Donald W. Hughes

A vancomycin photoprobe identifies the histidine kinase VanSsc as a vancomycin receptor

Inducible resistance to the glycopeptide antibiotic vancomycin requires expression of vanH, vanA and vanX, controlled by a two-component regulatory system consisting of a receptor histidine kinase, VanS, and a response regulator, VanR. The identity of the VanS receptor ligand has been debated. Using a synthesized vancomycin photoaffinity probe, we show that vancomycin directly binds Streptomyces coelicolor VanS (VanSsc) and this binding is correlated with resistance and required for vanH, vanA and vanX gene expression.

Rifamycin antibiotic resistance by ADP-ribosylation: Structure and diversity of Arr

The rifamycin antibiotic rifampin is important for the treatment of tuberculosis and infections caused by multidrug-resistant Staphylococcus aureus. Recent iterations of the rifampin core structure have resulted in new drugs and drug candidates for the treatment of a much broader range of infectious diseases. This expanded use of rifamycin antibiotics has the potential to select for increased resistance. One poorly characterized mechanism of resistance is through Arr enzymes that catalyze ADP-ribosylation of rifamycins. We find that genes encoding predicted Arr enzymes are widely distributed in the genomes of pathogenic and nonpathogenic bacteria. Biochemical analysis of three representative Arr enzymes from environmental and pathogenic bacterial sources shows that these have equally efficient drug resistance capacity in vitro and in vivo. The 3D structure of one of these orthologues from Mycobacterium smegmatis was determined and reveals structural homology with ADP-ribosyltransferases important in eukaryotic biology, including poly(ADP-ribose) polymerases (PARPs) and bacterial toxins, despite no significant amino acid sequence homology with these proteins. This work highlights the extent of the rifamycin resistome in microbial genera with the potential to negatively impact the expanded use of this class of antibiotic.

Studies of phospholipid hydration by high-resolution magic-angle spinning nuclear magnetic resonance.

A sample preparation method using spherical glass ampoules has been used to achieve 1.5-Hz resolution in 1H magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of aqueous multilamellar dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), serving to differentiate between slowly exchanging interlamellar and bulk water and to reveal new molecular-level information about hydration phenomena in these model biological membranes. The average numbers of interlamellar water molecules in multilamellar vesicles (MLVs) of DOPC and POPC were found to be 37.5 +/- 1 and 37.2 +/- 1, respectively, at a spinning speed of 3 kHz. Even at speeds as high as 9 kHz, the number of interlamellar waters remained as high as 31, arguing against dehydration effects for DOPC and POPC. Both homonuclear and heteronuclear nuclear Overhauser enhancement spectroscopy (NOESY and HOESY) were used to establish the location of water near the headgroup of a PC bilayer. 1H NMR comparisons of DOPC with a lipid that can hydrogen bond (monomethyldioleoylphosphatidylethanolamine, MeDOPE) showed the following trends: 1) the interlamellar water resonance was shifted to lower frequency for DOPC but to higher frequency for MeDOPE, 2) the chemical shift variation with temperature for interlamellar water was less than that of bulk water for MeDOPE MLVs, 3) water exchange between the two lipids was rapid on the NMR time scale if they were mixed in the same bilayer, 4) water exchange was slow if they were present in separate MLVs, and 5) exchange between bulk and interlamellar water was found by two-dimensional exchange experiments to be slow, and the exchange rate should be less than 157 Hz. These results illustrate the utility of ultra-high-resolution 1H MAS NMR for determining the nature and extent of lipid hydration as well as the arrangement of nuclei at the membrane/water interface.

Structure and Mechanism of the Lincosamide Antibiotic adenylyltransferase LinB

Lincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate- and binary product-bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve.

Induction of antimicrobial activities in heterologous streptomycetes using alleles of the Streptomyces coelicolor gene absA1

The bacterial genus Streptomyces is endowed with a remarkable secondary metabolism that generates an enormous number of bioactive small molecules. Many of these genetically encoded small molecules are used as antibiotics, anticancer agents and as other clinically relevant therapeutics. The rise of resistant pathogens has led to calls for renewed efforts to identify antimicrobial activities, including expanded screening of streptomycetes. Indeed, it is known that most strains encode >20 secondary metabolites and that many, perhaps most of these, have not been considered for their possible therapeutic use. One roadblock is that many strains do not express their secondary metabolic gene clusters efficiently under laboratory conditions. As one approach to this problem, we have used alleles of a pleiotropic regulator of secondary metabolism from Streptomyces coelicolor to activate secondary biosynthetic gene clusters in heterologous streptomycetes. In one case, we demonstrate the activation of pulvomycin production in S. flavopersicus, a metabolite not previously attributed to this species. We find that the absA1-engineered strains produced sufficient material for purification and characterization. As a result, we identified new, broad-spectrum antimicrobial activities for pulvomycin, including a potent antimicrobial activity against highly antibiotic-resistant Gram-negative and Gram-positive pathogens.

ApoA-I mimetic peptides with differing ability to inhibit atherosclerosis also exhibit differences in their interactions with membrane bilayers.

