High throughput identification of bacteriophages and their hosts with single-cell transcriptomics

The National Institute of Allergy and Infectious Diseases awarded The Washington University $179,114 on November 1, 2025, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to develop a high-throughput tool for identifying bacteriophage-host pairs using single-cell RNA sequencing in complex bacterial communities.

The project will adapt and implement single-cell RNA-sequencing (scRNA-seq) protocols to screen complex communities for individual bacterial cells carrying phage-derived RNA transcripts, establishing a method to link bacteriophages with their bacterial hosts. Current phage-host pairing tools lack the throughput and efficiency needed to rapidly screen thousands of bacterial cells in naturally occurring communities such as the human enteric microbiome, preventing mechanistic investigation of connections between phages, bacteria, and human disease. The work will proceed in multiple aims, beginning with adaptation of published scRNA-seq protocols for phage-infected, multi-species bacterial mixtures, an application not previously demonstrated in the literature.

Performance occurs in St. Louis, Missouri through December 31, 2027. This is a project grant, an assistance type supporting research by named principal investigators at eligible public and private nonprofit institutions.

Abstract

Bacteriophages (PHAGES) are bacterial viruses that can influence human health by modulating bacterial populations. As such, PHAGES have been associated with a number of diseases linked to bacterial dysbioses like inflammatory bowel disease. The number of known DNA and RNA PHAGES has exponentially increased by more than 10,000-fold over the past 10 years due to rapid advances in metagenomics. However, since this culture- independent method does not preserve phage-host relationships, the hosts of many PHAGES are unknown. This prevents experimental investigation of the mechanistic connections between PHAGES, bacteria, and human disease. A number of phage-host pairing tools are available but lack the throughput and efficiency to rapidly screen thousands of bacterial cells in complex communities like the human enteric microbiome. The overall goal of this proposal is to define phage-host pairs in the context of complex communities. I will achieve this goal by establishing an innovative, high throughput phage-host pairing tool for complex, naturally occurring communities. Specifically, I will use an innovative strategy based on prosingle-cell (SC) RNA-sequencing (SCRNA-SEQ) to rapidly screen complex communities for individual cells carrying phage-derived RNA transcripts, which are generated by DNA and RNA PHAGES. However, current prokaryotic SCRNA-SEQ protocols are not designed for profiling phage infections or multi-species scenarios. My preliminary data demonstrate that I can successfully recover the transcriptomes of noninfected and phage-infected bacterial species in a 10-species mixture using a SCRNA-SEQ protocol originally intended for noninfected monocultures, which has not been previously shown in the literature. Therefore, to establish and implement a SCRNA-SEQ-based, phage-host pairing tools for complex communities, I will first (aim 1) adapt a published prokaryotic SCRNA-SEQ protocol to identify RNA and DNA phage-infected cells in a synthetic community of bacteria representative of the human enteric microbiome to demonstrate proof of concept. (aim 2) I will then apply my optimized SCRNA-SEQ protocol to human stool samples to identify phage- host pairs. I will use multiple approaches to validate a subset of phage-host pairs (aim 3) identified in aim 2, including fluorescent in situ hybridization combined with whole genome sequencing and establishment of culture models. Collectively, this work will be instrumental in advancing our ability to functionally study phage-host interactions in the context of human health and disease.