Functional characterization of novel phage satellites in mycobacteria

The National Institute of General Medical Sciences awarded the College of William and Mary $571,875 on September 5, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to conduct functional characterization of novel phage satellites in mycobacteria.

The research will advance fundamental understanding of mycobacterium phage satellite biology—mobile genetic elements that parasitize helper phages to complete their life cycles. The college's laboratory discovered extracellular prophage-inducing particles (EPIPs), the first phage satellites identified in the mycobacterial genus, which consistently trigger excision and activation of the helper prophage HerbertWM from Mycobacterium aichiense. The project will quantify temporal gene expression dynamics of the host, helper phage, and satellite during infection through RNA sequencing and mass spectrometry; determine satellite gene functions through overexpression and targeted knockouts; and characterize host range and prevalence in natural environments. Work will be performed in Virginia and runs through August 31, 2029. This is a Project Grant, the assistance type NIGMS uses for investigator-initiated basic research spanning approximately nine months of review and award decision time.

Abstract

Phage satellites are mobile genetic elements that depend on helper phage for replication and assembly. They exist as plasmids, integrated chromosomal elements, or independent infectious particles. Lacking essential genes, they parasitize helper phage to complete their life cycles, creating dynamic interactions among host bacteria, helper phages, and satellites. Phage satellites can benefit their bacterial hosts by conferring immunity to non-helper phages, enhancing virulence, and disrupting helper phage reproduction. Once overlooked as degraded phage, phage satellites are now recognized as major drivers of virus-virus interactions, horizontal gene transfer, microbial evolution, and bacterial population dynamics. They also offer promising applications in biotechnology and phage therapy, however the vast majority of phage satellites remain unexplored. Only a single instance of a phage satellite has been identified in the entire actinobacteria phylum, and none have been reported in mycobacteria until the discovery of extracellular prophage-inducing particles (epips) by our laboratory-the first phage satellites discovered to infect the mycobacterium genus. Epips consistently trigger excision and activation of the helper prophage herbertwm from mycobacterium aichiense, establishing a novel satellite-helper system and the first reported in the mycobacterial genus. The overarching goal of this proposal is to advance fundamental understanding of mycobacterium phage satellite biology by delineating patterns of gene expression during infection, determining satellite gene functions through overexpression and targeted knockouts, and characterizing their host range and prevalence in natural environments through the following specific aims. Aim 1 will quantify temporal gene expression dynamics of the host, helper phage, and satellite during infection by performing RNA sequencing and mass spectrometry-based proteomics at multiple time points following infection with the satellite. Aim 2 will elucidate molecular functions of epip-encoded genes through inducible overexpression, targeted gene knockouts, and integrative structural bioinformatics to predict and test mechanistic roles in replication, excision, and helper activation. Aim 3 will delineate the host range of epips by testing host specificity, cross-species mobilization, and prevalence across mycobacterium and related actinobacteria using infection assays, qpcr-based excision tracking, and computational genome mining with satellite discovery software developed in our laboratory. This project will enhance our understanding of fundamental principles governing satellite-helper interactions in actinobacteria and molecular determinants of viral parasitism. The resulting insights will expand mechanistic understanding of phage regulation, inform evolutionary models of horizontal gene transfer, and provide a foundation for future applications in biotechnology, controlling bacterial infections, and phage therapy.