Development of Antibiotic Activity Profile Screening for the Classification and Discovery of Natural Product Antibiotics

Section: Screen To Identify T3ss Inhibitorsmentioning

confidence: 99%

An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

Duncan

Wong

Dupzyk

et al. 2014

“…In summary, our ChIPS approach offers a fast and simple way to characterize unknown protein synthesis inhibitors and among other advantages helps to address a significant impediment to antibiotic discovery-the rediscovery of known compounds. The tools we developed can be used in a stand-alone mode or in combination with other techniques aimed at streamlining the small molecule triaging process (6). Finally, although the method is particularly valuable for natural product extracts, it is fully applicable to synthetic protein synthesis inhibitors identified in generalized translation inhibition screens for which the ribosomal target and mode of action are unknown.…”

Section: Discussionmentioning

confidence: 99%

“…In order to close this gap, new technologies are being developed that allow researchers to access natural and synthetic products that have been previously overlooked by traditional screening programs. In particular, developing new antibiotic assays has been one strategy to quickly screen for small molecules that act through novel mechanisms of action (MOA) (4)(5)(6).…”

mentioning

confidence: 99%

Tools for Characterizing Bacterial Protein Synthesis Inhibitors

Orelle

Carlson

Kaushal

et al. 2013

131

Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is timeconsuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol.

“…Streptomyces sp. strain 1675 was isolated from a marine sediment sample collected at Westport Jetty, WA, under permit number 10- (22)(23)(24). For PCR amplification of the 16S rRNA gene, primers 8F (5=-AGAGTTT GATCCTGGCTCAG-3=) and 1492R (5=-GGTTACCTTGTTACGACTT-3=) were used.…”

Section: Methodsmentioning

confidence: 99%

Image-Based 384-Well High-Throughput Screening Method for the Discovery of Skyllamycins A to C as Biofilm Inhibitors and Inducers of Biofilm Detachment in Pseudomonas aeruginosa

Navarro

Cheng

Peach

et al. 2014

dTo date, most antibiotics have primarily been developed to target bacteria in the planktonic state. However, biofilm formation allows bacteria to develop tolerance to antibiotics and provides a mechanism to evade innate immune systems. Therefore, there is a significant need to identify small molecules to prevent biofilm formation and, more importantly, to disperse or eradicate preattached biofilms, which are a major source of bacterial persistence in nosocomial infections. We now present a modular high-throughput 384-well image-based screening platform to identify Pseudomonas aeruginosa biofilm inhibitors and dispersal agents. Biofilm coverage measurements were accomplished using non-z-stack epifluorescence microscopy to image a constitutively expressing green fluorescent protein (GFP)-tagged strain of P. aeruginosa and quantified using an automated image analysis script. Using the redox-sensitive dye XTT, bacterial cellular metabolic activity was measured in conjunction with biofilm coverage to differentiate between classical antibiotics and nonantibiotic biofilm inhibitors/dispersers. By measuring biofilm coverage and cellular activity, this screen identifies compounds that eradicate biofilms through mechanisms that are disparate from traditional antibiotic-mediated biofilm clearance. Screening of 312 natural-product prefractions identified the cyclic depsipeptide natural products skyllamycins B and C as nonantibiotic biofilm inhibitors with 50% effective concentrations (EC 50 s) of 30 and 60 M, respectively. Codosing experiments of skyllamycin B and azithromycin, an antibiotic unable to clear preattached biofilms, demonstrated that, in combination, these compounds were able to eliminate surface-associated biofilms and depress cellular metabolic activity. The skyllamycins represent the first known class of cyclic depsipeptide biofilm inhibitors/dispersers.