Banks, Emma Jane
(2022)
Dual modification of predator and prey cell shapes during predation by Bdellovibrio bacteriovorus.
PhD thesis, University of Nottingham.
Abstract
Bdellovibrio bacteriovorus HD100 is a vibrioid-shaped Gram-negative bacterium with an unusual predatory lifestyle. B. bacteriovorus attaches to, invades, and then replicates within Gram-negative prey bacteria including multi-drug resistant bacterial pathogens. Due to an extensive range of Gram-negative prey targets which are also incapable of developing genetic resistance to the invading predator, B. bacteriovorus presents itself as a potential novel antibacterial therapeutic within the worsening global antibiotic resistance crisis.
The predatory lifecycle of B. bacteriovorus involves numerous different effector proteins including peptidoglycan (PG)-modifying enzymes which remodel the PG cell walls of both predators and prey. Following attachment to prey via type IV pili, B. bacteriovorus secretes hydrolytic PG enzymes into the prey periplasm to sculpt an entry ‘porthole’ within the prey wall. The predator cell also secretes DD-endopeptidase (DacB) PG enzymes into the prey periplasm which cut crosslinks in the prey PG, causing it to become more malleable. This results in the transformation of rod-shaped prey cells into spherical prey bdelloplasts. B. bacteriovorus enters its prey through the porthole, reseals it, and then consumes the DNA and protein nutrients of the prey, elongating as a filament within the inner periplasmic compartment. When prey nutrients are exhausted, the predator filament synchronously divides to give variable numbers of daughter cells which lyse the prey cell and escape to seek out new prey.
During my PhD I worked on two projects, characterising new proteins that are involved in the modification of prey cell shape and the generation of vibrioid-shaped B. bacteriovorus predator cells. I discovered that the lytic transglycosylase Bd3285 is naturally secreted into the prey bdelloplast by B. bacteriovorus during predation and, when heterologouslyexpressed in Escherichia coli, localises to the septum of dividing E. coli cells. In the absence of bd3285, prey bdelloplasts form three different shapes: spheres, rods, and ‘dumbbells’, in comparison to wild-type bdelloplasts which are all spherical. Dumbbell-shaped prey bdelloplasts are derived from E. coli prey which were in the process of dividing at the moment of invasion by B. bacteriovorus. Pre-labelling of E. coli prey PG with a fluorescent D-amino acid dye revealed that the dumbbell bdelloplasts observed in the Dbd3285 mutant contained an intact PG septum, indicating that Bd3285 cleaves septal PG. It is probable that Bd3285 cutting of septal PG is required to allow DD-endopeptidase DacB access to PG crosslinks at the mid-cell and facilitate rounding of prey bdelloplasts. Bd3285 is a homologue of the E. coli lytic transglycosylase MltA. There are two other MltA homologues in B. bacteriovorus strain HD100: Bd0599 and Bd0519. During my PhD I characterised Bd0599, showing that although it is also secreted into the prey bdelloplast and can localise to the E. coli septum, it is dispensable for prey shape transformation as all prey bdelloplasts were spherical in shape.
In the second part of my thesis, I followed up my work from my short MRes project in which I identified the cell curvature-determinant of B. bacteriovorus strain HD100. In my PhD, I characterised this shape determinant, Bd1075, further, discovering that it is broadly conserved across Bdellovibrio strains, including a rod-shaped strain: strain 109J. Through cross-complementation experiments and analysis of PG wall composition (with collaborators), I discovered that a truncation of 171 bp within the bd1075109J gene renders strain 109J unable to generate cell curvature. Collaborating with Prof Waldemar Vollmer’s group at Newcastle University, I determined that Bd1075 generates cell curvature by exerting LD-carboxypeptidase activity upon the B. bacteriovorus cell wall. My co-supervisor and collaborator Prof Andrew Lovering and his group solved the crystal structure of Bd1075, revealing novel properties unique to this shape enzyme. I also discovered that Bd1075 specifically localises to the outer convex face of B. bacteriovorus cells and that the protein is targeted by its cryptic nuclear transport factor 2-like domain. Finally, I discovered benefits for cell curvature during B. bacteriovorus predation: faster invasion of curved predators into prey bacteria and potentially more optimal growth of curved predators within spherical prey bdelloplasts.
These two studies revealed important new insights into the generation and remodelling of predator and prey cell shapes during bacterial predation by B. bacteriovorus.
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