Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

The activation of the oxidative stress response transcription factor SKN-1 in Caenorhabditis elegans by mitis group streptococci.

The mitis group, a member of the genetically diverse viridans group streptococci, predominately colonizes the human oropharynx. This group has been shown to cause a wide range of infectious complications in humans, including bacteremia in patients with neutropenia, orbital cellulitis and infective endocarditis. Hydrogen peroxide (H2O2) has been identified as a virulence factor produced by this group of streptococci. More importantly, it has been shown that Streptococcus oralis and S. mitis induce epithelial cell and macrophage death via the production of H2O2. Previously, H2O2 mediated killing was observed in the nematode Caenorhabditis elegans in response to S. oralis and S. mitis. The genetically tractable model organism C. elegans is an excellent system to study mechanisms of pathogenicity and stress responses. Using this model, we observed rapid H2O2 mediated killing of the worms by S. gordonii in addition to S. mitis and S. oralis. Furthermore, we observed colonization of the intestine of the worms when exposed to S. gordonii suggesting the involvement of an infection-like process. In response to the H2O2 produced by the mitis group, we demonstrate the oxidative stress response is activated in the worms. The oxidative stress response transcription factor SKN-1 is required for the survival of the worms and provides protection against H2O2 produced by S. gordonii. We show during infection, H2O2 is required for the activation of SKN-1 and is mediated via the p38-MAPK pathway. The activation of the p38 signaling pathway in the presence of S. gordonii is not mediated by the endoplasmic reticulum (ER) transmembrane protein kinase IRE-1. However, IRE-1 is required for the survival of worms in response to S. gordonii. These finding suggests a parallel pathway senses H2O2 produced by the mitis group and activates the phosphorylation of p38. Additionally, the unfolded protein response plays an important role during infection.