Single-cell deconvolution of host-pathogen interactions

Microbes exhibit remarkable diversity in form and function. Some have evolved complicated developmental programs to infect and persist inside host cells, causing widespread and devastating human disease. On the other hand, host cells have evolved many mechanisms to limit the persistence of intracellular microbes. To understand the origin of intracellular microbial defenses, we apply single-cell and computational approaches to explore the cellular diversity underlying parasite development and host immune functions. In addition, we explore the effect of temperature adaptation by characterizing the activity and fidelity of DNA polymerases identified from a wide range of extremophilic microbes. In our work on an ubiquitous apicomplexan parasite, Toxoplasma gondii, we constructed the transcriptional landscape of its asexual development with scRNA- seq analysis. We discovered a striking pattern in its expression of surface antigens that may contribute to host immunity evasion. Based on our scRNA-seq analysis, we show that a novel transcription factor AP2-IX1 can trigger antigenic switch when transiently over-expressed. To better understand the heterogeneous nature of infection response, we also present efforts to characterize individual host cells that are infected by Toxoplasma at different developmental stages. We highlight some of the host transcriptional differences in reponse to tachyzoite and bradyzoite infection, as well as some of the technical challenges that we encountered in single-cell co- transcriptomic analysis. In a second project, we improved single-cell analysis by developing a novel soft feature learning algorithm called Self-Assembling Manifolds (SAM). We demonstrate that SAM outperforms state-of-the-art methods in discerning subtle transcriptional differences between cells based on extensive computational benchmark and experimental validation. In a third project, we explore the functional diversity of macrophages during persistent infection with intracellular bacteria. By leveraging scRNA-seq and a genetic mutant of Salmonella enterica Typhimurium, we resolved a vast continuum of macrophage states in the spleen during persistent bacterial infection of mice. We identified compositional variations of immune populations and macrophage states that correlated with the host bacterial burden. Furthermore, we demonstrate that angiotensin converting enzyme (Ace) expression defines a novel granuloma macrophage state that is non- permissive for intracellular bacteria, which suggests modulation of Ace+ macrophages may present a new therapeutic approach to control intracellular bacterial persistence. In a fourth project, we explore the temperature dependence of activity and fidelity in DNA polymerases that are adapted to extreme temperatures. We developed a high- throughput sequencing-based assay to measure polymerase replication errors with single-molecule resolution and comprehensively characterized the error spectrum for several DNA polymerases of distinct evolutionary origins. Furthermore, we recombinantly purified a novel psychrophilic DNA polymerase, PIPI, that can replicate DNA below the freezing temperature of water at down to -19◦C. Together, we show that sequencing analysis and single-cell measurement can unveil remarkable insights into enzyme fidelity, microbial development, and host immune functions. In particular, we reveal that cellular identity in both host cells and pathogens is much more heterogeneous than previously appreciated. Our results can inform novel ways to combat infectious diseases

Keywords: Bioengineering.
Advisor: Boothroyd John C.
Quake Stephen Ronald
Wang Bo (Researcher in bioengineering)