Here we show that a new class of antibiotics-acyldepsipeptides-has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA Phe ), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by highthroughput 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 g/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 tRNA Phe 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 phenylthiazolylurea-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.

Oxazolidinones are antibacterial agents that act primarily against gram-positive bacteria by inhibiting protein synthesis. The binding of oxazolidinones to 70S ribosomes from Escherichia coli was studied by both UV-induced cross-linking using an azido derivative of oxazolidinone and chemical footprinting using dimethyl sulphate. Oxazolidinone binding sites were found on both 30S and 50S subunits, rRNA being the only target. On 16S rRNA, an oxazolidinone footprint was found at A864 in the central domain. 23S rRNA residues involved in oxazolidinone binding were U2113, A2114, U2118, A2119, and C2153, all in domain V. This region is close to the binding site of protein L1 and of the 39 end of tRNA in the E site. The mechanism of action of oxazolidinones in vitro was examined in a purified translation system from E. coli using natural mRNA. The rate of elongation reaction of translation was decreased, most probably because of an inhibition of tRNA translocation, and the length of nascent peptide chains was strongly reduced. Both binding sites and mode of action of oxazolidinones are unique among the antibiotics known to act on the ribosome.

Changes in cell viability and in factors affecting metabolic integrity were examined after exposure of Escherichia coli LP1092 to human serum. Antibodydependent classical pathway activity accounted for the rapid killing of strain LP1092 by complement. Removal of serum lysozyme by bentonite absorption or by neutralization with anti-human lysozyme immunoglobulin G resulted in a reduction in the rate of killing; optimal activity could be restored by the addition of physiological amounts of egg-white lysozyme. The pattern of 86Rb+ and alkaline phosphatase release obtained after serum treatment did not support the view that complement simultaneously disrupts cytoplasmic and outer membrane integrity. Macromolecular synthesis was affected late in the reaction sequence; complete inhibition of precursor incorporation into RNA, DNA, and protein occurred only after almost total loss of bacterial colony-forming ability. Addition of chloramphenicol, an inhibitor of protein synthesis, to the bactericidal system resulted in a marked reduction in the rate of serum killing. Killing was completely inhibited by an inhibitor (KCN) and an uncoupler (2,4-dinitrophenol) of oxidative phosphorylation. Exposure of LP1092 cells to serum was followed by a rapid and large increase in intracellular ATP levels; ATP synthesis did not occur when bacteria were exposed to dialyzed serum, which killed LP1092 cells at a much reduced rate. Addition of glucose or serum ultrafiltrate to dialyzed serum restored optimal bactericidal activity. We suggest that optimal killing of gram-negative bacteria is an energy-dependent process requiring an input of bacterially generated ATP.

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Serum resistance is a major virulence factor of gram-negative bacteria, and K-1 polysaccharide has been shown to contribute to serum resistance in selected strains. To obtain further information about the role of K-1 in serum resistance and to find out whether loss of the ability to produce K-1 can induce loss of serum resistance, we studied the serum resistance of mutants derived from completely serum-resistant, K-i-positive blood culture isolates ofEscherichia coli by selection for resistance to infection with K-1 specific bacteriophages. The amounts of K-1 polysaccharide produced by wild-type strains and mutants were measured, and outer membrane protein and lipopolysaccharide (LPS) patterns were analyzed. In each group of mutants, several highly serum-sensitive strains were found. All mutant strains expressed less K-1 than did the corresponding wild-type strains. Mutants that became highly serum sensitive always had less K-1 than did mutants with less-pronounced changes of serum resistance. A few mutants derived from different wild-type strains showed increased expression of outer membrane proteins with molecular weights of about 46,000 and 67,000. All of the wild-type strains examined had smooth-type LPS, and only two mutants had altered LPS structures; alterations of mutants in outer membrane proteins and LPS could not be correlated with alterations of serum resistance. The results indicate that for K-i-positive blood culture strains of E. coli, K-1 expression is a prerequisite for serum resistance, and loss of ability to synthesize K-1 leads to loss of serum resistance.

were studied. The phospholipid/amino acid ratio was reduced in almost all OM preparations from antibiotic-treated bacteria. In one strain, antibiotic treatment increased the lipopolysaccharide/amino acid ratio. There are many hints, which have been reported by many different authors (1,7,21,24,26,29,30), indicating that such influences on the cell surface are closely related to the influence of antibiotics on various host-parasite interaction processes, such as adherence, phagocytosis, serum resistance, or immune response to OM components. There are, however, not enough data about the influence of antibiotics on the qualitative and, especially, the quantitative composition of the cell envelope to establish firmly the connection between changes of the OM and biological function.In a series of experiments which demonstrated the influence of antibiotics on the immunogenicity of the OM, we could not detect changes in qualitative OM properties, such as OM protein pattern, LPS chain length, and phospholipid (PL) composition (21). Therefore, we performed quantitative analyses of the cell envelope of E. coli. We prepared OM vesicles from bacteria that had been grown in the presence or absence of subinhibitory concentrations of cephaloridine, imipenem, moxalactam, or ciprofloxacin. We determined the amounts of LPS and PL in the OM, performed an amino acid (AA) analysis of the preparations, and measured the amount of peptidoglycan bound to the OM preparations. Because it was our aim to relate these data as closely as possible to our earlier findings on the influence of antibiotics on OM im-*

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