Dissecting Individual Interactions between Pathogenic and Commensal Bacteria within a Multispecies Gut Microbial Community.

Interactions of commensal bacteria within the gut microbiota and with invading pathogens are critical in determining the outcome of an infection. While murine studies have been valuable, we lack in vitro models to monitor community responses to pathogens at a single-species level. We have developed a multispecies community of nine representative gut species cultured together as a mixed biofilm and tracked numbers of individual species over time using a quantitative PCR (qPCR)-based approach. Introduction of the major nosocomial gut pathogen, Clostridioides difficile, to this community resulted in increased adhesion of commensals and inhibition of C. difficile multiplication. Interestingly, we observed an increase in individual Bacteroides species accompanying the inhibition of C. difficile Furthermore, Bacteroides dorei reduced C. difficile growth within biofilms, suggesting a role for Bacteroides spp. in prevention of C. difficile colonization. We report here an in vitro tool with excellent applications for investigating bacterial interactions within a complex community.IMPORTANCE Studying interactions between bacterial species that reside in the human gut is crucial for gaining a better insight into how they provide protection from pathogen colonization. In vitro models of multispecies bacterial communities wherein behaviors of single species can be accurately tracked are key to such studies. Here, we have developed a synthetic, trackable, gut microbiota community which reduces growth of the human gut pathogen Clostridioides difficile We report that Bacteroides spp. within this community respond by multiplying in the presence of this pathogen, resulting in reduction of C. difficile growth. Defined in vitro communities that can be tailored to include different species are well suited to functional genomic approaches and are valuable tools for understanding interbacterial interactions.

Probing Clostridium difficile Infection in Complex Human Gut Cellular Models.

Interactions of anaerobic gut bacteria, such as Clostridium difficile, with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins, and bacterial filaments. This system was used to examine C. difficile interactions with the commensal Bacteroides dorei, and interestingly, C. difficile growth is significantly reduced in the presence of B. dorei. Subsequently, we have developed novel models containing a myofibroblast layer, in addition to the epithelium, grown on polycarbonate or three-dimensional (3D) electrospun scaffolds. In these more complex models, C. difficile adheres more efficiently to epithelial cells, as compared to the single epithelial monolayers, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host-anaerobe and pathogen-commensal interaction studies in vitro.

Design and Characterization of Topological Small RNAs.

RNA can self-assemble into complex structures through base pairing, as well as encode information and bind with proteins to induce enzymatic activity. Furthermore, RNA can possess intrinsic enzymatic-like (ribozymatic) activity, a property that, if necessary, can be activated only upon the binding of a small molecule or another RNA (as is the case in aptazymes). As such, RNA could be of use in nanotechnology as a programmable polymer capable of self-assembling into complex topological structures. In this chapter we describe a method for designing advanced topological structures using self-circulating RNA, exemplified by three tiers of topologically manipulated self-assembling synthetic RNA systems. The first tier of topological manipulation, the RNA knot is a physically locked structure, formed by circularizing one monomer of knotted single-stranded RNA left with loose ends (an "open" knot). The second tier, a two interlocking ring system, is made by interlocking two circular RNA components: a circular RNA target, and an RNA lasso designed to intercalate the target before circularizing. The third tier naturally extends this system into a string of topologically locked circular RNA molecules (an RNA chain). We detail the methodology used for designing such topologically complex RNAs, including computational predictions of secondary structure, and where appropriate, RNA-RNA interactions, illustrated by examples. We then describe the experimental methods used for characterizing such structures, and provide sequences of building blocks that can be used for topological manipulation of RNA.