Analysis of assembly and function of bacterial type III secretion systems

Abstract

Gram-negative bacterial pathogens utilize virulence-associated type III secretion systems (T3SS) to inject effector proteins into eukaryotic host cells. These effector proteins modulate the host cell biology to promote colonization and infection. Hence, T3SS are essential bacterial pathogenicity factors. The transmembrane export apparatus at the core of T3SS contains a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU. These components comprise several highly conserved charged residues within their hydrophobic domains. Based on the closed state structure of the core complex SctR5S4T1, we know several of these residues form both inter- and intramolecular salt bridges, which should break to facilitate pore opening. Mutagenesis of individual residues compromises the assembly or secretion of both the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Hence, to understand the role of these residues in the T3SS of Salmonella Typhimurium, we performed an in-depth investigation. Our investigation included mutagenesis, blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these conserved salt bridges are not critical for the assembly of the respective protein but rather aid in incorporating the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctSE44 and SctTR204 and the cytoplasmic domain of SctU in gating the T3SS injectisome. T3SS includes various proteins, and the exact interactions amongst constituent proteins and the complex assembly are not fully understood. The interaction amidst the inner ring protein and the sorting platform components is critical for the principal activity of type III secretion systems. SctK is one of the least studied cytoplasmic component and hence lack of information poses a major impediment in advancing our understanding of its interactions. Additionally, many aspects related to the organization, configuration and function of the sorting platform in the needle complex are still unclear. We performed an in-depth analysis of the different positions of SctD with in vivo photocrosslinking, luciferase-based secretion assays and different functional assays. Our results provide the plausible orientation model of SctD protein highlighting the interacting interface of SctD-SctK where the cytoplasmic component SctK establishes itself as the interacting partner of SctD in the inner membrane. Our newly established functional assays could successfully elucidate that low external pH and blockage of PMF can lead to a temporary dissociation and change in dynamics of the cytosolic components from T3SS respectively. Our investigation also characterizes the effect of single-gene deletions on the SctD-SctK interactions confirming that different secretion states have no substantial effect on the interaction of the ring protein with the cytosolic protein.