The Streptococcus Anginosus/Milleri Group (SMG) is made up of three closely related but distinct bacterial species: Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. The SMG are recovered from about one-third of healthy, asymptomatic individuals. Despite this, the SMG cause more incidences of invasive streptococcal disease than Group A and Group B Streptococcus combined. Members of this group are somehow able to live a dual lifestyle. Little work has been conducted on the molecular pathogenicity of the SMG and host factors that contribute to host susceptibility to this group have been under-investigated. My research works towards discovering how the host recognizes the SMG as well as what enables the SMG to evade clearance by the immune system. I hypothesize that: 1) recognition of the SMG by toll-like receptor 2 (TLR2) plays a key role in triggering a cytokine response by the innate immune branch (which coordinates the immune response to the SMG), 2) the expression of cytolysins and extracellular polysaccharides by members of the SMG enables evasion of innate immune recognition and cytokine responses. hTLR2 reporter and monocyte-like cell lines as well as human blood samples from healthy donors were used to investigate the host factors that contribute to SMG infection. Five clinical reference SMG strains and a transposon mutant library were used to probe the contributing bacterial factors. It was found that TLR2 activation plays an important role in the cytokine response to the SMG, but there is heterogeneity between strains in their ability to activate TLR2. It was also found that intermedilysin expression by S. intermedius strains enables evasion of recognition; however, different hosts display varying susceptibility to this cytolysin. This study reveals that investigation of both host and microbial factors is essential to build an understanding of the mechanisms of SMG transition from commensalism to pathogenicity. / Thesis / Master of Science (MSc) / The Streptococcus Miller/Anginosus Group (SMG) is a group of bacteria comprised of three species. Members of this group are recovered from roughly one-third of healthy individuals. However, the SMG are also found in samples collected from patients with invasive disease. It is not well understood why some human-SMG relationships are pathogenic and others are not. However, it is likely that the combination of both human and SMG factors determine the nature of the relationship formed between the two. In this study, the human and SMG factors that contribute to infection were investigated. The ways by which human cells recognize members of the SMG and defend themselves from damage was explored. Additionally, SMG factors that potentially contribute to infection were probed to discover their effect on human cells. By investigating both the bacterial and host factors that lead to infection, disease treatments and preventative strategies can be tailored to individual cases.

Primary objective of my MS thesis project is to characterize and develop a B cell ablation model for investigating the pathogenesis of human diseases such as hepatitis and systemic lupus erythematosus (SLE). Conditional and targeted cell ablation is a powerful approach for studying cellular functions in vivo. However, currently available cell ablation models still have some limitations and therefore limit their broader application in biomedical research. For example, two of the most common cell ablation methods currently employed utilize the herpes simplex virus 1 thymidine kinase (HSVtk) or diphtheria toxin (DT) receptor combined with their respective transgenic strategies. The ablation using HSVtk transgenic mice eliminates dividing cells, but does not affect non-dividing cells. In addition, because of its extremely high potency (a single molecule of DT-A, the active cleavage product of DT is sufficient to induce apoptosis), dose dependent responses are difficult to achieve and off-target effects are frequently observed. These facts highlight the unmet need to develop alternative methods of targeted cell ablation, which our model very successfully addresses. Our recently established approach using intermedilysin (ILY)-mediated cell ablation that is specific for human CD59 (hCD59) expressing cells, obviates these problems and provides an excellent and significantly improved alternative approach to the existing cell ablation methodologies. Intermedilysin (ILY), a toxin secreted by Streptococcus intermedius, exclusively binds to the human cell membrane protein CD59 (hCD59) but not to CD59 of any other species. Once bound, ILY rapidly and potently lyses the cells. Using genetic engineering, animal models can be created that express hCD59 in a spatially regulated manner. Administration of ILY will then selectively ablate only those cells in the animals that express hCD59 without any non-specific effect. To expand and facilitate the application of this newly generated model, we recently generated a Cre-inducible floxedSTOP-hCD59 transgenic mouse line (ihCD59), where specific hCD59 expression occurs following Cre-mediated recombination. By crossing ihCD59 mice with specific immune cell (T cells or macrophage) Cre transgenic lines, we obtained double transgenic mice expressing hCD59. ILY administration mediated specific cell ablation in these target cell populations in a dose dependent manner. Based on these results, I wanted to establish a new B cell ablation model for further studying B cell functionality in the pathogenesis of human diseases. CD19-Cre mice expressing the Cre-recombinase in B cell population were crossed with ihCD59 mice to generate the double positive transgenic mice (ihCD59+/-/CD19-Cre+/-). In Aim 1, I have demonstrated that hCD59 is specifically expressed in the B cell populations. In Aim 2, I have documented that 1) ILY has a large pharmacological window, and 2) ILY injection to ihCD59+/-/CD19-Cre+/- mice resulted in a rapid cell ablation of the B cell cells with off-target effect. Further, I have demonstrated that the specific ablation of B cells did not prevent the immune (Con A)-mediated hepatitis. In the future, I will apply this conditional B cell ablation model for investigating the functionality of B cells in the pathogenesis of human disease such as SLE. / Biomedical Sciences