immune response, using the purified fimbriae as antigens in an enzyme linked immunosorbent assay (ELISA) and immunoblot assay. An anti-fimbrial response was evident in sera of 7 of 10 volunteers fed the diarrheagenic parent strain E2348 but also in 8 of 9 individuals fed the attenuated, plasmid-cured, derivative MAR20. The antibody response appeared specific in that the sera failed to react in an ELISA and by immunoblot assay with type 1 fimbriae from other E. coli. These findings suggest that the type 1 fimbriae of this representative EPEC strain are antigenically distinct. The results of this investigation provide the first evidence of seroconversion to type 1-like fimbriae in infections caused by diarrheagenic E. coli.

N‐acetylglucosaminyl N‐acetylmuramyl‐dipeptide, a novel murein building block formed during the cell division cycle of Proteus mirabilis

The murein (peptidoglycan) of Gram-negative bacteria consists of glycan chains containing alternatingN-acetylated residues of glucosamine and muramic acid. The muramic acid residues are substituted by tetrapeptide units consisting of Lalanyl-y-D-glutamyl(L)-meso-diaminopimelyl-(L)-D-alanine, part of which form crosslinkages between neighbouring glycan chains [ 11. Proteus mirubilis murein is unique in regard to the O-acetylation of a portion of its N-acetylmuramic acid residues [ 2,3 J . Working with synchronized cells of this organism and labelling the murein with radioactive N-acetyl-D[ 1 -14C]glucosamine in vivo, we detected 2 new uncrosslinked building blocks after enzymatic digestion of the isolated murein with endo-N,O-diacetylmuramidase from C’halaropsis. Here, we present analytical data which identify these new compounds as 0-acetylated and non-O-acetylated N-acetylglucosaminyl-N-acetylmuramyl-dipeptides.

Stable insertion of C5b-9 complement complexes into the outer membrane of serum treated, susceptible Escherichia coli cells as a prerequisite for killing.

Escherichia coli 17, a K12 derivative, was rapidly killed by human serum following a short lag period of 10 min. Stable binding of terminal C5b-9 complement complexes was investigated in time course experiments. Serum treated E. coli cells were lysed osmotically and the resulting outer and cytoplasmic membrane vesicles separated by sucrose gradient centrifugation. Exposure of E. coli 17 to serum rapidly reduced the degree of recoverability of cytoplasmic membrane vesicles. Electron microscopy revealed no interaction of C5b-9 complexes with CM vesicles. In contrast there was a clear time-dependent deposition of terminal complement complexes onto OM-vesicles. Very few complexes were detected during the prekilling phase of the reaction; initiation of the active killing phase was accompanied by a large increase in complement lesions. In contrast, no C5b-9 complexes could be visualised on outer or cytoplasmic membrane vesicles of a smooth, serum-resistant E. coli strain. We conclude that complement-mediated killing is a consequence of stable binding of C5b-9 complexes to the outer membrane of susceptible strains.