Two homologous apoA-I mimetic peptides, 3F-2 and 3F14, differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404-51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 μg/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 ± 1000 μm2, n = 29), whereas 3F14 does not (lesion area 20,400 ± 1000 μm2, n = 26) compared with control saline administered (19,900 ± 1400 μm2, n = 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F14 preferentially associates with apoB-containing particles. After intraperitoneal injection of 14C-labeled peptides, 3F14 reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F14. In contrast, only 3F14 affects the terminal methyl group of the acyl chain, decreasing the 2H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F14 can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.

15N NMR Study of the Ionization Properties of the Influenza Virus Fusion Peptide in Zwitterionic Phospholipid Dispersions

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.

The COOH Terminus of Aminoglycoside Phosphotransferase (3′)-IIIa Is Critical for Antibiotic Recognition and Resistance

The aminoglycoside phosphotransferases (APHs) are widely distributed among pathogenic bacteria and are employed to covalently modify, and thereby detoxify, the clinically relevant aminoglycoside antibiotics. The crystal structure for one of these aminoglycoside kinases, APH(3′)-IIIa, has been determined in complex with ADP and analysis of the electrostatic surface potential indicates that there is a large anionic depression present adjacent to the terminal phosphate group of the nucleotide. This region also includes a conserved COOH-terminal α-helix that contains the COOH-terminal residue Phe264. We report here mutagenesis and computer modeling studies aimed at examining the mode of aminoglycoside binding to APH(3′)-IIIa. Specifically, seven site mutants were studied, five from the COOH-terminal helix (Asp261, Glu262, and Phe264), and two additional residues that line the wall of the anionic depression (Tyr55 and Arg211). Using a molecular modeling approach, six ternary complexes of APH(3′)-IIIa·ATP with the antibiotics, kanamycin, amikacin, butirosin, and ribostamycin were independently constructed and these agree well with the mutagenesis data. The results obtained show that the COOH-terminal carboxylate of Phe264 is critical for proper function of the enzyme. Furthermore, these studies demonstrate that there exists multiple binding modes for the aminoglycosides, which provides a molecular basis for the broad substrate- and regiospecificity observed for this enzyme.

INACTIVATION OF THE LIPOPEPTIDE ANTIBIOTIC DAPTOMYCIN BY HYDROLYTIC MECHANISMS

ABSTRACT The lipopeptide daptomycin is a member of the newest FDA-approved antimicrobial class, exhibiting potency against a broad range of Gram-positive pathogens with only rare incidences of clinical resistance. Environmental bacteria harbor an abundance of resistance determinants orthologous to those in pathogens and thus may serve as an early-warning system for future clinical emergence. A collection of morphologically diverse environmental actinomycetes demonstrating unprecedented frequencies of daptomycin resistance and high levels of resistance by antibiotic inactivation was characterized to elucidate modes of drug inactivation. In vivo studies revealed that hydrolysis plays a key role, resulting in one or both of the following structural modifications: ring hydrolysis resulting in linearization (in 44% of inactivating isolates) or deacylation of the lipid tail (29%). Characterization of the mechanism in actinomycete WAC4713 (a Streptomyces sp. with an MIC of 512 μg/ml) demonstrated a constitutive resistance phenotype and established daptomycins circularizing ester linkage to be the site of hydrolysis. Characterization of the hydrolase responsible revealed it to be likely a serine protease. These studies suggested that daptomycin is susceptible to general proteolytic hydrolysis, which was further supported by studies using proteases of diverse origin. These findings represent the first comprehensive characterization of daptomycin inactivation in any bacterial class and may not only presage a future mechanism of clinical resistance but also suggest strategies for the development of new lipopeptides.

The Wall Teichoic Acid Polymerase TagF Efficiently Synthesizes Poly(glycerol phosphate) on the TagB Product Lipid III

Our understanding of the function of cell-wall teichoic acid polymerases such as TagF from Bacillus subtilis has been limited by the tools available for a functional assay. Teichoic acid polymerase activity has previously been studied by using crude membrane preparations as a source of substrate(s). Thus, an understanding of the most basic features of the teichoic acid polymerization has eluded characterization. Here we make use of a soluble synthetic glycolipid to provide the first demonstration that TagF polymerizes glycerol phosphate directly on the product of TagB—teichoic acid lipid III—at a rate approximately 100 times higher than observed with crude membrane preparations. Interestingly, polymer length was determined by the ratio of glycolipid acceptor to CDP-glycerol, implying that polymerization occurs in a distributive manner. This work provides new insights into the reaction catalyzed by TagF, a prototypic teichoic acid polymerase. The bacterial cell wall has been a popular target for the design of antibacterial agents. Nevertheless, cell wall-active antibiotics have exclusively targeted peptidoglycan synthesis and thus overlook other cell wall components. In Gram-positive bacteria, cell wall teichoic acids are a chemically diverse group of phosphate-rich polymers that are covalently linked to peptidoglycan. Wall teichoic acid accounts for up to 60 % of the Gram-positive cell-wall dry weight. [1] Indeed, wall teichoic acid has recently been shown to be essential to the proper rodshaped morphology of Bacillus subtilis [2] and a key virulence determinant for the human pathogen Staphylococcus aureus. [3, 4] Wall teichoic acid synthesis is thus an emerging