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Development of Peptide-Based Probes to Target Quorum Sensing in Streptococcus pneumoniae
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The microbial world existed from the beginning of the evolution process on Earth. Only after its discovery in the 17th century did much of the unknown facts surrounding this bacterial world start to be elucidated. It took three more centuries to discover that these tiny microorganisms can actually communicate with each other. This mechanism of communication is termed quorum sensing (QS). QS has since been shown to play a crucial role in different phenotypic behaviors like competence, virulence, and biofilm formation, and these bacterial behaviors contribute to survival. In the studies composing this dissertation, we structurally evaluate and modulate the QS mechanism in Streptococcus pneumoniae in order to attenuate its pathogenicity. S. pneumoniae is a commensal bacterium that resides in the nasopharynx of humans and can be deadly if it spreads throughout the body. It can cause conditions ranging from mild respiratory disease to life threating diseases like pneumonia, bacteremia and meningitis. The main problem associated with this pathogen is that it has developed resistance to conventional antibiotic drugs, making it critical to develop alternative avenues to fight this deadly pathogen. Targeting QS could be an alternative approach because it doesn’t involve killing the bacteria, rather it is aimed at “disarming” them. QS in S. pneumoniae centers around a 17-mer signaling peptide molecule known as competence stimulating peptide (CSP). The main aim of my Ph.D. work has been to modulate the QS circuit by interfering with the interaction between the signal (CSP) and the receptor (ComD). We have designed several CSP-mimicking peptide analogs aiming to competitively inhibit the signal-receptor interaction. First, we have conducted thorough structure-activity relationship (SAR) studies of the CSP signal and identified several key residues that are critical for receptor binding, activation, and specificity. We further evaluated the secondary structures of CSPs and their analogs using circular dichroism (CD) to correlate between the structure and function of these peptides. The CD analysis results suggest that an α-helix conformation is required for effective binding to the cognate ComD receptor. Moreover, a double mutant analog, CSP2 E1Ad10, was found to effectively inhibit ComD2-mediated pneumolysin release in vitro and to attenuate pherotype-2 pneumococcus infectivity in a mouse model of acute pneumonia, as well as exhibiting high stability against enzymatic degradation, making this analog a lead scaffold for the design of potential therapeutics.