Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion

Abstract

To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

Keywords: CD-NTases; cyclic GMP-AMP; folate; phage; quorum sensing.

Fig 1

The VSP islands do not contribute to common V. cholerae biotyping phenotypes or virulence in a murine model of cholera. Differential biotyping phenotypes between classical O395, El Tor C6706, and C6706 VSP island mutants (ΔVSP-1, ΔVSP-2, and ΔVSP-1/2) demonstrating strain-specific (A) proteolysis of casein on milk agar, (B) hemolytic activity on blood agar, (C) growth on citrate minimal medium agar, (D) growth on matched LB and MacConkey agar plates, and (E) production of acetoin detected by Voges-Proskauer Assay. All images are representative of three independent experimental replicates. (F) In vivo competition between a 1:1 mixture of C6706 ΔVSP1/2 and C6706 ΔlacZ in an infant mouse model of cholera. Intestinal colony-forming units (CFUs) were enumerated using blue-white screening 20 h after oral gavage. N = 8 mice and statistical significance was determined using a one-sample t test and a hypothetical mean log₁₀ C.I. = 0. The hypothetical mean is represented by a dotted line. The calculated mean log₁₀ C.I. is represented by a solid line. ns = not significant.

Fig 2

VSP-1 encoded CBASS is responsible for V. cholerae biotype-specific SMX sensitivity. Twenty-four hours planktonic antibiotic sensitivity assays were performed in a variety of SMX concentration gradients. Heatmaps (A), (B), and (D) represent relative growth for each strain calculated using culture optical densities and the equation (OD₆₀₀ SMX treatment/OD₆₀₀ untreated) and reported as a color-coded mean % of N = 3 biological replicates. The IC₅₀ for all strains in (A) are presented in Table S1. Scatter plots corresponding to (A), (B), and (D) are presented in (Fig. S2A through F). (C) Cartoon depiction of the VSP-1 genomic island. Dotted lines indicate partial VSP-1 truncations. Gray chevrons highlight the four gene VSP-1 CBASS operon. Numerals 1–5 indicate the location of a mutation identified in each of the five respective V. cholerae isolates found to have spontaneously evolved SMX resistance.

Fig 3

Culture density-dependent sensitivity to SMX reveals quorum sensing regulation of VSP-1 CBASS. (A) Growth curves (OD₆₀₀, left y-axis) of WT C6706 and ΔcapV cultures treated without (+DMSO) and with 100 µg/mL SMX (+SMX). Intracellular cGAMP (µM, right y-axis) measured by UPLC-MS/MS in the SMX treated and untreated ΔcapV cultures. Growth curves of (B) quorum fluent ΔvpsL, (C) LCD-locked ΔvpsLΔhapR, and (D) HCD-locked ΔvpsLΔluxO cultures treated without (+DMSO) and with 100 µg/mL SMX (+SMX). Gray arrows indicate addition of 100 µg/mL SMX or DMSO. N = 3 biological replicates and error bars represent standard deviation. Statistical significance calculated using an unpaired T test with the Holm-Šídák method (*P < 0.05, **P < 0.005, ***P < 0.0005), n.s. = not significant.

Fig 4

QS regulates expression of V. cholerae CBASS. (A) Growth curve (OD₆₀₀, left y-axis, solid line) of V. cholerae C6706 and the corresponding fold-change in transcript abundance (right-axis) of capV and dncV, relative to the initial 2 h time point, measured using RT-qPCR. N = 2 biological replicates and error bars represent standard deviation. (B) Relative transcript abundance of capV, dncV, and hapR measured by qRT-PCR in ΔcsqAΔluxS grown in the presence (+) and absence (−) of exogenous autoinducers (AIs). N = 3 biological replicates and error bars represent standard error of the mean. (C) % relative luminescence units of the indicated strains normalized to the mean lum/OD₆₀₀ of ∆vpsL∆luxO maintaining the luminescent transcriptional reporter pP_CBASS::lux. Population densities are low-cell density (LCD) and high-cell density (HCD). N = 3 biological replicates and error bars represent standard deviation. Statistical significance calculated using an unpaired t test with the Holm-Šídák method (*P < 0.05). This method was only used between strains at the same population density and the lack of graphical comparison indicates no statistical difference observed. (D) Relative luminescence (lum/OD₆₀₀) of E. coli maintaining the luminescent transcriptional reporter pP_CBASS::lux and the P_tac inducible hapR plasmid (pHapR) or a vector control (pVector2) grown in the presence of 6.25 µM IPTG. N = 3 biological replicates and error bars represent standard deviation. Statistical significance calculated using an unpaired t test with the Holm-Šídák method (**P < 0.005, ***P < 0.0005), n.s. = not significant. (E) Intracellular cGAMP measured using UPLC-MS/MS in the quorum fluent ΔcapV and LCD-locked ΔcapVΔcsqAΔluxS strains grown ~24 h in the presence of 100 µg/mL SMX. N = 4 biological replicates and error bars represent standard deviation. Statistical significance calculated using an unpaired t test (*P < 0.05).

Fig 5

HapR enhances VSP-1 mediated phage defense in E. coli. (A) Growth of E. coli containing either pHapR induced with 10 µM IPTG and pVSP-1 with their associated vector controls after overnight growth with T2 phage is shown as a mean percent of the uninfected culture from N = 3 biological replicates. mean % presented numerically and by heatmap for each condition. Scatter plot of data presented in Fig. S4. (B) Model of folate (by SMX) and QS (by HapR) regulation of CBASS activity and expression. At HCD, HapR induces transcription of the CBASS operon. Inhibition of folate biosynthesis by SMX alleviates the folate-dependent non-competitive inhibition of DncV leading to synthesis of cGAMP and activation of the phospholipase CapV. CBASS activity ultimately culminates in abortive infection that thwarts phage predation by limiting phage replication. Solid arrows and brakes indicate regulatory mechanisms that influence CBASS activity addressed in this study. Black hatched arrows and brakes indicate mechanisms known to contribute to CBASS activity but were not found to significantly contribute to QS or folate mechanisms illuminated in this study. Red brake represents a hypothesized phage-dependent disturbance in folates which is sensed by DncV to initiate abortive infection by the V. cholerae CBASS. Created with BioRender.com.