Bacterial pathogens deliver effector proteins into host cells by deploying sophisticated secretion systems. This effector translocation during host‐pathogen interactions is a prerequisite for the manipulation of host cells and organisms and is important for pathogenesis. Analyses of dynamics and kinetics of translocation, subcellular localization, and cellular targets of effector proteins lead to understanding the mode of action and function of effector proteins in host‐pathogen interplay. This review provides an overview of biochemical and genetic tools that have been developed to study (...) protein effector translocation qualitatively or quantitatively. After introducing the challenges of analyses of effector translocation during host‐pathogen interaction, we describe various methods ranging from static visualization in fixed cells to dynamic live‐cell imaging of effector protein translocation. We show the main findings enabled by the approaches, emphasize the advantages and limitations of the methods, describe recent approaches that allow real‐time tracking of effector proteins in living cells on a single molecule level, and highlight open questions in the field to be addressed by application of new methods. (shrink)

The type III secretion system (T3SS) is a specialized nanomachine that enables bacteria to secrete proteins in a specific order and directly deliver a specific set of them, collectively known as effectors, into eukaryotic organisms. The core structure of the T3SS is a syringe‐like apparatus composed of multiple building blocks, including both membrane‐associated and soluble proteins. The cytosolic components organize together in a chamber‐like structure known as the sorting platform (SP), responsible for recruiting, sorting, and initiating the substrates (...) destined to engage this secretion pathway. In this article, we provide an overview of recent findings on the SP's structure and function, with a particular focus on its assembly pathway. Furthermore, we discuss the molecular mechanisms behind the recruitment and hierarchical sorting of substrates by this cytosolic complex. Overall, the T3SS is a highly specialized and complex system that requires precise coordination to function properly. A deeper understanding of how the SP orchestrates T3S could enhance our comprehension of this complex nanomachine, which is central to the host‐pathogen interface, and could aid in the development of novel strategies to fight bacterial infections. (shrink)