Study of the bacterial communication in Pseudomonas aeruginosa: a systems and synthetic biology perspective.

The major public health concern worldwide is the antibiotic resistance which is making difficult to treat diseases caused by the pathogenic microbiome carrying multiple resistance genes. The increase in frequency of these diseases has led to the new therapeutic approaches as alternative to traditional antibiotics. One of the innovative therapeutic approaches is the examination of bacterial cell communication system, known as Quorum sensing. These systems are laborious to study as they are regulated by a complex network of genes which are multifactorial and interconnected. The genes associated with quorum sensing is also related to toxin production and the formation of extracellular structures i.e., biofilms which is capable to form a physical barrier against antibiotics. Pseudomonas aeruginosa, a Gram-negative bacterium and one of the “superbugs”, is known for evading the effects of multiple drugs due to its innate antibiotic resistance. This serves as a case study in this thesis work.The main objective of this thesis is to study the LasR, RhlI - RhlR systems, of the QS mechanisms present in P.aeruginosa. This is done by creating engineered systems for ease of study. In this we addressed and resolves issues in the Las system by optimizing ribosome binding sites (RBSs). Constructs, including pLasA RFP34, pLasB RFP34, and pLasI RFP34, were designed and transformed into E. coli cells with LasR receptor and ÿD transcription factor. The exploration of pLasR3 promoter and its processing paralleled the study. Co-transformed systems were evaluated with LasR and RhlR receptors in the presence of auto-inductors. Bacterial growth in the presence of 3OC12-HSL revealed nutrient dependence in P. aeruginosa supernatant, impacting E. coli growth. Fluorescence assays indicated suboptimal sensitivity, limiting the system's practicality for screening applications, with pRhlR promoter exhibiting unique behavior. Thorough examination of LasR receptor promoters under varying 3OC12-HSL concentrations revealed distinct expression patterns. In the realm of Synthetic Biology, a groundbreaking approach emerges, aiming to streamline the exploration of intricate systems by untangling the involved gene components. This deliberate reduction of variables serves as a powerful method to dissect individual actors within communication networks. The overarching objective is twofold: firstly, to meticulously identify potential therapeutic targets, and secondly, to engineer systems that facilitate rapid and cost-effective screening of molecules with promising therapeutic effects. This methodology, with its precision and efficiency, becomes especially crucial in the battle against antibiotic resistance, offering a strategic arsenal to confront the challenges posed by resilient bacterial strains.