A polymorphic helix of a Salmonella needle protein relays signals defining distinct steps in type III secretion

Abstract

Type III protein-secretion machines are essential for the interactions of many pathogenic or symbiotic bacterial species with their respective eukaryotic hosts. The core component of these machines is the injectisome, a multiprotein complex that mediates the selection of substrates, their passage through the bacterial envelope, and ultimately their delivery into eukaryotic target cells. The injectisome is composed of a large cytoplasmic complex or sorting platform, a multiring base embedded in the bacterial envelope, and a needle-like filament that protrudes several nanometers from the bacterial surface and is capped at its distal end by the tip complex. A characteristic feature of these machines is that their activity is stimulated by contact with target host cells. The sensing of target cells, thought to be mediated by the distal tip of the needle filament, generates an activating signal that must be transduced to the secretion machine by the needle filament. Here, through a multidisciplinary approach, including solid-state NMR (SSNMR) and cryo electron microscopy (cryo-EM) analyses, we have identified critical residues of the needle filament protein of a Salmonella Typhimurium type III secretion system that are involved in the regulation of the activity of the secretion machine. We found that mutations in the needle filament protein result in various specific phenotypes associated with different steps in the type III secretion process. More specifically, these studies reveal an important role for a polymorphic helix of the needle filament protein and the residues that line the lumen of its central channel in the control of type III secretion.

Activation of Shigella flexneri type 3 secretion requires a host-induced conformational change to the translocon pore

“…Whereas we were unable to observe an intermediate filament-dependent conformational change to SipC that was similar to the one we observed for IpaC, recent investigations of the S . Typhimurium needle suggest that secretion is associated with defined conformational states [16]. This finding suggests that, like Shigella and Pseudomonas [10], conformational changes in the Salmonella pore may also occur, even though they were not observed with the approach we used here.…”

“…The sorting platform coordinates the secretion of effector proteins out of the bacterial cytoplasm and into the needle [12, 18]. A mounting body of evidence suggests that conformational changes in the T3SS needle and base are required to activate type 3 secretion [15, 16, 31, 32]. Imaging studies that are sufficiently sensitive to detect subtle changes in conformation of the T3SS needle and base show these structures are altered by host contact [11, 32].…”

“…We and others hypothesize that host contact generates a series of conformational changes along the T3SS needle that constitute a signal from the extracellular environment into the bacterial cytoplasm [7, 15–17], where effector secretion is orchestrated [12, 18, 19]. Because the T3SS translocon pore is situated in the host membrane [6, 8, 20–22], its interactions with the host could be the initial sensor that instigates this signaling cascade [10, 16, 23]; however, how the translocon pore would generate such a signal and specifically whether conformational changes that might contribute to such a signal occur in the translocon pore and/or needle are unknown.…”

Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation

“…Likewise, also N-termini of previously unresolved PrgI1,4-5 protomers cross the EA/InvG gap to interact with SpaP, PrgJ, and the basal body component InvG, with each of these PrgI N-termini forming unique, plastic interactions with its respective environment, providing a rationale as to why some mutations localizing to the PrgI N-terminus abrogate filament assembly in cellulo but not in vitro ( Supplementary Fig. 23) 24 .…”

“…per turn compared to ~23.8Å (6dwb), ~23.3Å (6ofh) and ~23.1Å (2lpz) in models obtained by helical reconstruction cryo-EM and NMR, respectively 7,8,24 . The quality of our map allowed us to model 72 PrgI subunits, covering a distance of ~36 nm and therefore our filament accumulates a total size difference of at least ~5.5Å.…”