Selective Inhibition ofNeisseria gonorrhoeaeby a Dithiazoline in Mixed Infections withLactobacillus gasseri

ABSTRACTThe Gram-negative human pathogenNeisseria gonorrhoeaehas progressively developed resistance to antibiotic monotherapies, and recent failures of dual-drug therapy have heightened concerns that strains resistant to all available antibiotics will begin circulating globally. Targeting bacterial cell wall assembly has historically been effective at treating infections withN. gonorrhoeae, but as the effectiveness of β-lactams (including cephalosporins) is challenged by increasing resistance, research has expanded into compounds that target the numerous other enzymes with roles in peptidoglycan metabolism. One example is the dithiazoline compound JNJ-853346 (DTZ), which inhibits the activity of anEscherichia coliserine proteasel,d-carboxypeptidase (LdcA). Recently, the characterization of an LdcA homolog inN. gonorrhoeaerevealed localization and activity differences from the characterizedE. coliLdcA, prompting us to explore the effectiveness of DTZ againstN. gonorrhoeae. We found that DTZ is effective at inhibitingN. gonorrhoeaein all growth phases, unlike the specific stationary-phase inhibition seen inE. coli. Surprisingly, DTZ does not inhibit gonococcal LdcA enzyme activity, and DTZ sensitivity is not significantly decreased inldcAmutants. While effective against numerousN. gonorrhoeaestrains, including recent multidrug-resistant isolates, DTZ is much less effective at inhibiting growth of the commensal speciesLactobacillus gasseri. DTZ treatment during coinfections of epithelial cells resulted in significant lowering of gonococcal burden and interleukin-8 secretion without significantly impacting recovery of viableL. gasseri. This selective toxicity presents a possible pathway for the use of DTZ as an effective antigonococcal agent at concentrations that do not impact vaginal commensals.

Purification of large peptides using Chemoselective tags

In solid phase peptide synthesis (SPPS), deletion sequences are generated at each addition of amino acid due to non-quantitative coupling reactions. Their concentration increases exponentially with the length of the peptide chain, and after many cycles not only do they represent a large proportion of the crude preparation, but they can also exhibit physicochemical characteristics similar to the target sequence. Thus, these deletion-sequence contaminants present major problems for removal, or even detection. In general, purification of synthetic peptides by conventional chromatography is based on hydrophobicity differences (using RP-HPLC) and charge differences (using ion-exchange chromatography). For short sequences, the use of one or both techniques is in general sufficient to obtain a product with high purity. However, on increasing the number of amino acid residues, the peptide secondary and progressively tertiary and quaternary structures begin to play an important role and the conformation of the largest peptides can decisively affect their retention behaviour. Furthermore, very closely related impurities such as deletion sequences lacking one or few residues can be chromatographically indistinguishable from the target sequence. Therefore, purification of large synthetic peptides is a complex and time-consuming task that requires the use of several separation techniques with the inevitable dramatic reduction in yields of the final material. Permanent termination (capping) of unreacted chains using a large excess of an acylating agent after each coupling step prevents the formation of deletion sequences and generates N-truncated peptides. However, even under these more favourable conditions, separation of the target sequence from chromatographically similar N-capped polypeptides requires extensive purification. If the target sequence could be specifically and transiently labelled so that the resulting product were selectively recognized by a specific stationary phase, then separation from impurities should be facilitated. This chapter deals with such an approach and in particular with the purification of large polypeptides, assembled by solid phase strategy, using lipophilic and biotin-based 9-fluorenylmethoxycarbonyl (Fmoc) chromatographic probes. Assuming that the formation of deletion sequences is prevented by capping unreacted chains, a reciprocal strategy can be applied that involves functional protection of all polymer-supported peptide chains that are still growing, with a specially chosen affinity reagent or chromatographic probe.