Cell-Based Assay to Determine Type 3 Secretion System Translocon Assembly in Pseudomonas aeruginosa Using Split Luciferase.

Multi-drug-resistant Pseudomonas aeruginosa poses a serious threat to hospitalized patients. This organism expresses an arsenal of virulence factors that enables it to readily establish infections and disseminate in the host. The Type 3 secretion system (T3SS) and its associated effectors play a crucial role in the pathogenesis of P. aeruginosa, making them attractive targets for the development of novel therapeutic agents. The T3SS translocon, composed of PopD and PopB, is an essential component of the T3SS secretion apparatus. In the properly assembled translocon, the N-terminus of PopD protrudes into the cytoplasm of the target mammalian cell, which can be exploited as a molecular indicator of functional translocon assembly. In this article, we describe a novel whole-cell-based assay that employs the split NanoLuc luciferase detection system to provide a readout for translocon assembly. The assay demonstrates a favorable signal/noise ratio (13.6) and robustness (Z' = 0.67), making it highly suitable for high-throughput screening of small-molecule inhibitors targeting T3SS translocon assembly.

Multiple Stimuli-Responsive Supramolecular Hydrogels Constructed by Decamethylcucurbit[5]uril-para-phenylenediamine Exclusion Complex.

The hydrogels composed of decamethylcucurbit[5]uril (Me10 Q[5]) and para-phenylenediamine (p-PDA) are first reported herein. They are the first Q[5]-based supramolecular hydrogels, the formation of which is driven by portal exclusion between Me10 Q[5] and p-PDA. The composition, structure, and properties of the Me10 Q[5]/p-PDA-based hydrogels are investigated by various techniques. Since the 1D supramolecular chain forms via portal exclusion between Me10 Q[5] and p-PDA is the key to the formation of the hydrogels, any competitive species, such as metal ions, organic molecules, and amino acids, which can affect the portal exclusion, can change the behavior of the Me10 Q[5]/p-PDA-based hydrogels. Hence, the hydrogels can be used for various applications. Importantly, the results may provide a new research direction for the preparation of Q[n]-based hydrogels via portal exclusion of Q[n]s with guests.

Topological analysis of type 3 secretion translocons in native membranes.

PFPs (Pore-forming proteins) perforate cellular membranes to create an aqueous pore and allow the passage of ions and polar molecules. The molecular mechanisms for many of these PFPs have been elucidated by combining high resolution structural information of these proteins with biochemical and biophysical approaches. However, some PFPs do not adopt stable conformations and are difficult to study in vitro. An example of these proteins are the bacterial Type 3 Secretion (T3S) translocators. The translocators are secreted by the bacterium and insert into the target cell membrane to form a translocon pore providing a portal for the passage of T3S toxins into eukaryotic cells. Given the important role that the T3S systems play in pathogenesis, methods to study these translocon pores in cellular membranes are needed. Using a combination of protein modifications and methods to selectively permeate and solubilized eukaryotic membranes, we have established an experimental procedure to analyze the topology of the Pseudomonas aeruginosa T3S translocon using P. aeruginosa strain variants and HeLa cell lines.