Characterizing Cooperative and competitive interactions involving Streptococcus intermedius

Abstract

The Streptococcus Anginosus/Milleri group (SMG) colonize mucosal surfaces in humans but are also associated with numerous respiratory and invasive infections. These infections are often polymicrobial in nature, with obligate anaerobes often being isolated. The group consists of three species, S. anginosus, S. constellatus and S. intermedius. SMG are considered to be lactic acid bacteria, producing acids such as lactate, formate and acetate as byproducts of their metabolism. Their genomes have been recently sequenced but little is known about their metabolism. Understanding the basis of their metabolism is beneficial in determining optimal growth conditions and mechanisms associated with their pathogenicity. The isolation of obligate anaerobes from SMG polymicrobial infections suggests that they have anoxic microenvironments. There is also some evidence for synergy between SMG species and anaerobes. While cooperation might be occurring with certain anaerobes, streptococci also produce inhibitors such as hydrogen peroxide and short peptides called bacteriocins. These give streptococci a competitive advantage in polymicrobial commensal communities such as the oral cavity. The Streptococcus invasion locus controls bacteriocin production in Group A streptococci and has been identified in SMG species as well. It is unknown if SMG have mechanisms to compete with closely related streptococci. The goal of my thesis is to characterize the cooperative and competitive interactions of S. intermedius with other species. In chapter 2, we characterized the in vitro metabolism of S. intermedius under aerobic (5% CO2) and anaerobic conditions. Using a transcriptomic and metabolomic approach, we mapped the pathways involved in S. intermedius B196 metabolism. We found that there was a minimal upregulation of core pathways including carbohydrate metabolism under anaerobic conditions. Under aerobic conditions, oxidative stress genes were induced. An increased growth rate was also observed anaerobically. In chapter 3, I demonstrated that Streptococcus strains, including S. intermedius, can deplete oxygen and create an anaerobic environment. Certain strains could support the viability of the obligate anaerobe Prevotella melaninogenica in broth cultures under hypoxic conditions, while others inhibited Prevotella by producing hydrogen peroxide. S. intermedius B196 has an alkylhydroperoxidase system (ahpCF), which is thought to endogenously detoxify peroxides. An S. intermedius ahpCF mutant produced hydrogen peroxide and inhibited P. melaninogenica in coculture. Complementation in S. intermedius restored P. melaninogenica viability in coculture. I demonstrated that the ahpCF peroxide detoxification system directly protects S. intermedius from peroxides and indirectly affects a polymicrobial community. In chapter 4, we used a subcutaneous abscess model in BALB/c mice to demonstrate that S. intermedius promotes P. melaninogenica survival during co-infection in comparison to a P. melaninogenica mono-infection. S. intermedius induced abscesses appeared to induce apoptosis, necrosis and NETosis in neutrophils that infiltrated the site of infection. Our results demonstrate the complexity of SMG infections. In chapter 5, I demonstrated that S. intermedius B196 produces inhibitors of other SMG in response to stimulation with the pheromone peptide SilCR. This is the first case of S. intermedius inhibiting a closely related SMG strain. A bioinformatic analysis was done on the sil system in SMG. The system is associated with a genetically heterogeneous bacteriocin cluster which can carry any combination of sixteen putative open reading frames, six of which are putative bacteriocins. Together, my thesis outlines that S. intermedius has specific mechanisms of cooperation and competition. These allow it to cooperate with obligate anaerobes such as P. melaninogenica and inhibit other SMG species. Oxygen depletion, hydrogen peroxide production and bacteriocin production are only three factors addressed in this thesis. However, there are many factors involved in shaping a polymicrobial environment with SMG species. More research in SMG polymicrobial interactions is required to fully understand SMG pathogenicity.