Examining Sporadic Cancer Mutations Uncovers a Set of Genes Involved in Mitochondrial Maintenance.

Mitochondria are key organelles for cellular health and metabolism and the activation of programmed cell death processes. Although pathways for regulating and re-establishing mitochondrial homeostasis have been identified over the past twenty years, the consequences of disrupting genes that regulate other cellular processes, such as division and proliferation, on affecting mitochondrial function remain unclear. In this study, we leveraged insights about increased sensitivity to mitochondrial damage in certain cancers, or genes that are frequently mutated in multiple cancer types, to compile a list of candidates for study. RNAi was used to disrupt orthologous genes in the model organism Caenorhabditis elegans, and a series of assays were used to evaluate these genes' importance for mitochondrial health. Iterative screening of ~1000 genes yielded a set of 139 genes predicted to play roles in mitochondrial maintenance or function. Bioinformatic analyses indicated that these genes are statistically interrelated. Functional validation of a sample of genes from this set indicated that disruption of each gene caused at least one phenotype consistent with mitochondrial dysfunction, including increased fragmentation of the mitochondrial network, abnormal steady-state levels of NADH or ROS, or altered oxygen consumption. Interestingly, RNAi-mediated knockdown of these genes often also exacerbated α-synuclein aggregation in a C. elegans model of Parkinson's disease. Additionally, human orthologs of the gene set showed enrichment for roles in human disorders. This gene set provides a foundation for identifying new mechanisms that support mitochondrial and cellular homeostasis.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem.

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.