The bacterial flagellum is a rotary motor that propels motile bacteria through their surroundings via swimming motility, or on surfaces via swarming motility. The flagellum is a key virulence factor for motile pathogenic bacteria. Viruses that infect bacteria via this appendage are known as flagellotropic or flagellum-dependent bacteriophages. Much like other phages, flagellotropic phages are of interest for clinical applications as antibacterial agents, particularly against multidrug resistant (MDR) bacteria. Bacteriophage χ is a flagellotropic phage that infects multiple species of motile pathogens. In the projects described below, we characterized several aspects of the complex interactions between χ and two of its hosts: Salmonella enterica and Serratia marcescens. In Chapter I, we describe in detail the existing knowledge on flagellum-dependent bacteriophages, pathogenic bacteria, and the flagellar motility system. We also expand significantly on flagellotropic phage χ. In Chapter II, we describe our discovery of S. enterica cellular components other than motility that are crucial for bacteriophage χ infection, making the key discovery that the AcrABZ-TolC multi-drug efflux system is required for infection to proceed. We additionally found that the host molecular chaperone trigger factor is important for the χ phage lifecycle. In Chapter III, we outline our characterization of the initial binding interaction between χ and the flagellum, determining that of flagellin's seven domains, C-terminal domain D2 is the most important for χ adsorption. In Chapter IV, we expand on this by discussing our work that determined that the χ tail fiber protein is encoded by the gene CHI_31, purification of this recombinantly-expressed protein, and demonstration of its direct interaction with the flagellar filament. Lastly, in Chapter V, our findings indicate that S. marcescens is able to detect χ infection and lysis in the surroundings and alter gene expression, resulting in an increase in the production of the red pigment prodigiosin. Overall, our hypothetical model for χ infection is as follows: χ binds to the flagellum of its host using its single tail fiber, composed of monomers of the CHI_31 gene product gp31. This tail fiber interacts with CTD2 of flagellin, and the rotation of the flagellum brings the phage to the cell surface, where it interacts with AcrABZ-TolC to inject its genetic material into the host cytoplasm. At some point during the process of production of phage particles and subsequent cell lysis, the host molecular chaperone trigger factor likely assists with proper folding of χ proteins. After cell lysis, cells in the surroundings are capable of detecting lysis and responding accordingly, at least in the case of S. marcescens. This research is clinically relevant for a number of reasons. Phage therapy, the use of bacteriophages as antibacterial agents, requires knowledge of phage infection pathways for optimal implementation. The fact that the flagellum and a complex mediating MDR are both essential for χ infection leads to particular interest in χ for this application. Knowledge of the host-determining factors between χ and Salmonella may lead to the ability to alter the χ phage genome to target specific pathogenic Salmonella or Escherichia coli strains while avoiding disruption of beneficial bacterial communities. / Doctor of Philosophy / Bacteriophages (phages) are viruses that only infect bacteria. They do not harm animal cells or the human body, despite being highly effective predators of bacteria. As such, they have applications in the medical field as antibacterial agents, similar to antibiotics. Phages that infect pathogenic bacteria like Salmonella are of particular interest for scientific research. Bacteriophage χ (Chi) infects bacteria by binding to their flagella, propeller-like appendages that a bacterial cell uses to swim through its surroundings. In many bacterial species, flagella and the ability to swim are closely involved in human infection. Due to this, flagellotropic (flagellum-dependent) phages like χ may be particularly useful as antibiotics. Throughout this project, we characterized the χ phage infection process, including exploring how it attaches to flagella, interactions it has on the surface of and inside Salmonella cells, and the largely unexplored relationship with Serratia marcescens, another bacterial species that causes illness in humans and is highly antibiotic resistant. Overall, our research contributes to the medical field, and indicates that χ may serve as a highly effective antibacterial treatment.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/118603 |
| Date | 15 April 2024 |
| Creators | Esteves, Nathaniel Carlos |
| Contributors | Biological Sciences, Scharf, Birgit, Hsu, Bryan, Caswell, Clayton Christopher, Jutras, Brandon L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International, http://creativecommons.org/licenses/by-nc-sa/4.0/ |
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