Investigating the assembly of the bacterial type III secretion system injectisome by in vivo photocrosslinking

Abstract

Virulence-associated type III secretion systems serve the injection of bacterial effector proteins into eukaryotic host cells. These effector proteins modulate host cell biology in order to promote colonization and infection, hence type III secretion systems are often essential bacterial pathogenicity factors. The core of type III secretion systems is a cell envelope-spanning macromolecular machine called injectisome. It consists of almost twenty different components in a stoichiometry of one to more than one hundred. Assembly of this 6 MDa complex requires the coordinated integration of components from the cytoplasm, the inner membrane, the periplasm, the outer membrane and even the extracellular space of Gram-negative bacteria. Here, we review injectisome assembly with an emphasis on the techniques that were employed towards its investigation. In particular, we focus on in vivo photocrosslinking, a technique that exploits the encoding of the artificial UV-inducible crosslinking amino acid p-benzoyl-phenylalanine to identify protein-protein interactions and to delineate assembly pathways.

Introduction

The export of proteins is instrumental for the interaction of bacteria with their environment. Bacteria export proteins of diverse functions, like toxins, adhesion factors, effector proteins, transcription factors, or proteases. Protein export across the cell envelope of Gram-positive bacteria is relatively simple as only one membrane needs to be passed. Export occurs mostly by means of the Sec and Tat translocons that translocate unfolded and folded substrate proteins across the cytoplasmic membrane, respectively (Hamed et al., 2018). Protein export across the two membranes of the cell envelope of Gram-negative bacteria requires more elaborate mechanisms. To date, nine different protein secretion systems, termed type I to type IX, have been identified that differ in the buildup of their machines, energization of secretion (ATP vs. PMF, or both), secretion signal, and folding state of their substrates (unfolded vs. folded), and whether they secrete proteins in one step or in two steps across the two membranes of the cell envelope (Costa et al., 2015). Three of these secretion systems (type III, type IV, and type VI) even serve the introduction of bacterial proteins directly into the cytosol of target cells (Fig. 1A) (Galán and Waksman, 2018). These injection machines constitute large protein complexes of usually more than 100 subunits, several megadalton in size. Often, they consist of cytosolic, inner membrane, periplasmic, outer membrane, and extracellular components. Assembly of these machines is an intricate and costly task that requires precise orchestration and is often highly regulated at multiple levels. In this review, we focus on the current state of our understanding of the assembly of the type III secretion injectisome (Fig. 1B), whose secretion system is also found in bacterial flagella. We will particularly emphasize the methodology that has been used towards elucidating assembly mechanisms. More elaborate reviews of assembly, structure and function of T3SS can be found elsewhere (Deng et al., 2017; Diepold and Wagner, 2014; Galán et al., 2014; Wagner et al., 2018). We will refer to the unified nomenclature of type III secretion systems (T3SS) throughout (Fig. 1C) (Hueck, 1998; Portaliou et al., 2016).