Bacteria constantly face threats from bacteriophages, viruses that hijack their cellular machinery for replication. To counter these infections, bacteria have evolved sophisticated immune mechanisms, including the Zorya anti-bacteriophage defense system. This research aims to unravel the molecular foundations of Zorya-mediated immunity, shedding light on how bacterial cells detect, respond to, and neutralize phage infections. This project is carried out within the DFG Priority Programme SPP 2330 - New Concepts in Prokaryotic Virus-host Interactions.
A key focus of this study is enhancing the our understanding of the sensing mechanisms that activate the Zorya system in response to phage invasion as we have propsed it recently Hu, Popp et al. 2024. Nature. This includes investigating how bacterial cells recognize phage infections and trigger the recruitment of Zorya components to infection sites. The research will also examine how these components interfere with phage DNA injection and replication, thereby preventing viral propagation.
Beyond elucidating its immediate molecular functions, this study will explore the evolutionary conservation of the Zorya system across its three known types (I, II, and III). A critical question is whether Zorya acts by directly degrading or immobilizing phage DNA or whether it employs an abortive infection-like strategy, where infected cells undergo dormancy or death to protect the bacterial population. Additionally, the role of the ZorAB complex, particularly the rotary mechanism and conserved cytoplasmic tail of ZorA, will be analyzed to determine their contributions to phage defense.
Another essential aspect of this research is identifying the activation signals that trigger the Zorya system. Specifically, we will investigate how cell envelope damage, such as peptidoglycan disruption caused by phages, serves as a molecular cue for Zorya activation. The study will also compare the functional diversity of Zorya subtypes across Gram-negative and Gram-positive bacteria, assessing their adaptation to different cell envelope architectures.
To achieve these objectives, we will employ advanced microscopy techniques, including TIRF and STED microscopy, alongside genetic approaches such as RNA sequencing, HaloTag/SNAP-tag labeling, single-molecule tracking, and proximity labeling. These tools will provide high-resolution insights into the dynamic localization, interactions, and recruitment patterns of Zorya proteins during phage infection. Additionally, large-scale bacteriophage screening will be conducted to map subtype-specific resistance profiles and further refine our understanding of Zorya's protective mechanisms.
By integrating molecular, genetic, and imaging techniques, this research will provide a comprehensive understanding of the Zorya defense system. These findings will not only advance our knowledge of bacterial immunity but could also inform new strategies for combating phage infections in industrial and clinical settings.