Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity

Abstract

Bacteria utilize CRISPR-Cas adaptive immune systems for protection from bacteriophages (phages), and some phages produce anti-CRISPR (Acr) proteins that inhibit immune function. Despite thorough mechanistic and structural information for some Acr proteins, how they are deployed and utilized by a phage during infection is unknown. Here, we show that Acr production does not guarantee phage replication when faced with CRISPR-Cas immunity, but instead, infections fail when phage population numbers fall below a critical threshold. Infections succeed only if a sufficient Acr dose is contributed to a single cell by multiple phage genomes. The production of Acr proteins by phage genomes that fail to replicate leave the cell immunosuppressed, which predisposes the cell for successful infection by other phages in the population. This altruistic mechanism for CRISPR-Cas inhibition demonstrates inter-virus cooperation that may also manifest in other host-parasite interactions.

Keywords: CRISPR-Cas immunity; CRISPR-Cas9; Pseudomonas aeruginosa; altruism; anti-CRISPR; bacteriophage; cooperation; host-pathogen interaction; virus.

Figure 1. Anti-CRISPRs are imperfect CRISPR-Cas inhibitors

(A) Efficiency of plaquing (EOP) of 5 related phages bearing distinct acrIF genes (JBD30_acrIF1, MP29_acrIF2, JBD88a_acrIF3, JBD24_acrIF4, LPB1_acrIF7) on Pseudomonas aeruginosa strain PA14. Plaque forming units (PFUs) were quantified on wild-type PA14 with 1–2 natural targeting spacers (WT + pEmpty) or on PA14 overexpressing 1 targeting spacer (WT + pSp1), then normalized to the number of PFUs measured on a non-targeting PA14 derivative (0sp). Data are represented as the mean of 3 biological replicates +/− SD. (B) EOP of isogenic DMS3m_acr phages with acrIF1-7 or acrIE3 in the DMS3m acr locus. EOP was calculated as PFU counts measured on WT PA14 with 1 targeting spacer (1sp) or a laboratory evolved PA14 derivative with 5 targeting spacers (5sp) normalized to PFU counts measured on non-targeting PA14 (0sp). Data are represented as the mean of 3 biological replicates +/− SD. ND, not detectable. (C) Plot of association (k_a) and dissociation (k_d) rates for AcrIF1 (data adapted from Chowdhury et al. 2017) and AcrIF4 binding the PA14 Csy complex. AcrIF1 rate constants: k_a = 5 × 10⁴ (1/Ms), k_d = 2 × 10⁻⁷ (1/s), K_D = 3 × 10⁻¹¹ M. AcrIF4 rate constants: k_a = 1 × 10³ (1/Ms), k_d = 5 × 10⁻⁴ (1/s), K_D = 4 × 10⁻⁷ (M). See Figure S2 for AcrIF4 SPR sensogram.

Figure 2. Anti-CRISPR success requires cooperative infections during lytic growth

(A–F) 12 hour growth curves of P. aeruginosa strain PA14 with 5 targeting spacers (+CRISPR, panels A–C) or no CRISPR-Cas function (ΔCRISPR, D–F) infected with virulent variants of DMS3m_acrIF1, DMS3m_acrIF4, or DMS3m_acrIE3 at multiplicities of infection (MOI) increasing in 10-fold steps from 2×10⁻⁵ to 2×10¹ (rainbow colors) or uninfected (black). Colors correspond to the MOI legend and growth curves. OD_600nm is represented as the mean of 3 biological replicates +/− SD (vertical lines). ND, not detectable. (G–I) Replication of virulent DMS3m_acr phages (acceptor phage) in the presence of 10⁶ PFU (MOI 0.2) hybrid phage (donor) in PA14 with 5 targeting spacers (5sp) expressing the JBD30 C repressor. Phages were harvested after 24 hours of co-culture and DMS3m_acr phage PFUs were quantified on PA14 0sp expressing the JBD30 C repressor. Phage output is represented as the mean of 3 biological replicates +/− SD. ND, not detectable. (J) Schematic of the experimental design in G–I, where a high MOI of non-replicative “donor” phages is used to rescue a low MOI infection of wild-type “acceptor” phages.

Figure 3. Immunosuppression facilitates acquisition of a marked prophage

(A,B) Acquisition of a marked DMS3m_{acrIF4 gp52::gent} or DMS3m_{acrIE3 gp52::gent} prophage by PA14 with 0 spacers (0sp, circles) or 5 targeting spacers (5sp, triangles). This experiment was performed in biological triplicate, and individual replicate values are displayed. LoD, limit of detection. (C,D) Efficiency of lysogeny (EOL) of DMS3m_{acrIF4 gp52::gent} and DMS3m_{acrIE3 gp52::gent} in the presence of CRISPR targeting. EOL was calculated by dividing the output lysogens forming units (LFUs) from the strain with 5 targeting spacers (5sp) to the number of LFUs in PA14 with 0 targeting spacers (0sp). Data are represented as the mean of 3 biological replicates +/− SD. ND, not detectable. (E,F) EOL of 10³ LFUs of DMS3m_{acrIF4 gp52::gent} and DMS3m_{acrIE3 gp52::gent} in the presence of 10⁷ PFU of the indicated DMS3m_acr phage. Data are represented as the mean of 3 biological replicates +/− SD. ND, not detectable. See Figure S4 for analysis of lysogen prophage content. (G) Schematic of the experimental design in E–F, where a high MOI of wild-type “donor” phages is used to rescue a low MOI infection of marked “acceptor” phages.

Figure 4. Cas9 anti-CRISPR AcrIIA4 requires cooperative infection to neutralize Type II-A CRISPR immunity

(A) 10-fold serial dilutions of DMS3m_acrIE3 or DMS3m_acrIIA4 plated on a lawn of Pseudomonas aeruginosa strain PAO1 expressing Streptococcus pyogenes Type II-A Cas9 (PAO1::SpyCas9) and single guide RNA (+ sgRNA) or non-targeting control (+ vector). (B) Efficiency of plaquing of DMS3m_acrIIA4 and DMS3m_acrIE3 was calculated by normalizing PFU counts on a targeting strain of PAO1::SpyCas9 (+sgRNA) to PFU counts on a non-targeting strain of PAO1::SpyCas9 (+vector). Data are represented as the mean of 3 biological replicates +/− SD. ND, not detectable. (C–F) 12 hour growth curves of PAO1::SpyCas9 expressing a targeting sgRNA (+ sgRNA, panels C–D) or a non-targeting vector control (+vector, E–F) that were infected with virulent DMS3m_acrIIA4 or DMS3m_acrIE3 at multiplicities of infection (MOI, rainbow colors) from 2×10⁻⁵ to 2×10⁻². Growth curves of uninfected cells are shown in black. OD_600nm values are represented as the mean of 3 biological replicates +/− SD (vertical lines). (G–H) Replication of virulent DMS3m_acr phages (acceptor phage) in the presence of 10⁷ PFU (MOI 2) hybrid phage (donor) in PAO1::SpyCas9 + sgRNA expressing the JBD30 C repressor. Phages were harvested after 24 hours and DMS3m_acr phage PFUs quantified on PAO1::SpyCas9 + vector expressing the JBD30 C repressor. Phage output is represented as the mean of 3 biological replicates +/− SD. ND, not detectable. (I) Schematic of the experimental design in G–H, where a high MOI of non-replicative “donor” phages is used to rescue a low MOI infection of wild-type “acceptor” phages.