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...

In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419-430, 1998). To define the role of ypfP in this strain of S. aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP. Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either L-or D-alanine. Moreover, viability of the mutant at 37°C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1% D-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.

The glycopeptide antimicrobials are a group of natural product and semisynthetic glycosylated peptides that show antibacterial activity against Gram-positive organisms through inhibition of cell-wall synthesis. This is achieved primarily through binding to the d-alanyl-d-alanine terminus of the lipid II bacterial cell-wall precursor, preventing cross-linking of the peptidoglycan layer. Vancomycin is the foundational member of the class, showing both clinical longevity and a still preferential role in the therapy of methicillin-resistant Staphylococcus aureus and of susceptible Enterococcus spp. Newer lipoglycopeptide derivatives (telavancin, dalbavancin, and oritavancin) were designed in a targeted fashion to increase antibacterial activity, in some cases through secondary mechanisms of action. Resistance to the glycopeptides emerged in delayed fashion and occurs via a spectrum of chromosome- and plasmid-associated elements that lead to structural alteration of the bacterial cell-wall precursor substrates.

In the cariogenic organism, Streptococcus mutans, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyllipoteichoic acid. Inactivation of the dltC gene, encoding the D-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in (14), as well as hydroxyapatite (9). These are important factors in the formation of dental plaque biofilms. Streptococcus mutans, an organism associated with dental caries, synthesizes considerable LTA (25), particularly under the low growth rates typical of the plaque biofilm (7).The biosynthesis of D-alanyl-LTA, studied in Lactobacillus rhamnosus (12,27,28,42) and Bacillus subtilis (46), requires the D-alanyl-carrier protein (Dcp) for incorporation of the activated D-alanine into membrane-associated LTA (mLTA). The activation and ligation of D-alanine to Dcp is catalyzed by D-alanine:Dcp ligase (D-alanine-Dcl) having a function similar to the acid thiol ligases (28). Unlike the first reaction, the transfer of activated D-alanine to mLTA is highly specific for D-alanine-Dcp. Recent genetic analysis (11, 42) has indicated that the proteins for D-alanine incorporation reside in the dlt operon comprised of four genes:dltA, encoding Dcl; dltC, encoding Dcp; dltB, encoding a putative transmembrane protein involved in the secretion of activated D-alanine; and dltD, a membrane-associated thioesterase for mischarged carrier protein.Two recent reports have indicated that oral streptococci possess the dlt operon comprised of dltA, dltB, dltC, and dltD having homology to the operons of L. rhamnosus and B. subtilis. In one study, Spatafora et al. (49) observed that the inactivation of the S. mutans UA130 dlt by transposon insertion into a site upstream of dltA resulted in the diminished synthesis of intracellular polysaccharide. Interestingly, the expression of the dlt operon was induced in the exponential phase when the cells were grown with sugars transported by the phosphoenolpyruvate-sugar phosphotransferase system (PTS) but was expressed constitutively when grown with the non-PTS sugars raffinose and melibiose. A mutant of S. mutans defective in PTS activity showed constitutive expression, suggesting a relationship between the dlt operon and sugar transport via th...

Antibiotic resistance is a growing public health crisis. To address the development of bacterial resistance, the use of antibiotics has to be minimized for nonsystemic applications in humans, as well as in animals and plants. Possible substitutes with low potential for developing resistance are active chlorine compounds that have been in clinical use for over 180 years. These agents are characterized by pronounced differences in their chlorinating and/or oxidizing activity, with hypochlorous acid (HOCl) as the strongest and organic chloramines as the weakest members. Bacterial killing in clinical practice is often associated with unwanted side effects such as chlorine consumption, tissue irritation, and pain, increasing proportionally with the chlorinating/oxidizing potency. Since the chloramines are able to effectively kill pathogens (bacteria, fungi, viruses, protozoa), their application as anti-infectives is advisable, all the more so as they exhibit additional beneficial properties such as destruction of toxins, degradation of biofilms, and anticoagulative and anti-inflammatory activities. Within the ample field of chloramines, the stable N-chloro derivatives of ß-aminosulfonic acids are most therapeutically advanced. Being available as sodium salts, they distinguish themselves by good solubility and absence of smell. Important representatives are N-chlorotaurine, a natural compound occurring in the human immune system, and novel mono-and dichloro derivatives of dimethyltaurine, which feature improved stability.