Dynamic Determinants of Quorum Quenching Mechanism Shared among N-terminal Serine Hydrolases

Due to the alarming global crisis of the growing microbial antibiotic resistance, investigation of alternative strategies to combat this issue has gained considerable momentum in the recent decade. A quorum quenching (QQ) process disrupts bacterial communication through so-called quorum sensing that enables bacteria to sense the cell density in the surrounding environment. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and consequently avoid inducing resistance. Although many enzymes are known to display the QQ activity towards various molecules used for bacterial signaling, the in-depth mechanism of their action is not well understood hampering their possible optimization for such exploitation. In this study, we compare the potential of three members of N-terminal serine hydrolases to degrade N-acyl homoserine lactones--signaling compounds employed by Gram-negative bacteria. Using molecular dynamics simulation of free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics molecular dynamics simulation of their initial catalytic steps, we explored molecular details behind their QQ activities. We observed that all three enzymes were able to degrade bacterial signaling molecules following an analogous reaction mechanism. For the two investigated penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), we confirmed their putative activities experimentally hereby extending the set of known quorum quenching enzymes by these representatives of biotechnologically well-optimized enzymes. Interestingly, we detected enzyme- and substrate-depended differences among the three enzymes caused primarily by the distinct structure and dynamics of acyl-binding cavities. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate exhibited an elevated energy barrier due to a too shallow acyl-binding site incapable of accomodating this molecule in a required configuration. Conversely, unfavorable energetics on both reaction steps were observed for aPGA in complex with both substrates, conditioned primarily by the increased dynamics of the residues gating the entrance to the acyl-binding cavity. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to distinct positioning of Arg297β. These discovered dynamic determinants constitute valuable guidance for further research towards designing robust QQ agents capable of selectively controlling the virulence of resistant bacteria species.

Photoluminescence-Based Bioassay With Cysteamine-Capped TiO2 Nanoparticles for the Selective Recognition of N-Acyl Homoserine Lactones

Currently available diagnostic procedures for infections are laborious and time-consuming, resulting in a substantial financial burden by increasing morbidity, increased costs of hospitalization, and mortality. Therefore, innovative approaches to design diagnostic biomarkers are imperative to assist in the rapid and sensitive diagnosis of microbial infections. Acyl homoserine lactones (AHLs) are ubiquitous bacterial signaling molecules that are found to be significantly upregulated in infected sites. In this pioneering work, we have developed a simple photoluminescence-based assay using cysteamine-capped titanium oxide (TiO2) nanoparticles for AHL detection. The PL intensity variation of the oxygen defect state of TiO2 was used for the biosensing measurements. The bioassays were validated using two well-studied AHL molecules (C4-HSL and 3-oxo-C12 HSL) of an important human pathogen, Pseudomonas aeruginosa. The developed system has a maximum relative response of 98%. Furthermore, the efficacy of the system in simulated host urine using an artificial urine medium showed a linear detection range of 10–160 nM. Also, we confirmed the relative response and specificity of the system in detecting AHLs produced by P. aeruginosa in a temporal manner.

The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions

The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown.

Weaving of bacterial cellulose by the Bcs secretion systems

Cellulose is the most abundant biological compound on Earth and while it is the predominant building constituent of plants, it is also a key extracellular matrix component in many diverse bacterial species. While bacterial cellulose was first described in the 19th century, it was not until this last decade that a string of structural works provided insights into how the cellulose synthase BcsA, assisted by its inner-membrane partner BcsB, senses c-di-GMP to simultaneously polymerize its substrate and extrude the nascent polysaccharide across the inner bacterial membrane. It is now established that bacterial cellulose can be produced by several distinct types of cellulose secretion systems and that in addition to BcsAB, they can feature multiple accessory subunits, often indispensable for polysaccharide production. Importantly, the last years mark significant progress in our understanding not only of cellulose polymerization per se, but also of the bigger picture of bacterial signaling, secretion system assembly, biofilm formation and host tissue colonization, as well as of structural and functional parallels of this dominant biosynthetic process between the bacterial and eukaryotic domains of life. Here we review current mechanistic knowledge on bacterial cellulose secretion with focus on the structure, assembly and cooperativity of Bcs secretion system components.