Showing papers by "Ian Chopra published in 2009"

XF-73, a novel antistaphylococcal membrane-active agent with rapid bactericidal activity

Abstract: Objectives XF-73 is a novel porphyrin antibacterial agent previously reported to inhibit a range of Gram-positive bacterial species, including Staphylococcus aureus. Its mode of action is unknown. Using S. aureus as a model organism we sought to examine the basis of its antibacterial activity. Methods The effects of XF-73 on the growth and survival of S. aureus SH1000 were investigated by viable count and culture absorbance techniques. Inhibition of macromolecular synthesis and disruption of membrane integrity after exposure to XF-73 were examined by radiolabelling experiments, the BacLight fluorescent dye assay and measurement of K+ and ATP leakage from the cell. The effect of XF-73 on a staphylococcal coupled transcription–translation system was also investigated. Results XF-73 was rapidly bactericidal against S. aureus SH1000 and demonstrated more rapid killing kinetics than all other comparator agents when tested at an equivalent multiple (4×) of the MIC. Exposure of S. aureus to XF-73 for 10 min completely inhibited DNA, RNA and protein synthesis. XF-73 had no effect on transcription and translation in vitro. Cells exposed to XF-73 gave a positive response in the BacLight assay, which detects membrane damage. The drug also caused substantial loss of K+ and ATP from the cell, but did not promote bacterial lysis. Conclusions XF-73 exhibited rapid membrane-perturbing activity, which is likely to be responsible for inhibition of macromolecular synthesis and the death of staphylococci exposed to the drug.

The nature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors

Abstract: Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes (murA and murZ) encoding UDP-N-acetylglucosamine enolpyruvyl transferase activity for catalysing the first committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro, consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA, but remained almost unchanged following inactivation of murZ. Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA. Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.

Analysis of mutational resistance to trimethoprim in Staphylococcus aureus by genetic and structural modelling techniques

Abstract: Received 12 November 2008; returned 27 December 2008; revised 18 February 2009; accepted 23 February 2009 Objectives: This study sought to expand knowledge on the molecular mechanisms of mutational resistance to trimethoprim in Staphylococcus aureus, and the fitness costs associated with resistance. Methods: Spontaneous trimethoprim-resistant mutants of S. aureus SH1000 were recovered in vitro, resistance genotypes characterized by DNA sequencing of the gene encoding the drug target (dfrA) and the fitness of mutants determined by pair-wise growth competition assays with SH1000. Novel resistance genotypes were confirmed by ectopic expression of dfrA alleles in a trimethoprim-sensitive S. aureus strain. Molecular models of S. aureus dihydrofolate reductase (DHFR) were constructed to explore the structural basis of trimethoprim resistance, and to rationalize the observed in vitro fitness of trimethoprim-resistant mutants. Results: In addition to known amino acid substitutions in DHFR mediating trimethoprim resistance (F99Y and H150R), two novel resistance polymorphisms (L41F and F99S) were identified among the trimethoprim-resistant mutants selected in vitro. Molecular modelling of mutated DHFR enzymes provided insight into the structural basis of trimethoprim resistance. Calculated binding energies of the substrate (dihydrofolate) for the mutant and wild-type enzymes were similar, consistent with apparent lack of fitness costs for the resistance mutations in vitro. Conclusions: Reduced susceptibility to trimethoprim of DHFR enzymes carrying substitutions L41F, F99S, F99Y and H150R appears to result from structural changes that reduce trimethoprim binding to the enzyme. However, the mutations conferring trimethoprim resistance are not associated with fitness costs in vitro, suggesting that the survival of trimethoprim-resistant strains emerging in the clinic may not be subject to a fitness disadvantage.

Functional and Biochemical Analysis of the Chlamydia trachomatis Ligase MurE

Abstract: Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid (UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm ligase activity. Recombinant MurE from C. trachomatis (MurE(Ct)) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-L-Ala-D-Glu as the nucleotide substrate, MurE(Ct) demonstrated ATP-dependent meso-A(2)pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the ll and dd isomers of A(2)pm, D-lysine) were also recognized by MurE(Ct.) However, the activities for these amino acid substrates were weaker than that for meso-A(2)pm. The specificity of MurE(Ct) for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k(cat)/K(m) ratios for UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ser-D-Glu, and UDP-MurNAc-Gly-D-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A(2)pm. However, due to the lack of specificity of MurE(Ct) for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.

Transcriptional signature following inhibition of early-stage cell wall biosynthesis in Staphylococcus aureus.

Abstract: To facilitate mode of action studies on antibacterial inhibitors of early-stage cell wall biosynthesis (CWB), we determined the transcriptional response of Staphylococcus aureus to depletion/inhibition of enzymes in this pathway by DNA microarray analysis. We identified a transcriptional signature distinct from that previously observed following exposure to inhibitors of late-stage CWB.