The emergence and spread of multidrug-resistant gram-positive bacteria represent a serious clinical problem. Telavancin is a novel lipoglycopeptide antibiotic that possesses rapid in vitro bactericidal activity against a broad spectrum of clinically relevant gram-positive pathogens. Here we demonstrate that telavancin's antibacterial activity derives from at least two mechanisms. As observed with vancomycin, telavancin inhibited latestage peptidoglycan biosynthesis in a substrate-dependent fashion and bound the cell wall, as it did the lipid II surrogate tripeptide N,N-diacetyl-L-lysinyl-D-alanyl-D-alanine, with high affinity. Telavancin also perturbed bacterial cell membrane potential and permeability. In methicillin-resistant Staphylococcus aureus, telavancin caused rapid, concentration-dependent depolarization of the plasma membrane, increases in permeability, and leakage of cellular ATP and K ؉ . The timing of these changes correlated with rapid, concentration-dependent loss of bacterial viability, suggesting that the early bactericidal activity of telavancin results from dissipation of cell membrane potential and an increase in membrane permeability. Binding and cell fractionation studies provided direct evidence for an interaction of telavancin with the bacterial cell membrane; stronger binding interactions were observed with the bacterial cell wall and cell membrane relative to vancomycin. We suggest that this multifunctional mechanism of action confers advantageous antibacterial properties.The emergence and spread of bacterial resistance to vancomycin, an important antibiotic used to treat serious infections caused by gram-positive bacteria, has prompted active research to discover new glycopeptides and semisynthetic analogs with improved antimicrobial properties. Vancomycin and related glycopeptide antibiotics inhibit cell wall synthesis in susceptible bacteria by binding with high specificity to peptidoglycan precursors containing the C-terminal D-alanyl-D-alanine (D-Ala-DAla) motif (8). The peptide portion of glycopeptide antibiotics forms a carboxylate binding pocket that imparts, through a combination of five hydrogen bonds plus favorable hydrophobic interactions, strong affinity for the D-Ala-D-Ala-containing terminus of lipid II (8,46,54). Rational approaches toward the design of glycopeptides with improved antimicrobial activities have been described previously (for reviews, see references 35 and 36). One promising approach has been the discovery of lipoglycopeptides, analogs containing hydrophobic groups substituted at the amine position of the disaccharide moiety (20,39,40,45).Telavancin, a semisynthetic derivative of vancomycin possessing a hydrophobic (decylaminoethyl) side chain appended to the vancosamine sugar and a hydrophilic [(phosphonomethyl)aminomethyl] group on the resorcinol-like 4Ј position of amino acid 7 (33), is in late-stage clinical development for the treatment of serious gram-positive infections. Telavancin and other lipoglycopeptides exhibit superior in vitro activity compa...

The increasing prevalence of serious infections caused by gram-positive bacteria, including those caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), highlights the need for new agents with enhanced antimicrobial properties (2,10,21,26,35). One promising approach has been the development of lipoglycopeptide antibiotics, semisynthetic derivatives of glycopeptides that contain hydrophobic substituents and that possess improved antimicrobial properties (1,4,13,32,39). Telavancin, a lipoglycopeptide derivative of vancomycin, exhibits enhanced potency in vitro, concentration-dependent bactericidal activity, and activity both in vitro and in vivo against organisms that display reduced susceptibility to vancomycin (17,18,23,24,28,31,33,36,42). Telavancin has been evaluated in phase III clinical trials for the treatment of complicated skin and skin structure infections and hospitalacquired pneumonia (46,53).The bactericidal action of telavancin results from a mechanism that includes the inhibition of cell wall synthesis and the disruption of essential membrane barrier functions (25). Telavancin possesses the glycopeptide core of vancomycin, which binds with a high affinity to the acyl-D-alanyl-D-alanine (DAla-D-Ala) terminus of cell wall precursors through a network of hydrogen bonds and hydrophobic packing interactions (3, 45). Inhibition of cell wall synthesis by telavancin therefore involves binding to late-stage peptidoglycan precursors, including membrane-embedded lipid II. These interactions prevent both the polymerization of the precursor into peptidoglycan and subsequent cross-linking events. Telavancin also binds to bacterial membranes and causes membrane depolarization and increased membrane permeability. The mechanism by which telavancin binds to and disrupts the function of the bacterial membrane has not been determined.The present study was undertaken to further explore the interaction of telavancin with the bacterial membrane. Using a flow cytometry assay optimized for the accurate measurement of membrane potential in bacteria, we demonstrate that telavancin causes pronounced, concentration-dependent depolarization in S. aureus cells. Isolates of S. aureus expressing important and emerging resistance phenotypes, such as MRSA, heterogeneous vancomycin-intermediate S. aureus (hVISA), vancomycin-intermediate S. aureus (VISA), and daptomycinnonsusceptibile MRSA, are equally susceptible to depolarization by telavancin. We provide evidence, through multiple lines of investigation, that membrane disruption by telavancin requires binding to the bacterial specific target, lipid II. Finally, we demonstrate that telavancin does not lyse bacteria during the time course that membrane effects are assayed. Importantly, the latter observation indicates that telavancin-induced membrane depolarization is not a consequence of a weakened * Corresponding author. Mailing address:

