Electrophysiological Analysis and Characterization of Two Microbial Channels: The PapC Usher of Upec and the NSP4 Viroporin of Rotavirus
Pham, Thieng Thanh 1987-
MetadataShow full item record
Microbes, such as bacteria and viruses, utilize channel proteins that play an important role in the infection process, and thus are considered virulence factors. With the rise in antibiotic resistance and despite the existence of vaccines, there is a need for alternative drug therapies, and these virulence factors can be used as new drug targets. In this work, we examined the molecular properties of a bacterial pore-forming protein (PapC) and a viral ion channel (NSP4) using electrophysiological techniques. The PapC usher is produced by uropathogenic E. coli, the causative agent of urinary tract infections, and catalyzes the assembly and surface expression of P pili used to mediate host cell attachment. At rest, the PapC channel is closed by a plug domain that is displaced to allow the passage of the growing pilus. We showed that key residues involved in allosteric and electrostatic networks within the channel govern gating of the usher pore. In particular, a region localized to the periplasmic base of PapC appears to stabilize the usher in a closed configuration. In addition, we showed that the complex of the first pilus subunit associated with its chaperone activates the usher, and partially engages within the channel. Altogether, our work has provided mechanistic insight into usher gating and a basis for alternative drug design. The second part of our work is centered on the non-structural protein 4 (NSP4) of rotavirus. The expression of NSP4 alone was previously shown to increase cytoplasmic calcium, by releasing calcium stores from the endoplasmic reticulum, and thus NSP4 has been proposed to be a calcium channel. To test this hypothesis, we investigated the electrophysiological properties of the putative, channel-forming viroporin domain (VPD) of NSP4. We demonstrated that NSP4 VPD is indeed a bona fide ion channel, capable of conducting potassium, calcium and barium ions. This property sets it apart from most other viroporins studied to date. The channel is not strictly selective to calcium ions, but appears sensitive to inhibition by classical calcium channel inhibitors, such as cadmium and nickel ions. Altogether, our work established the foundations for future characterization of NSP4 properties using electrophysiology.