Protocol for analyzing the function of the type VI secretion system of the oral symbiont Aggregatibacter aphrophilus in targeting pathobionts

Here, we present a protocol for evaluating type VI secretion system (T6SS)-dependent fitness of the oral symbiont A. aphrophilus using biofilm competition assays and metaproteomics. We describe steps for designing T6SS-specific mutants. We then detail procedures for using them in competition assays with the pathobiont A. actinomycetemcomitans and in biofilm models, analyzing metaproteomes to assess the impact of the T6SS on multiple pathobionts. The biofilm model is designed to mimic the oral plaque ecosystem and includes seven species. For complete details on the use and execution of this protocol, please refer to Oscarsson et al.1.