Combination Therapy Strategy of Quorum Quenching Enzyme and Quorum Sensing Inhibitor in Suppressing Multiple Quorum Sensing Pathways of P. <i>aeruginosa</i>

Abstract The threat of antibiotic resistant bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Many bacteria employ quorum sensing (QS) to govern the production of virulence factors and formation of drug-resistant biofilms. Targeting the mechanism of QS has proven to be a functional alternative to conventional antibiotic control of infections. However, the presence of multiple QS systems in individual bacterial species poses a challenge to this approach. Quorum sensing inhibitors (QSI) and quorum quenching enzymes (QQE) have been both investigated for their QS interfering capabilities. Here, we first simulated the combination effect of QQE and QSI in blocking bacterial QS. The effect was next validated by experiments using AiiA as QQE and G1 as QSI on Pseudomonas aeruginosa LasR/I and RhlR/I QS circuits. Combination of QQE and QSI almost completely blocked the P. aeruginosa las and rhl QS systems. Our findings provide a potential chemical biology application strategy for bacterial QS disruption

Formation and breakdown of death domain oligomers

Innate immune signaling involves formation of large signaling complexes through death domain homotypic interactions, allowing rapid signal amplification to facilitate inflammatory signaling. The formation and breakdown of these immune complexes allow modulation of innate immune responses. Elucidating the structures of death domain filaments is an important step in understanding the signaling mechanisms of these pathways. Conversely, the hijacking of innate immune proteins by pathogens to carry out immune evasion is also well studied. However, there is little known information on direct degradation of death domain complexes by pathogens. In this thesis, the formation and breakdown of two innate immune adaptor filaments were studied. Firstly, the structure of the CARD domain filament of apoptosis- associated speck-like protein containing a CARD (ASC) was investigated. This was achieved by reconstituting ASC CARD filament in vitro by refolding bacteria expressed recombinant ASC CARD monomers. Cryoelectron microscopy was used to image the filaments to obtain micrographs and images were processed using RELION. Helical reconstruction yielded a 4.1 Å resolution structure of ASC CARD filament. Secondly, the bacterial modulation of death domain signaling was investigated. This was done by screening reconstituted myeloid differentiation primary response gene 88 (Myd88) DD filaments against bacterial secretions in vitro. Characterization of bacterial secretions indicated that pseudolysin secreted by Pseudomonas aeruginosa modulates mammalian inflammatory response through degradation of Myd88 DD. The findings of this thesis can contribute to the existing knowledge of both the formation and breakdown of innate immune signalosomes.Doctor of Philosoph

Structural basis of RIP2 activation and signaling

The pathogen recognition receptors NOD1/2 recognize bacterial cell wall components and signal through their downstream adapter kinase RIP2 via a CARD (Caspase activation and recruitment domain) mediated oligomerization process. Here the authors present the cryo-EM structure of the active RIP2-CARD filament and discuss implications for NOD1/2-RIP2 signalling

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a Delta wspF mutant strain (high c-di-GMP levels), and a p(lac)-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo Delta wspF mutant compared to the in vivo p(lac)-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The Delta wspF mutant was less susceptible to host clearance than the p(lac)-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. Delta wspF mutant with a defective T3SS was more susceptible to host clearance than Delta wspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.ISSN:0019-9567ISSN:1098-552

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells—a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes

A three dimensional immersed smoothed finite element method (3D IS-FEM) for fluid–structure interaction problems

A three-dimensional immersed smoothed finite element method (3D IS-FEM) using four-node tetrahedral element is proposed to solve 3D fluid–structure interaction (FSI) problems. The 3D IS-FEM is able to determine accurately the physical deformation of the nonlinear solids placed within the incompressible viscous fluid governed by Navier-Stokes equations. The method employs the semi-implicit characteristic-based split scheme to solve the fluid flows and smoothed finite element methods to calculate the transient dynamics responses of the nonlinear solids based on explicit time integration. To impose the FSI conditions, a novel, effective and sufficiently general technique via simple linear interpolation is presented based on Lagrangian fictitious fluid meshes coinciding with the moving and deforming solid meshes. In the comparisons to the referenced works including experiments, it is clear that the proposed 3D IS-FEM ensures stability of the scheme with the second order spatial convergence property; and the IS-FEM is fairly independent of a wide range of mesh size ratio