Mechanism of type‐III protein secretion: Regulation of FlhA conformation by a functionally critical charged‐residue cluster

Abstract

The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram-negative pathogens to export virulence factors into host cells. This mode of protein export is termed type-III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton-binding groups that might function in transport. Conserved proton-binding residues in all the membrane components were tested. The results identify residues R147, R154, and D158 of FlhA as most critical. These lie in a small, well conserved cytoplasmic domain of FlhA, located between trans-membrane segments 4 and 5. Two-hybrid experiments demonstrate self-interaction of the domain, and targeted cross-linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton-actuated movements in the cytoplasmic domains of FlhA.

The substrate specificity switch FlhB assembles onto the export gate to regulate type three secretion

“…Interestingly, this network of charged residues in FlhB includes D208. The D208A mutation disrupts motility, but this defect can be rescued by overexpression of FlhA 27 , reinforcing the functional link between the charge network in FlhB and FlhA. As FlhB C is thought to interact with substrates just before they pass through the export gate 28 , it is possible that transition of FlhA between the different conformations pulls on FlhB C , either directly or via substrate, thereby pulling on the FlhB TM network, leading to an opening of the complex.…”

Type‐III secretion pore formed by flagellar protein FliP

“…Previous estimates of FliP stoichiometry suggested the presence of 4-5 copies per basal body (Fan et al, 1997), and structural studies of a sub-domain of FliP similarly indicate the likelihood of FliP multimers (Fukumura et al, 2014). To further test the proposal that the apparatus contains multiple, jointly functioning copies of FliP, we examined inter-subunit complementation between FliP mutations that were previously found to cause fairly severe motility impairments individually (Erhardt et al, 2017). Selected fliP alleles were transferred from the Cm R plasmid onto a plasmid encoding Km R , then transformed into the DfliP strain in pairs and tested for function in motility plates.…”

“…S1). In a mutational study of conserved charged residues of the apparatus, replacements of Asp197 and Lys222 caused a substantial reduction in function as assayed on motility plates (Erhardt et al, 2017). The strong sequence conservation in TM3 and TM4 and the significant motility defects of Asp197 and Lys222 mutants indicate that these parts of FliP contribute in an important way to the transport process.…”

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility

“…However, it is not known how flagellar structural proteins such as FlhA can contribute to flagellar type III secretion-independent regulation events of earlier expressed genes (class 2 flagellar genes). FlhA is known to harbor a critical patch of charged amino acid residues in a cytoplasmic loop which can influence its conformation (Erhardt et al, 2017). In H. pylori, the expression of intermediate (class 2 and class 3) flagellar genes is regulated by the cytoplasmic TCS FlgS-FlgR and its impact on σ54-dependent transcription (Niehus et al, 2004).…”