Microbiology

Reagents such as Amplex^® Red have been developed for detecting hydrogen peroxide (H₂O₂) and are used to measure the release of H₂O₂ from biological samples such as mammalian leukocytes undergoing the oxidative burst. Caenorhabditis elegans is commonly used as a model host in the study of interactions with microbial pathogens and releases reactive oxygen species (ROS) as a component of its defense response. We adapted the Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit to measure H₂O₂ output from live Caenorhabditis elegans exposed to microbial pathogens. The assay differs from other forms of ROS detection in the worm, like dihydrofluorescein dyes and genetically encoded probes such as HyPer, in that it generally detects released, extracellular ROS rather than intracellular ROS, though the distinction between the two is blurred by the fact that certain species of ROS, including H₂O₂, can cross membranes. The protocol involves feeding C. elegans on a lawn of the pathogen of interest for a period of time. The animals are then rinsed off the plates in buffer and washed to remove any microbes on their cuticle. Finally, the animals in buffer are distributed into 96-well plates and Amplex^® Red and horseradish peroxidase (HRP) are added. Any H₂O₂ released into the buffer by the worms will react with the Amplex^® Red reagent in a 1:1 ratio in the presence of HRP to produce the red fluorescent excitation product resorufin that can be measured fluorometrically or spectrophotometrically, and the amount of H₂O₂ released can be calculated by comparison to a standard curve. The assay is most appropriate for studies focused on released ROS, and its advantages include ease of use, the ability to use small numbers of animals in a plate reader assay in which measurements can be taken either fluorometrically or spectrophotometrically.

The Gram-positive bacterium Staphylococcus aureus is a human pathogen that displays virulence towards the nematode Caenorhabditis elegans. This property can be used to discover genes that are important for virulence in humans, because S. aureus possesses common virulence factors that are used in C. elegans and in humans to cause disease. S. aureus colonizes the C. elegans intestine, establishes an infection, and causes pathogenesis of the intestinal epithelium that ultimately kills the infected animal after 3 to 4 days (Sifri et al., 2003; Irazoqui et al., 2008; Irazoqui et al., 2010). The protocol described here is used to establish the rate of S. aureus-induced C. elegans death, which allows the comparison of wild type and mutant strains and thus ultimately aids in the identification of genes required either for S. aureus virulence or for C. elegans host defense. The assay can also be applied for antimicrobial drug discovery.