| The type III secretion system (T3SS) is a powerful virulence-associated machine used by bacteria to deliver specific proteins, termed effectors, from the cytosol of a bacterial cell directly into a eukaryotic host cell. Several Gram-negative bacteria express these virulence systems, including the intestinal pathogen, Salmonella enterica serovar Typhimurium. In addition to mediating virulence, the T3SS has also been engineered to deliver vaccine antigens. These T3SS delivered antigens are targeted to the class I antigen presentation pathway and have been shown to elicit antigen-specific CD8+ T cells. Furthermore, antigens delivered through the T3SS have been shown to confer protection to subsequent infectious challenge or even lead to tumor regression when used as a therapeutic vaccine. One limitation of using this powerful antigen delivery machine is that it requires a live bacterial vector. Due to some toxicity issues associate with live, virulence-attenuated vaccine vectors, the use of these vaccines in certain populations such as the immunocompromised, elderly, or young children is limited. Given the complexity of the T3SS and the energy requirements for its proper function, the assembly of a fully functioning T3SS in an alternate synthetic platform is presently not possible. Moreover, a synthetic platform would lack many of the potent innate immune stimulators found in a bacterial cell. Therefore, to address these limitations, I have developed a functional T3SS in an achromosomal, non-replicative vehicle, the bacterial minicell. Minicells are achromosomal daughter cells which form the poles of bacterial rods during asymmetric cell division. Minicells retain several of the potent innate immune stimulators in their envelope yet are not capable of replication and therefore are less toxic than live, virulence attenuated bacterial vectors. In the following study, I have demonstrated that a functional T3SS partitions to minicells generated from Salmonella enterica serovar Typhimurium. Furthermore, I demonstrated that minicells are capable of T3SS-dependent protein translocation and delivery of antigens to the class I antigen presentation pathway both in vitro and in an animal model. |
