Single-cell RNA-seq reveals a key role for Vibrio cholerae Mak toxins in Tetrahymena pyriformis killing and bacterial survival

Abstract

In the environment, Vibrio cholerae employs multiple strategies to resist predation by heterotrophic protozoa. For example, V. cholerae biofilms release toxic compounds, such as ammonium and pyomelanin, which can kill protists, such as Tetrahymena pyriformis. V. cholerae has also been shown to survive intracellularly and can escape as viable cells inside protozoan-expelled food vacuoles (EFVs). We previously reported that V. cholerae encased in EFVs are hyperinfectious, establishing an important link between anti-protozoal strategies and bacterial virulence. Although the intracellular resistance and escape of V. cholerae in EFVs have been reported, the molecular mechanisms behind this remain poorly understood. Here, we used single-cell transcriptomics of V. cholerae exposed to T. pyriformis and captured a total of 5,344 bacterial cells with heterogeneous gene expression. Cells with the same pattern of gene expression were grouped, resulting in 11 clusters of cells with a unique gene expression profile. Genes encoding outer membrane proteins, F₁F₀-Na⁺/H⁺ ATPase, metabolites, and toxins showed differential expression among the clusters. Furthermore, the motility-associated killing factor (Mak) toxins were differentially expressed. The V. cholerae mutants ΔmakA, ΔmakB, and ΔmakE were not capable of killing T. pyriformis, and ΔmakA and ΔmakE showed reduced survival inside EFVs compared to the wild type. These findings identify Mak toxins as key mediators of V. cholerae resistance to protozoan grazing and survival within EFVs. More broadly, our results provide mechanistic insight into grazing resistance, reveal factors facilitating persistence in EFVs, and underscore the interplay between environmental survival strategies and virulence in pathogenic bacteria.

Keywords: Vibrio cholerae; anti protozoal; protozoa; toxins; transcriptomics.

Figure 1

Single-cell RNA-seq of V. cholerae reveals heterogeneous gene expression in the presence of T. pyriformis. (a) Number of cells captured in the ungrazed (control) and grazed (experimental) conditions. (b) Uniform manifold approximation and projection (UMAP) of two-dimensional plots of the 11 clusters of cells displaying differential gene expression within the grazed population. (c) Number of mRNA UMI counts per cell. (d) Number of mRNA UMI per cluster of gene expression. The results are shown as median with 95% CI.

Figure 2

Heatmap of gene expression (z-score of log-transformed values) from 5,344 V. cholerae cells organized into 11 clusters (0–10). Upregulation of genes is indicated by yellow, and downregulation is indicated by magenta. On the left side, there is a selection of genes indicated.

Figure 3

Differential expression of HapR-regulated toxins. Single-cell gene expressions of makA, makB, makC, and hapA (VCA0865) are highlighted in the UMAP plots. The left column corresponds to the expression in the grazing condition, while the right column shows the ungrazed condition (control).

Figure 4

Importance of Mak toxins in V. cholerae for survival within EFVs. All complemented mutants described here are notated as Δmak𝑥-mak𝑥⁺, where 𝑥 is the specific gene. (a) ΔmakA; (b) ΔmakB; (c) ΔmakE. Bacterial survival in EFVs was calculated as the number of colonies of Δmak strains divided by the number of colonies of WT after 24 h incubation of V. cholerae and T. pyriformis in ASW. Co-incubation of V. cholerae WT (ΔlacZ) or Δmak strains was independently performed with T. pyriformis at an infectious dose of 10,000. The initial inoculum of both strains was mixed 50:50 and plated on X-gal LB plates at 30 °C to assess blue (mutant) and white (WT) number of colonies (T₀). After overnight incubation, equal amount of EFVs produced independently with the WT and Δmak strains were mixed and digested with Triton-X100 to release the bacterial cells inside. The mixture was then plated on X-gal LB plates at 30 °C to assess blue (mutant) and white (WT) number of colonies (T₂₄). Data are from three independent biological replicates and are shown as the average. Significant differences were determined using one-way ANOVA with Tukey’s multiple comparisons test. ****p < 0.0001.

Figure 5

Percentage of T. pyriformis cells killed by V. cholerae. All complemented mutants described here are notated as Δmak𝑥-mak𝑥⁺ where xis the specific gene. WT, Δmak strains, ΔhapR, and ΔflaA were incubated with T. pyriformis for ~5 h of co-incubation in ASW at RT. The percentage of killing was assessed by counting the number of dead cells at the bottom of the well, divided by the total number of cells, multiplied by 100. Data are from three independent biological replicates and are shown as the average ± s.d. Significant differences were determined using an unpaired t-test with Welch’s correction (*p < 0.05, **p < 0.01).