Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense

Abstract

Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.

Fig. 1.. Csx28 enhances Cas13b-mediated immunity against λ-phage by inducing slow-growing phenotype that helps prevent the establishment and maintenance of infection.

(A) Schematic of the Type VI-B2 CRISPR-Cas system from P. buccae (B) Schematic of the phage λ genome in its circular form showing the location of crRNA-1 to −3 target sites. (C) Plasmid schematics for phage interference experiments in which Cas13b and Csx28 are expressed on two separate plasmids. Cas13b-crRNA-X also contains a synthetic CRISPR-Cas array. (D) Efficiency of Plating (EOP) assays measuring λ-phage infection susceptibility of untransformed (untrans.) E. coli, or E. coli the carrying the indicated plasmids. (E-H). Growth curves of E. coli carrying the indicated plasmids, as measured using OD₆₀₀ after the addition of λ-phage at an MOI of 0.2. (I) Efficiency of Center of Infection (ECOI) assays measuring λ-phage infective center formation of E. coli strains carrying the indicated plasmids infected with λ-phage at an MOI of 0.1. (J) Phage growth assays measuring the λ-phage production over time for E. coli strains carrying the indicated plasmids infected with λ-phage at an MOI of 0.1. Data in D-J are shown as mean ± s.e.m for three biological replicates. One-way ANOVA and Dunnett’s multiple comparisons test for data in D and I; Repeated Measures (RM) one-way ANOVA using the Geisser-Greenhouse correction and Dunnett’s multiple comparisons test for data in J, comparing strains with plasmids to the untransformed control. No significance was detected, unless indicated (*p ≤ 0.05).

Fig 2.. Cryo-EM reveals that Csx28 forms an octameric detergent-embedded pore-like structure with a unique protomer interface.

(A) Static light scattering coupled with size-exclusion chromatography analysis of Csx28 heavy fraction. See fig. S5 for full three detector traces of Csx28 and a BSA standard. (B) High resolution (3.65Å) cryo-EM reconstruction of Csx28 (each protomer colored is uniquely) embedded in a DDM micelle, which is displayed as a composite high-resolution cryo-EM map superimposed with an 8 Å low-pass filtered version of the same map to display lower resolution features, such as the DDM micelle and transmembrane helices. (C) Bottom and side views of the atomic model of the Csx28 octamer. The dimensions of the octamer and the diameter at the constriction of the pore are shown. (D) Atomic model of an isolated Csx28 protomer with each helix of the four alpha-helical bundle labeled. (E) Electrostatic surface representations of the bottom and side of Csx28. The red-blue color gradient represents negative to positive electrostatic potential (± 5kT/e). (F) A magnified view of the Csx28 protomer-protomer interface. Amino acids residues of interest are shown as sticks and labeled (G) Efficiency of Plating (EOP) assays measuring the effect of amino acid mutations at the Csx28 protomer-promoter interface on λ-phage infection susceptibility of E. coli strains carrying the indicated plasmids. Data is shown as mean ± s.e.m for three biological replicates. Statistical significance was calculated using one-way ANOVA and Dunnett’s multiple comparisons test, comparing mutant Csx28 strains to wild type Csx28. No significance was detected, unless indicated (*p ≤ 0.05).

Fig. 3.. Csx28 localizes to the inner membrane in E. coli regardless of Cas13b expression or λ-phage infection, and is required for membrane depolarization and a loss of metabolic activity upon Cas13b-sensing of λ-phage infection.

(A) Western blot to detect the localization of Cas13 and Csx28 in cytosolic vs. detergent-soluble and detergent-insoluble fractions obtained from E. coli expressing HA-tagged Cas13b-crRNA1 and/or V5-tagged Csx28. (B) Western blot to detect the localization of Csx28 in inner membrane or outer membrane fractions from E. coli expressing ΔCas13b and V5-tagged Csx28. (C-D) Western blot to detect the localization of Csx28 in inner membrane or outer membrane fractions from E. coli expressing Cas13b-crRNA1 and V5-tagged Csx28 in the absence or presence of λ-phage infection (MOI: 0.1), respectively. In all cases, blots were first probed with either anti-HA or anti-V5 antibodies to detect HA-Cas13 and Csx28-V5, respectively, then probed for DnaK and OmpC as cytosolic and outer membrane fractionation controls, respectively. (E) A schematic detailing the mechanism by which DiBAC₄(3) detects membrane polarization. Δψ: resting membrane potential and pmf: proton motive force. (F) Flow cytometry histograms of a DiBAC₄(3) staining assay measuring membrane depolarization of WT E. coli or E. coli possessing the indicated plasmids over the course of a λ-phage infection (MOI of 1). A Polymyxin B (Poly. B) treated E. coli sample was used as positive control for membrane depolarization. (G) Quantification of the percentage of depolarized cells in (F), determined by calculating the area under the curve of the depolarized cell subpopulation as a percentage of the total population. (H) A schematic detailing the mechanism by which resazurin acts as a readout of cellular respiration. (I) Resazurin assay for untransformed E. coli or E. coli strains carrying the indicated plasmids in the absence or presence of λ-phage infection (MOI of 2). Data is shown as mean ± s.e.m for three biological replicates.