Resolving rare invasive pathogen states with scalable dual single-cell transcriptomics

The National Institute of Allergy and Infectious Diseases awarded the Institute for Systems Biology $235,000 on August 12, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855, Project Grant R21AI202154).

The award funds development of a dual single-cell transcriptomics technology to measure both host and pathogen gene expression programs across hundreds of thousands of individual infected cells, with enhanced bacterial transcript detection to identify and characterize rare cell states that lead to bacterial persistence. The research investigates how Salmonella typhimurium persister cells arise through complex interplay between genetic programs activated in both the host cell and the invasive pathogen, addressing the poorly understood mechanisms by which a small fraction of infected bacteria enter a persistence state that survives antibiotic treatment without genetic mutation. After developing and validating this technology in Salmonella-infected macrophages, the work will employ computational analyses to characterize persistence mechanisms.

Work is performed in Seattle, Washington. The period of performance runs through July 31, 2028.

Abstract

Infection with the intracellular pathogen salmonella typhimurium results in distinct outcomes for the host cells, ranging from the infection clearance to long-term survival. A small fraction of infected cells becomes colonized by bacteria in a state called persistence. Bacterial persister cells arise from a phenotypic shift in gene expression and survive antibiotic treatment without undergoing genetic change such as mutations. The mechanisms leading to persister formation are poorly understood, since only a minor fraction of bacteria become persisters during infection. Therefore, measuring all genes across a population of cells is insufficient to provide insight into persistence formation. The cornerstone of this proposal is the hypothesis that a small number of intracellular bacteria become persisters as a result of a complex interplay between the genetic programs activated in both the host cell and the invasive pathogen. IT is well established that genes are expressed non-uniformly across both human and bacterial cells even in the same cell type and in absence of genomic differences. Each interaction of a human cell with a bacterial cell is then slightly different for each individual encounter, due to the interactions of two variable gene expression programs in the host and the pathogen. Some of these combinations ultimately lead to persistence, while others to alternative fates such as death of the pathogen or the host. Here, i propose to develop a novel technology for measurement of both host and pathogen gene expression programs in hundreds of thousands of individual infected cells. IT will feature enhanced bacterial transcript detection which is crucial for identifying and characterizing rare cell states that lead to persistence. After developing and applying this technology to salmonella-infected macrophages, we will employ computational analyses to characterize the variable genetic programs of the pathogen and the host (aim 1). We will then apply this technology to salmonella- infected macrophages after antibiotic treatment to characterize the systems-level drivers of persistence (aim 2). The resulting technology will be an important tool in our arsenal to combat the worldwide rise of antimicrobial resistance. Crucially, this approach could be implemented to study single-cell infection outcomes of other intracellular pathogens.