Identifying virulence factors that enable Salmonella Typhi replication in human macrophages

Bacterial pathogens use a variety of mechanisms to promote their own replication within a single host and dissemination to new hosts. Salmonella enterica serovar Typhi (S. Typhi) is a bacterial pathogen that causes typhoid fever in humans and is not known to cause disease in any other host. Because this pathogen is human-restricted, the mechanisms underlying S. Typhi replication in humans specifically have been indirectly inferred by studying proxy animal models. Although models such as S. Typhimurium infections in mice have yielded valuable insights into typhoid fever pathogenesis, directly studying S. Typhi is required to uncover typhoidal-specific mechanisms of pathogenesis. The focus of this dissertation is to identify virulence factors that S. Typhi requires to replicate inside of human cells. Chapter 1 highlights the current understanding of S. Typhi pathogenesis, from the few S. Typhi studies available and insights gleaned from S. Typhimurium studies. The evolution and genetics of typhoidal Salmonella serovars is reviewed. The two Type III Secretion Systems (T3SS), which injects bacterial proteins into host cells, and known T3SS-dependent effectors present in the S. Typhi genome are reviewed. In Chapter 2, I investigate the roles of S. Typhi's two T3SSs during human macrophage infection and demonstrate that S. Typhi uses two T3SSs to replicate in human macrophages. Further, I demonstrate that both T3SSs contribute to virulence in a humanized mouse model of systemic typhoid fever. In Chapter 3, I identify which of S. Typhi's known T3SS effectors contribute to S. Typhi replication in human macrophages. I accomplish this by creating a collection of single effector knock-out strains of S. Typhi then performing a time-lapse microscopy screen of this strain collection to assess intracellular replication in THP-1 macrophages over time. This targeted screen of T3SS effectors reveals that PipB2 and SifA are required for S. Typhi replication in THP-1 macrophages. This study is the first demonstration of PipB2 being required for Salmonella intracellular replication to the best of my knowledge. PipB2 is known to be translocated through both T3SS-1 and -2 in S. Typhimurium, but SifA is only known to be translocated through T3SS-2. Given that both T3SSs are required for replication, and that SifA has a replication defect that is more severe and appears earlier than a T3SS-2 knockout, I then assess whether S. Typhi SifA translocation depends on T3SS-1 at 8 hours post-infection (h.p.i.), a time point in infection where a T3SS-2-null S. Typhi mutant does not have a significant intracellular replication defect. While previous studies have demonstrated that SifA is required for S. Typhimurium intracellular replication, this is the first study to directly demonstrate that S. Typhi also requires this effector, and further that SifA translocation into human macrophages is T3SS-1-dependent in this context. In Chapter 4, I leverage two machine-learning based algorithms to identify novel T3SS substrates from the S. Typhi genome. I identify six putative T3SS substrates that are present in typhoidal Salmonella serovars but absent in the commonly studied serovar S. Typhimurium. Using methods described in chapters 2 and 3, I find that one of these six putative effectors, T_RS14515, termed "TirA", contributes to replication in THP-1 macrophages. Further, I demonstrate that TirA is translocated into THP-1 macrophages as early as 2 h.p.i. and that TirA translocation is dependent on both T3SS-1 and -2. Finally, Chapter 5 discusses the gaps in the field addressed in this dissertation, summarizes the findings presented here, and highlights the significance of these findings towards advancing our understanding of Salmonella enterica serovar Typhi virulence

Keywords: Medicine.
Microbiology and Immunology.
Advisor: Huang Kerwyn Casey
Bhatt Ami (Ami Siddharth)
Boothroyd John C.
Idoyaga Juliana
Monack Denise M.