Abstract
Type III secretion (TTS) is an essential virulence function for Shigella flexneri that delivers effector proteins that are responsible for bacterial invasion of intestinal epithelial cells. The Shigella TTS apparatus (TTSA) consists of a basal body that spans the bacterial inner and outer membranes and a needle exposed at the pathogen surface. At the distal end of the needle is a "tip complex" composed of invasion plasmid antigen D (IpaD). IpaD not only regulates TTS, but is required for the recruitment and stable association of the translocator protein IpaB at the TTSA needle tip in the presence of deoxycholate or other bile salts. This phenomenon is not accompanied by induction of TTS or the recruitment of IpaC to the Shigella surface. We now show that IpaD specifically binds fluorescein-labeled deoxycholate and, based on energy transfer measurements and docking simulations, this interaction appears to occur where the N-terminal domain of IpaD meets its central coiled-coil, a region that may also be involved in needle-tip interactions. TTS is initiated as a series of distinct steps and that small molecules present in the bacterial milieu are capable of inducing the first step of TSS through interactions with the needle tip protein IpaD. Furthermore, the amino acids proposed to be important for deoxycholate binding by IpaD appear to have significant roles in regulating tip complex composition and pathogen entry into host cells.
Highlights
18646 JOURNAL OF BIOLOGICAL CHEMISTRY where it is an infectious agent in child daycare centers, nursing homes, and in any situation where sanitation procedures become compromised [1]
Deoxycholate-induced Recruitment of IpaB to the Shigella Surface Appears to Correlate with Deoxycholate Binding by invasion plasmid antigen D (IpaD)—We have previously shown that S. flexneri grown to early log phase in tryptic soy broth (TSB) has IpaD present on its surface
It has been recently shown that the essential virulence protein IpaD works primarily as a the type III secretion apparatus (TTSA) needle tip protein [9] with bile salts acting as environmental signals for the stable recruitment of IpaB onto the Shigella needle tip complex [11]
Summary
Materials—Antibodies to IpaB, IpaC, and IpaD were provided by E. Growth of S. flexneri in the Presence of Deoxycholate—S. flexneri is routinely grown to early or mid log phase in TSB after inoculation from a trypticase soy agar plate containing Congo red These growth conditions do not lead to detectable levels of IpaB on the bacterial surface. Forster Resonance Energy Transfer (FRET) Analyses—FRET measurements were obtained for CPM-labeled IpaD or CPMIpaD (donor) and FITC-deoxycholate (acceptor). A RO value (the theoretical distance that would give 50% energy transfer efficiency) was determined to be 52 Å [19], which was used with the calculated energy transfer maximum to determine the distance between the coumarin on Cys-322 of CPM-IpaD to the fluorescein on FITC-deoxycholate according to E ϭ RO6/(RO6 ϩ R6) (Equation 2), where R is the calculated distance separating the donor and acceptor fluorophores [20]. The centrifugation step normally used for monitoring Shigella invasiveness was omitted
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