Type III secretion (TTS) is an essential virulence function forShigella 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.Shigella flexneri is the etiologic agent of shigellosis, a potentially life-threatening bacillary dysentery in humans. Although shigellosis is typically considered a disease of the developing world, it is an underreported problem in industrialized nations where it is an infectious agent in child daycare centers, nursing homes, and in any situation where sanitation procedures become compromised (1). Shigellosis is spread by the fecal-oral route with larger outbreaks linked to contaminated water, which makes it a serious public health problem anywhere treatment regimens are inadequate. Following ingestion, Shigella travels to the colon where it crosses M cells and kills macrophages to gain access to the basal side of the colonic epithelium. S. flexneri then promotes its own uptake into epithelial cells by inducing membrane ruffling at the site of pathogen contact.Shigella invasiveness is the product of a 31-kb segment on its large virulence plasmid, which encodes the components of a type III secretion system (TTSS). 4 The Shigella TTSS is used to subvert the normal host cell mechanisms that control the actin cytoskeleton to promote invasion (2, 3). Within this genetic region, the mxi/spa operons encode the type III secretion apparatus (TTSA) and the ipa/ipg operon encodes the type III secreted protein effectors/translocators, IpaA-D, and IpgC, the cytoplasmic chaperone for IpaB and IpaC (4). The TTSA is a nanomachine that provides a conduit for secretion from the bacterial cytoplasm to the membrane and cytoplasm of target cells. It is composed of two main parts: an external needle that allow...

Three new trifluoromethylated p-hydroxyphenacyl (pHP) caged γ-aminobutyric acid (GABA) and glutamate (Glu) derivatives have been examined for their efficacy as photoremovable protecting groups in aqueous solution. By replacing hydrogen with fluorine, e.g., a m-trifluoromethyl or a m-trifluoromethoxy vs. m-methoxy substituents on the pHP chromophore, modest increases in the quantum yields for release of the amino acids GABA and glutamate were realized as well as improved lipophilicity. The pHP triplet undergoes a photo-Favorskii rearrangement with concomitant release of the amino acid substrate. Deprotonation competes with the rearrangement from the triplet excited state and yields the pHP conjugate base that, upon reprotonation, regenerate the starting ketoester, a chemically unproductive or “energy wasting” process. Employing picosecond pump–probe spectroscopy, GABA derivatives 2 – 5 are characterized by short triplet lifetimes, a manifestation of their rapid release of GABA. The bioavailability of released GABA at the GABAA receptor improved when the release took place from m-OCF3 (2) but decreased for m-CF3 (3) when compared with the parent pHP derivative. These studies demonstrate that pKa and lipophilicity exert significant but sometimes opposing influences on the photochemistry and biological activity of pHP phototriggers.

Using model (R)-2-acetyl-2-phenyl acetate esters of (S)- or (R)-α-substituted-p-hydroxybutyrophenones (S,R)-12a and (R,R)-12b, we have shown that a highly efficient photo-Favorskii rearrangement proceeds through a series of intermediates to form racemic rearrangement products. The stereogenic methine on the photoproduct, rac-2-(p-hydroxyphenyl)propanoic acid (rac-9), is formed by closure of a phenoxy-allyloxy intermediate 17 collapsing to a cyclopropanone, the “Favorskii” intermediate 18. These results quantify the intermediacy of a racemized triplet biradical 316 on the major rearrangement pathway elusively to the intermediate 18. Thus, intersystem crossing from the triplet biradical surface to the ground state generates a planar zwitterion prior to formation of a Favorskii cyclopropanone that retains no memory of its stereochemical origin. These results parallel the mechanism of Dewar and Bordwell for the ground state formation of cyclopropanone 3 that proceed through an oxyallyl zwitterionic intermediate. The results are not consistent with the stereospecific SN2 ground state Favorskii mechanism observed by Stork, House, and Bernetti. Interconversion of the diastereomeric starting esters of (S,R)-12a and (R,R)-12b during photolysis did not occur thus ruling out leaving group return prior to rearrangement.

To further explore the nature of the photo-Favorskii rearrangement and its commitment to substrate photorelease from p-hydroxyphenacyl (pHP), an array of ten new fluorinated pHP γ-aminobutyric acid (GABA) derivatives was synthesized and photolyzed. The effects of fluorine substitution on the chromophore and the photophysical and photochemical properties of these new chromophores were shown to be derived primarily from the changes in the ground state pK a of the phenolic groups. The quantum yields and rate constants for release are clustered around Φ dis = 0.20 ± 0.05 and k r = 8 ± 7 × 10 7 s −1 (H 2 O), respectively. The triplet lifetimes of the pHP GABA derivatives were concentrated in the range of 0.4-6.0 ns (H 2 O). The corresponding deprotonated conjugate bases displayed reduced efficiencies by 50% or more (one exception) and exhibited a weak fluorescence in pH 8.2 buffer. Pump-probe spectroscopy studies have further defined the rates of intersystem crossing and the lifetimes of the reactive triplet state of the fluoro pHP chromophore.

Gold surface attachment of altitudinal molecular rotors provided with ten -HgSCH 2 CH 2 SCH 3 "tentacles" has been monitored with ellipsometry, scanning tunneling microscopy, and X-ray photoelectron spectroscopy (XPS). The rotors appear to adsorb on the gold surface in the intended orientation, with rotor axle parallel to the surface, without any inclination for multilayer growth. According to XPS data, the sulfur-containing tentacles start to be detectably oxidized within hours of exposure to air and can be ultimately removed by washing. The rotor molecules nevertheless remain firmly attached in the desired orientation, apparently due to a direct interaction of their Hg atoms with the gold surface. When the tentacles are simplified to -HgOCOCF 3 substituents, the molecules adhere to the surface as well, but not always in the desired orientation.