A Pro-Drug Approach for Selective Modulation of AI-2-Mediated Bacterial Cell-to-Cell Communication

The universal quorum sensing autoinducer, AI-2, is utilized by several bacteria. Analogs of AI-2 have the potential to modulate bacterial behavior. Selectively quenching the communication of a few bacteria, in the presence of several others in an ecosystem, using analogs of AI-2 is non-trivial due to the ubiquity of AI-2 processing receptors in many bacteria that co-exist. Herein, we demonstrate that when an AI-2 analog, isobutyl DPD (which has been previously shown to be a quorum sensing, QS, quencher in both Escherichia coli and Salmonella typhimurium) is modified with ester groups, which get hydrolyzed once inside the bacterial cells, only QS in E. coli, but not in S. typhimurium, is inhibited. The origin of this differential QS inhibition could be due to differences in analog permeation of the bacterial membranes or ester hydrolysis rates. Such differences could be utilized to selectively target QS in specific bacteria amongst a consortium of other species that also use AI-2 signaling

Embedding Explicit Metacognitive Instruction into the General Chemistry Curriculum: Equipping Community College Students with 21st Century STEM Competencies

This work describes the development, implementation, and assessment of a curriculum- embedded metacognition instruction model. The Talking About Learning is Key (TALK) module, a 10-week, discussion-based instruction module was initially integrated into a general chemistry course at a community college in Spring 2020. This case study was assessed qualitatively using a thematic analysis to characterize student posts and replies to the Learning Management System discussion board. Course outcomes between Spring 2020 – Fall 2021 were analyzed to determine what effect participation had on course completion and final grade.Finally, the generalizability of the module was investigated using a focused case comparison between implementation in introductory, general, and organic chemistry. Assessment of the module included the characterization of student and instructor perspectives which were collected using semi-structured interviews. Results of the initial case study indicate that participation in the TALK module results in students adapting their metacognitive knowledge and adjusting their study strategies. The aggregated outcome analysis suggests that participation in the module is correlated with higher rates of course completion and improved outcomes. Also, the case comparison suggests that these results are generalizable, with similar thematic patterns resulting from the analysis of introductory, general, and organic chemistry discussion posts and replies. Student perspectives support student buy in, as those interviewed enthusiastically described successfully using skills and concepts that they learned in the module. Student comments also implicate intimidation due to social comparison (with peers viewed as being more skilled) and fear of failure (beyond failing the course) as potential barriers to engagement in the online module. Perspectives from different instructors who implemented the module confirm that both students and instructors saw value in the instruction, and that implementing the module was not cumbersome. The qualitative nature of this work allowed for the simultaneous characterization and assessment of metacognitive awareness and skillfulness. Consistent with the literature, significant numbers of students lacked awareness of critical metacognitive strategies and possessed a fixed view on intelligence. Importantly, students describe benefiting from this instruction and report adjustments in their behaviors in subsequent semesters. This work represents an effective metacognition model implemented in the community college setting and demonstrates that a more comprehensive metacognitive curriculum can be implemented alongside course content instruction. This is especially relevant for the community college community as the module structure allows for a simple yet effective method for delivering supplemental instruction. Future studies will be directed towards increasing student participation and faculty adoption

Structural Variants of AI-2 Analogs to Probe Quorum Sensing in Diverse Bacteria

Bacterial infections which were once easily managed with antibiotics are now reemerging as a serious threat to human health. The difficulty in managing infectious diseases is arising out of bacterial resistance to front line antibiotics. A new paradigm for fighting bacterial infection via the inhibition of quorum sensing has emerged. Quorum sensing is the process by which small diffusible molecules (autoinducers) are used to sense population density and upregulate genes. Notably, genes for virulence production and biofilm formation have been found to be controlled by this process. Thus, quorum sensing, offers an alternative target for the treatment of bacterial infections. One autoinducer which has been identified across many bacterial species is AI-2. The goals of this thesis were to make more hydrolytically stable analogs of AI-2 as potent inhibitors of quorum sensing, as well as, exploring the effects of AI-2 analogs on QS in P. aeruginosa. In this study, the processing of bis ester protected AI-2 analogs was examined. Also, two long chain AI-2 analogs were synthesized and tested for their ability to inhibit QS in P.aeruginosa. It was found that bis protected analogs are processed different across bacterial species. Also, long chain AI-2 analogs were found to be inhibitors of QS in P. aeruginosa, specifically, by inhibiting a LasR receptor which typically responds to a different class of autoinducer

Altering the Communication Networks of Multispecies Microbial Systems Using a Diverse Toolbox of AI-2 Analogues

There have been intensive efforts to find small molecule antagonists for bacterial quorum sensing (QS) mediated by the “universal” QS autoinducer, AI-2. Previous work has shown that linear and branched acyl analogues of AI-2 can selectively modulate AI-2 signaling in bacteria. Additionally, LsrK-dependent phosphorylated analogues have been implicated as the active inhibitory form against AI-2 signaling. We used these observations to synthesize an expanded and diverse array of AI-2 analogues, which included aromatic as well as cyclic C-1-alkyl analogues. Species-specific analogues that disrupted AI-2 signaling in <i>Escherichia coli</i> and <i>Salmonella typhimurium</i> were identified. Similarly, analogues that disrupted QS behaviors in <i>Pseudomonas aeruginosa</i> were found. Moreover, we observed a strong correlation between LsrK-dependent phosphorylation of these acyl analogues and their ability to suppress QS. Significantly, we demonstrate that these analogues can selectively antagonize QS in single bacterial strains in a physiologically relevant polymicrobial culture

Small Molecule Inhibitors of AI-2 Signaling in Bacteria: State-of-the-Art and Future Perspectives for Anti-Quorum Sensing Agents

Bacteria respond to different small molecules that are produced by other neighboring bacteria. These molecules, called autoinducers, are classified as intraspecies (i.e., molecules produced and perceived by the same bacterial species) or interspecies (molecules that are produced and sensed between different bacterial species). AI-2 has been proposed as an interspecies autoinducer and has been shown to regulate different bacterial physiology as well as affect virulence factor production and biofilm formation in some bacteria, including bacteria of clinical relevance. Several groups have embarked on the development of small molecules that could be used to perturb AI-2 signaling in bacteria, with the ultimate goal that these molecules could be used to inhibit bacterial virulence and biofilm formation. Additionally, these molecules have the potential to be used in synthetic biology applications whereby these small molecules are used as inputs to switch on and off AI-2 receptors. In this review, we highlight the state-of-the-art in the development of small molecules that perturb AI-2 signaling in bacteria and offer our perspective on the future development and applications of these classes of molecules