Direct activation of an innate immune system in bacteria by a viral capsid protein

Abstract

Bacteria have evolved sophisticated and diverse immunity mechanisms to protect themselves against a nearly constant onslaught of bacteriophages. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns (PAMPs), many bacterial immune systems that respond to bacteriophage infection require a phage-specific trigger to be activated. However, the identities of such triggers and the mechanistic basis of sensing remain almost completely unknown. Here, we discover and investigate the anti-phage function of a fused toxin-antitoxin (TA) system called CapRelSJ46 that protects E. coli against diverse phages. Through genetic, biochemical, and structural analysis, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both an antitoxin element and a phage-infection sensor. Following infection by certain phages, the newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to then pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for TA-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a Red Queen conflict, revealing a new front in the intense coevolutionary battle being waged by phage and bacteria. With capsid proteins of some eukaryotic viruses known to stimulate innate immune signaling in mammalian hosts, our results now reveal an ancient, deeply conserved facet of immunity.

Accumulation of defense systems in phage resistant strains ofPseudomonas aeruginosa

“…The other 11 defense systems have morphotype-specific activities, targeting one (myophage-specific Druantia Type III and IetAS) or two phage morphotypes. This is in line with the sensing of specific conserved phage proteins, such as the capsid or terminase proteins, to trigger an immune response 26–28 .…”

Discovery of phage determinants that confer sensitivity to bacterial immune systems

“…A second major category of escape mutations involves mutations in phage structural genes (Figure 3, Figure 6). For example, mutations in the phage capsid protein enable various phages to overcome the defense systems SEFIR (Figure 3B, Table S2), Pycsar (Tal et al, 2021), some types of CBASS (Huiting et al, 2022), PifA (Molineux et al, 1989), Lit (Champness and Snyder, 1982), and CapRel SJ46 (Zhang et al, 2022). Most of these systems function via abortive infection, but at least in the case of CBASS, Pycsar and SEFIR, co-expression of the wild-type form of the major capsid protein with the defense system did not activate toxicity of the system (Figure S3A) (Huiting et al, 2022; Tal et al, 2021).…”

“…Although we show that expression of each individual phage protein triggers the respective defense system, our data does not reveal whether this triggering is by direct interactions of the phage protein and the defense system or by an indirect effect. Previous studies have identified defense systems that are activated by direct binding to a phage protein, as in the case of DSR2 (Garb et al, 2021) and CapRel (Zhang et al, 2022). On the other hand multiple defense systems are known to sense the effect of the phage protein and not the protein itself, for example retrons Ec48 and Se72 that sense RecBCD inhibition (Millman et al, 2020; this study), as well as PrrC that is triggered upon sensing the inhibition of a restriction enzyme by phages (Penner et al, 1995).…”

“…Alternatively, the system could be activated by the interaction of the capsid protein with other phage or host factors, as shown for the Lit abortive infection protein which is triggered when the major head protein of phage T4 interacts with the translation elongation factor EF-Tu (Bingham et al, 2000). In other cases, direct interaction of the phage structural protein with the defense system results in activation of abortive infection, as recently demonstrated for the DSR2 (Garb et al, 2021), and CapRel SJ46 (Zhang et al, 2022) systems. We note that it makes biological sense for abortive infection systems to sense phage structural genes, which are typically expressed at late stages of the phage infection cycle and are usually highly expressed.…”

Dual control of lysogeny and phage defense by a phosphorylation-based toxin/antitoxin system

“…The phage adsorption assay we performed as we reported recently with minor modification 53 . Briefly, 2 ml of culture was collected at OD 600 ∼ 1 and mixed with different amounts of phages (10 7 -10 9 PFU/ml) to reach a MOI of 0.01. Then the phage and cell mixtures were cultured at 37°C for 10 min to allow phages to adsorb to the cells, and supernatants were plated to quantify PFUs before and after adsorption.…”