A High-throughput, High-content, Liquid-based <em>C. elegans</em> Pathosystem

Abstract

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up. Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans-Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches. We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans-E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

Pyoverdine-Dependent Virulence of Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients

“…Liquid Killing assay was performed as previously described (Anderson et al, 2018). In brief, C. elegans were exposed to overnight cultures of P. aeruginosa diluted to OD 600 0.03 in Liquid Kill media [a 1:4 mix of SK media (0.3% w/v NaCl, 0.35% w/v Bacto-Peptone in water) and S-Basal (100 mM NaCl, 50 mM potassium phosphate, pH 6.0)] in 384-well plates.…”

Novel Pyoverdine Inhibitors Mitigate Pseudomonas aeruginosa Pathogenesis

“…The Liquid Killing assay was performed essentially as described (Anderson et al, 2018). In brief, glp-4(bn2ts) worms were synchronized by bleaching gravid adults and hatching in the absence of food at 15°C.…”

Novel Immune Modulators EnhanceCaenorhabditis elegansResistance to Multiple Pathogens

“…Enterococcus faecalis and Staphylococcus aureus are Gram-positive bacterial species frequently responsible for nosocomial infections [23,24]. These bacterial species readily infect C. elegans and liquidbased pathogenesis models have been developed for each of them [25][26][27]. Sterile, young adult glp- 4(bn2) worms were incubated with either E. faecalis or S. aureus and varying concentrations of the five compounds.…”