Guillaume Duret

Modulating Effects of the Plug, Helix, and N- and C-terminal Domains on Channel Properties of the PapC Usher

The chaperone/usher system is one of the best characterized pathways for protein secretion and assembly of cell surface appendages in Gram-negative bacteria. In particular, this pathway is used for biogenesis of the P pilus, a key virulence factor used by uropathogenic Escherichia coli to adhere to the host urinary tract. The P pilus individual subunits bound to the periplasmic chaperone PapD are delivered to the outer membrane PapC usher, which serves as an assembly platform for subunit incorporation into the pilus and secretion of the pilus fiber to the cell surface. PapC forms a dimeric, twin pore complex, with each monomer composed of a 24-stranded transmembrane β-barrel channel, an internal plug domain that occludes the channel, and globular N- and C-terminal domains that are located in the periplasm. Here we have used planar lipid bilayer electrophysiology to characterize the pore properties of wild type PapC and domain deletion mutants for the first time. The wild type pore is closed most of the time but displays frequent short-lived transitions to various open states. In comparison, PapC mutants containing deletions of the plug domain, an α-helix that caps the plug domain, or the N- and C-terminal domains form channels with higher open probability but still exhibiting dynamic behavior. Removal of the plug domain results in a channel with extremely large conductance. These observations suggest that the plug gates the usher channel closed and that the periplasmic domains and α-helix function to modulate the gating activity of the PapC twin pore.

Deoxycholic Acid Blocks Vibrio cholerae OmpT but Not OmpU Porin

OmpT and OmpU are general diffusion porins of the human intestinal pathogen Vibrio cholerae. The sole presence of OmpT in the outer membrane sensitizes cells to the bile component deoxycholic acid, and the repression of OmpT in the intestine may play an important role in the adaptation of cells to the host environment. Here we report a novel important functional difference between the two porins, namely the sensitivity to deoxycholic acid. Single channel recordings show that submicellar concentrations of sodium deoxycholate induce time-resolved blocking events of OmpT but are devoid of any effect on OmpU. The effects are dose-, voltage-, and pH-dependent. They are elicited by deoxycholate applied to either side of the membrane, with some asymmetry in the sensitivity. The voltage dependence remains even when deoxycholate is applied symmetrically, indicating that it is intrinsic to the binding site. The pH dependence suggests that the active form is the neutral deoxycholic acid and not the negatively charged species. The results are interpreted as deoxycholic acid acting as an open-channel blocker, which may relate to deoxycholic acid permeation.

Modulation of Vibrio cholerae porin function by acidic pH.

The outer membrane of Gram-negative bacteria contains porins, large pore-forming proteins which allow the traffic of hydrophilic compounds between the external medium and the periplasm. The oral mode of infection of Vibrio cholerae, the agent of cholera, implies that the bacteria must adapt to severe changes in the environment, such as acidic pH and the presence of bile. Because of their localization and the regulation of their expression in response to these external factors, the OmpU and OmpT porins of V. cholerae are thought to be involved in the adaptation of the bacteria to the host environment. Using patch clamp and planar lipid bilayer electrophysiology, we assessed the effect of pH on the channel properties of OmpU and OmpT. OmpT does not show any major modification in its activity between pH 4 and pH 7.2. In the case of OmpU, the effect of acidic pH is manifested by promoting single-step closures, whose duration, frequency and current size increase as pH is lowered, thereby producing a pH-dependent decrease in the channel open probability. Surprisingly, the increase in current size of this single-step closure is not coupled with an increase of the total current through the porin, indicating that the trimeric conductance remains unchanged. This observation suggests that coordinated events take place at the level of the trimer, and various explanations for this peculiar effect of acidic pH on porin gating and conductance are provided.

Size and Dynamics of the Vibrio cholerae Porins OmpU and OmpT Probed by Polymer Exclusion

The trimeric OmpU and OmpT porins form large, triple-barrel hydrophilic channels in the outer membrane of the pathogen Vibrio cholerae. They have distinct pore properties, such as conductance, block by deoxycholic acid, and sensitivity to acidic pH. Their three-dimensional structures are unknown, but they share significant sequence homologies. To gain insight into the molecular basis for the distinct functional properties of these two similar porins, we carried out polymer exclusion experiments using planar lipid bilayer and patch-clamp electrophysiology. By studying the partitioning of polyethylene glycols (PEGs) of different molecular weights into each porin, we determined an effective radius of 0.55 nm and 0.43 nm for OmpU and OmpT respectively, and found an increased OmpU effective radius at acidic pH. PEGs or high buffer ionic strength promotes the appearance of single step closures in OmpU similar to the acidic-pH induced closures we documented previously. In addition, these closing events can be triggered by nonpenetrating PEGs applied asymmetrically. We believe our results support a model whereby acidic pH, high ionic strength, or exposure to PEGs stabilizes a less conductive state that corresponds to the appearance of an additional resistive element on one side of the OmpU protein and common to the three monomers.

The TpsB Translocator HMW1B of Haemophilus influenzae Forms a Large Conductance Channel

The Haemophilus influenzae HMW1 adhesin is secreted via the two-partner secretion pathway and requires HMW1B for translocation across the outer membrane. HMW1B belongs to the Omp85-TpsB superfamily of transporters and consists of two structural domains, a C-terminal transmembrane β-barrel and an N-terminal periplasmic domain. We investigated the electrophysiological properties of the purified full-length HMW1B and the C-terminal domain using planar lipid bilayers. Both the full-length and the truncated proteins formed conductive pores with a low open probability, two well defined conductance states, and other substates. The kinetic patterns of the two conductance states were distinct, with rapid and frequent transitions to the small conductance state and occasional and more prolonged openings to the large conductance state. The channel formed by the full-length HMW1B showed selectivity for cations, which decreased when measured at pH 5.2, suggesting the presence of acidic residues in the pore. The C-terminal domain of HMW1B was less stable and required reconstitution into liposomes prior to insertion in the bilayer. It formed a channel of smaller conductance but a similar gating pattern as the full-length protein, demonstrating the ability of the last 312 C-terminal amino acids to form a pore and suggesting that the periplasmic domain is not involved in occluding the pore, nor in controlling the inherent basal kinetics of the channel. The HMW1 pro-piece containing the secretion domain, although binding to the channel with high affinity, did not induce channel opening.

Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli

TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain.

Diflunisal inhibits prestin by chloride-dependent mechanism

The motor protein prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on prestin. In a screen to identify other inhibitors of prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded prestin activity by whole-cell patch clamping HEK cells transiently expressing prestin and mouse outer hair cells. We monitored the impact of diflunisal on the prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two prestin-associated effects: a chloride independent increase in the surface area and the specific capacitance of the membrane, and a chloride dependent inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.