In the ongoing funding period of SNSF grant 31003A-173119 we are studying the cellular, molecular and evolutionary basis of subversion of host cell functions by type IV secretion (T4S) effector proteins during chronic bacterial infection by the related zoonotic pathogens Bartonella and Brucella. The three-year pro rata temporis extension in the frame of the excellence grant will allow us to extend our studies towards a better understanding (i) of the evolutionary origin and function of these host cell-targeted effector proteins and (ii) of the nature of their signal for T4S-dependent translocation. In Subproject A we will continue the functional characterization of Bartonella effector proteins (Beps) translocated by the VirB T4S system and the structure/function analysis of the BID domain as principle constituent of their T4S signal. Furthermore, we will extend our multidisciplinary studies to an unrelated class of newly identified T4S effectors in Bartonella with homology to a family of widespread type III secretion (T3S) effectors. Furthermore, we will study the role of the Bartonella Gene Transfer Agent (BaGTA) in facilitating adaptive evolution of Beps and associated virulence factors in processes like host adaptation. Subproject B will focus on the functional analysis of effector proteins translocated by the distinct VirB T4S system of Brucella with particular emphasis on delineating the unknown T4S signal. Together, these studies will contribute fundamentally to our molecular understanding of the widespread T4S mechanism in the context of chronic bacterial infections.