Abbreviations used in this paper:CsA, cyclosporin A; FKBP, FKS06-binding protein; Gal, 2% galactose supplemented with 0.2% sucrose; Glc, 2 % glucose; GST, Schistosomajaponicura glutathione S-transferase; HSE, heat shock response element; Hsp, heat shock protein; PPIases, peptidylprolyl-cis, trans-isomerases. in protein folding and protein assembly events in vivo (Parsell and Lindquist, 1993) because in vitro these enzymes catalyze interconversion of the cis and trans isomers of the bond between the carboxyl group of the preceding amino acid and the imino nitrogen of proline in peptide and protein substrates (Kofron et al., 1991;Stein, 1991). It was discovered several years ago that there are two separate types of PPIases. This realization was made possible by investigations into the molecular mode of action of antibiotics that act as immunosuppressants in humans. These drugs effectively block the specific signal transduction pathways required for activation and proliferation of T lymphocytes (for review see Schreiber et al., 1993).The first PPIase isolated (Fischer et al., 1984) was shown to be the intracellular receptor for an immunosuppressant, cyclosporin A (CsA); and hence, a member of this class of PPIase is called a cyclophilin (Fischer et al., 1989; Taka-

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

Telavancin is an investigational, rapidly bactericidal lipoglycopeptide antibiotic that is being developed to treat serious infections caused by gram-positive bacteria. A baseline prospective surveillance study was conducted to assess telavancin activity, in comparison with other agents, against contemporary clinical isolates collected from 2004 to 2005 from across the United States. Nearly 4,000 isolates were collected, including staphylococci, enterococci, and streptococci (pneumococci, beta-hemolytic, and viridans). Telavancin had potent activity against Staphylococcus aureus and coagulase-negative staphylococci (MIC range, 0.03 to 1.0 g/ml), independent of resistance to methicillin or to multiple agents. Telavancin activity was particularly potent against all streptococcal groups (MIC 90 s, 0.03 to 0.12 g/ml). Telavancin had excellent activity against vancomycin-susceptible enterococci (MIC 90 , 1 g/ml) and was active against VanB strains of vancomycinresistant enterococci (MIC 90 , 2 g/ml) but less active against VanA strains (MIC 90 , 8 to 16 g/ml). Telavancin also demonstrated activity against vancomycin-intermediate S. aureus and vancomycin-resistant S. aureus strains (MICs, 0.5 g/ml to 1.0 g/ml and 1.0 g/ml to 4.0 g/ml, respectively). These data may support the efficacy of telavancin for treatment of serious infections with a wide range of gram-positive organisms.Antibiotic resistance in gram-positive bacteria is a continuing health care problem, both in hospitals and in the community. Telavancin is a novel, once-daily, intravenously administered lipoglycopeptide that is being developed to treat serious infections caused by gram-positive bacteria. It has shown promising results in patients with complicated skin and skin structure infections (SSSIs) (i.e., cellulitis, major abscess, infected wound/ulcer, or burn complicated by a requirement for surgical intervention and/or involvement of deeper tissues), including those infected with methicillin-resistant Staphylococcus aureus (MRSA) (19,20,21). A U.S. Food and Drug Administration New Drug Application has been filed for telavancin based on two completed phase 3 clinical trials for the treatment of complicated SSSIs (20), and two phase 3 trials for the treatment of hospital-acquired pneumonia have finished patient enrollment.Like vancomycin and teicoplanin, telavancin inhibits the polymerization of cell wall peptidoglycan precursors by binding to their D-alanyl-D-alanine termini, but telavancin has greater activity than vancomycin in this interaction (50% inhibitory concentration, 0.14 M versus 2.0 M) (8). Additionally, the interaction of telavancin with peptidoglycan precursors facilitates the perturbation of bacterial plasma membrane function, which leads to concentration-dependent membrane depolarization and increases in membrane permeability (8). The second mode of action is likely responsible for the more rapid and extensive bactericidal activity of telavancin than vancomycin and teicoplanin.Telavancin exhibits potent in vitro antibacterial activity ...