Abstract Many pathogenic bacteria inject effector proteins into host cells in order to reprogram cellular behavior. Insight into function is typically gained from studying each effector in isolation. However, bacteria often employ multiple effectors and it is rarely considered how effector ensembles synergize to promote unique cellular behaviors that cannot be predicted from analyses of each effector in isolation. Thus, understanding the molecular basis of effector synergy is crucial for unraveling how cellular reprogramming contributes to pathogenesis and the selective pressures driving effector evolution. Effector synergy has emerged as an important concept for understanding the pathogenesis of the myriad diseases caused by Streptococcus pyogenes, whose virulence derives from its production of multiple effectors that reprogram host cell behavior. An important example is Cytolysin-Mediated Translocation (CMT), a process by which the cytolysin Streptolysin O (SLO) translocates an effector, the S. pyogenes NAD+ glycohydrolase (SPN), across the host cell membrane into its cytosol. Synergy continues post-translocation, as intracellular SPN modifies cellular reprogramming initiated by SLO. A complication, is that SPN has diverged into two widely distributed haplotypes, one of which lacks its signature NAD+ glycohydrolase activity (NADase-). Why both variants are under selection and how each reprograms cellular behaviors to promote pathogenesis is not understood. There are multiple significant gaps in our understanding of synergy in CMT, including how the two toxins interact with host cells to promote the translocation of SPN and how the biological activities of SLO and SPN act synergistically to promote cell signaling and cell death. To address these questions, this proposal has two overall goals: 1. To develop novel infection-free models of CMT for analysis of specific steps leading to membrane translocation and cellular toxicity using new insights gained from understanding that SLO has two distinct modes on binding to host cells, only one of which can support CMT; and 2. To gain mechanistic insight into translocation, toxicity and signaling by conducting an unbiased forward screen to identify host cell genes that support these activities. Identification of these genes and development of new CMT models has the potential to be transformative in our understanding of CMT and toxin synergy.