Infectious diseases develop when the host immune system fails to control and eradicate pathogens from tissues. Mammalian hosts use several cell types and hundreds of molecular factors to detect and eradicate pathogens, and to develop adaptive immunity to prevent re-infection. On the other hand, pathogens use dozens to hundreds of virulence factors to escape detection, interfere with host cell signaling, resist against host antimicrobial effector mechanisms, and/or induce death of attacking host cells. The interplay between these host and pathogen activities determine disease outcome.

Host-pathogen interactions occur in tissues with a large diversity of microenvironments such as inflammatory lesions, necrotic areas, or not yet inflamed regions. Some of these sites may play distinct roles in disease progression and control, but most cellular and molecular studies rely on bulk average measurements or cell culture experiments that disregard this diversity. To overcome this limitation, we have recently developed single-cell approaches to investigate Salmonella-host encounters in a mouse typhoid fever model. Our results show that infected spleen contains distinct regions in which at least nine different types of molecular host-Salmonella interactions occur. Depending on the type of interaction, the encounters have highly divergent outcomes ranging from killing of all local Salmonella to survival and even rapid proliferation of other Salmonella subsets. These divergent interactions explain apparently contradictory previous bulk average data, and resolve highly controversial interpretations of the role of fundamental host effector mechanisms such as generation of reactive oxygen and nitrogen species (ROS/RNS).

Our previous study was restricted to processes in spleen of systemically infected, genetically susceptible mice. In part A of this project, we will extend our investigations to additional tissues and stages that play key roles in salmonellosis. This includes Peyer's patches and mesenteric lymph nodes of orally infected mice, later stages of disease when mice can control some moderately virulent Salmonella strains, and infections of genetically resistant mice. In all these cases, Salmonella colonization kinetics and inflammation dynamics differ markedly from the previously studied conditions, suggesting differences in type and organization of microenvironments, molecular host-Salmonella interactions, and/or a higher proportion of encounters in which the host wins. To test these hypotheses, we will employ our already developed approaches that combine Salmonella and mouse mutants, Salmonella biosensors, flow cytometry, immunohistochemistry, confocal microscopy, and proteomics. The results will enable a comprehensive analysis of in vivo circumstances under which host mechanisms can successfully control/eradicate local Salmonella subsets. On the other hand, they will also reveal the localization and properties of successful Salmonella subsets that drive disease progression.

In part B of this project, we will develop complementary single-cell methods for in situ analysis of additional host antimicrobial effector mechanisms and corresponding Salmonella stress exposure and defense. We will primarily use these new methods to identify ROS/RNS-independent killing mechanisms in macrophages. Our previous research has indicated that such mechanisms play a major role for Salmonella control by resident macrophages in infected spleen, but it is unclear which of the many potential mechanisms act alone or in combination to overcome the versatile and partially redundant Salmonella stress defenses.

Together, this project will reveal key host-Salmonella encounters and their underlying cellular and molecular mechanisms at various stages and sites in infected mice. In addition to addressing fundamental questions in infection biology, this research will unravel Salmonella subsets that escape host control and drive disease progression. Our techniques for Salmonella ex vivo purification and in-depth proteome analysis might identify opportunities to specifically target these crucial subsets. On the other hand, we will learn how the host can successfully kill major subsets of this successful pathogen. Together, these results might provide a basis for development of urgently needed novel antimicrobials.