Förster Resonance Energy Transfer (FRET) as a Tool for Dissecting the Molecular Mechanisms for Maturation of the Shigella Type III Secretion Needle Tip Complex

Abstract

Förster resonance energy transfer (FRET) provides a powerful tool for monitoring intermolecular interactions and a sensitive technique for studying Å-level protein conformational changes. One system that has particularly benefited from the sensitivity and diversity of FRET measurements is the maturation of the Shigella type III secretion apparatus (T3SA) needle tip complex. The Shigella T3SA delivers effector proteins into intestinal cells to promote bacterial invasion and spread. The T3SA is comprised of a basal body that spans the bacterial envelope and a needle with an exposed tip complex that matures in response to environmental stimuli. FRET measurements demonstrated bile salt binding by the nascent needle tip protein IpaD and also mapped resulting structural changes which led to the recruitment of the translocator IpaB. At the needle tip IpaB acts as a sensor for host cell contact but prior to secretion, it is stored as a heterodimeric complex with the chaperone IpgC. FRET analyses showed that chaperone binding to IpaB’s N-terminal domain causes a conformational change in the latter. These FRET analyses, with other biophysical methods, have been central to understanding T3SA maturation and will be highlighted, focusing on the details of the FRET measurements and the relevance to this particular system.

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence

“…While the injected T3SS effector proteins are specific to each pathogen's infection mechanism and replicative niche, they are all secreted through a highly-conserved syringe and needle-like injectisome [10,11]. The injectisome consists of a basal body that spans the periplasmic space and anchors the apparatus to the bacterial inner and outer membranes, a hollow needle that extends from the basal body beyond the lipopolysaccharide layer, and a protein tip complex that serves as an environmental sensor and provides access to the host cell cytoplasm by penetrating the host membrane [10][11][12][13]. In addition, a multi-protein cytosolic sorting platform is located just below the apparatus basal body and is believed to play important roles in the recognition and unfolding of effector proteins prior to secretion [14][15][16].…”

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

“…Because F€ orster resonance energy transfer (FRET) provides a sensitive spectroscopic ruler with Å -level resolution, 28,29 it was also used to probe for protein/liposome interactions. By covalently linking a fluorescein FRET donor to the native cysteine of IpaB and including a TRITC acceptor probe conjugated to the polar head group of the phospholipid DHPE in the liposomes, energy transfer between the FRET pair could be examined to provide confirmation of the IpaB-liposome interaction.…”

Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation

“…Insertion of the T3SS translocator tip proteins IpaB and IpaC into the host cell membrane forms the translocon pore and ultimately allows secretion of effector proteins into eukaryotic host cell cytoplasm . Secretion of these effectors into the host cell supports invasion of the infected host cell, escape from the resulting vacuole, and evasion of host immune responses .…”