Antimicrobial resistance is a global threat that poses a rising concern. One underlying challenge is the limited number of targets in bacteria affected by the current pool of antibiotics. To potentially help find new targets, we studied a member of the class of antimicrobial natural products named glycocins. We examined the mode of action of sublancin, which contains an unusual and essential glucosylated Cys residue, by monitoring macromolecular synthesis. Sublancin negatively affected DNA replication, transcription, and translation without affecting cell wall biosynthesis. In addition, we confirmed that the presence of the PTS sugar glucose in the medium negatively impacted antimicrobial activity of sublancin. Additionally, sublancin analogues carrying different sugars retained their antimicrobial activity regardless of which sugar was attached to the peptide or the carbon source used. These data suggest a novel mechanism upstream of transcription and translation and are consistent with previous studies suggesting that the glucose uptake system is involved.

Sublancin is a 37-amino acid antimicrobial peptide belonging to the glycocin family of natural products. It contains two helices that are held together by two disulfide bonds, as well as an unusual S-glucosidic linkage to a Cys in a loop connecting the helices. We report the reconstitution of the biosynthetic pathway to this natural product in Escherichia coli. This technology enabled the evaluation of the structure-activity relationships of the solvent-exposed residues in the helices. The biosynthetic machinery proved tolerant of changes in both helices, and the bioactivity studies of the resulting mutants show that two residues in helix B are important for bioactivity, Asn31 and Arg33.

Glycocins (glycosylated bacteriocins) are a family of ribosomally synthesized and post-translationally modified peptides with antimicrobial activities against pathogens of interest, including methicillin-resistant Staphylococcus aureus, representing a promising source of new antibiotics. Glycocins are still largely underexplored, and thus far, only six glycocins are known. Here, we used genome mining to identify 50 putative glycocin biosynthetic gene clusters and then chose six of them with distinct features for further investigation. Through two rounds of plug-and-play pathway refactoring and expression in Escherichia coli BL21(DE3), four systems produced novel glycocins. Further structural characterization revealed that one of them, which belongs to the enterocin 96-type glycocins, was diglucosylated on a single serine. The other three compounds belong to the SunA/ThuA-type glycocins and exhibit a antimicrobial spectrum narrower than that of sublancin, the best characterized member in this group, even though they share a similar disulfide topology and glycosylation. Further evaluation of their bioactivities with free glucose at high concentrations suggested that their antimicrobial mechanisms might be both glycocin- and species-specific. These glycocins with distinct features significantly broaden our knowledge and may lead to the discovery of new classes of antibiotics.

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Antimicrobial resistance is a global challenge that is compounded by the limited number of available targets. Glycocins are antimicrobial glycopeptides that are believed to have novel targets. Previous studies have shown that the mechanism of action of the glycocin sublancin 168 involves the glucose uptake system. The phosphoenolpyruvate:sugar phosphotransferase system (PTS) phosphorylates the C6 hydroxyl group on glucose during import. Since sublancin carries a glucose on a Cys on an exposed loop, we investigated whether phosphorylation of this glucose might be involved in its mechanism of action by replacement with xylose. Surprisingly, the xylose analog was more active than wild-type sublancin and still required the glucose PTS for activity. Overexpression of the individual components of the PTS rendered cells more sensitive to sublancin, and their resistance frequency was considerably decreased. These observations suggest that sublancin is activated in some form by the glucose PTS or that sublancin imparts a deleterious gain-of-function on the PTS. Superresolution microscopy studies with fluorescent sublancin and fluorescently labeled PTS proteins revealed localization of both at the poles of cells. Resistant mutants raised under conditions that would minimize mutation of the PTS revealed mutations in FliQ, a protein involved in the flagellar protein export process. Overexpression of FliQ lead to decreased sensitivity of cells to sublancin. Collectively, these findings enforce a model in which the PTS is required for sublancin activity, either by inducing a deleterious gain-of-function or by activating or transporting sublancin.

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Biofilm is the sessile group of organisms that adhere irreversibly with the biotic and abiotic surface with the help of pilli and extracellular polymeric substance [EPS] which comprises of carbohydrates, proteins and nucleic acids that provides nutrition to the developing microcolonies and it also prevents the penetration of drug molecules upto the cells thus rendering antibiotic and antimicrobial resistance. Antibiotics are still considered to be one of the potent antimicrobial agents which helps in removing the biofilm. This results in the overuse of antibiotics thereby developing antibiotic resistance within the microorganisms. Thus an alternative pathway to develop compounds or materials having potent antimicrobial and antibiofilm properties is the need of present hour. Nanoparticles have at least one dimension and are extremely small in size ranging between 1-100 nm so it can easily penetrate in the cells of the microorganisms. But over use of this metallic or non-metallic nanoparticles can cause toxic effect on the living cells. Thus, green synthesized nanoparticles are very useful alternative because it is made up from natural sources and are less harmful to living beings. Green-synthesized silver nanoparticles has provided an alternate pathway in removing the biofilm being formed upon the biotic and abiotic surfaces. This review provides an overall concept upon the synthesis and the mode of action of Ag-NPs.