Single cell RNA-seq reveals that the Vibrio cholerae Mak toxins are required for killing of Tetrahymena pyriformis and for survival in protozoan expelled food vacuoles

Vibrio cholerae uses multiple strategies to resist predation by heterotrophic protozoa. For example, V. cholerae releases toxic compounds such as ammonium and pyomelanin, that can kill protists such as Tetrahymena pyriformis. V. cholerae also survives intracellularly and escapes 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 has 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 eleven 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 (makA, makB and makC) were differentially expressed. Individual V. cholerae ?makA, ?makB, and ?makE strains were not capable of killing T. pyriformis and ?makA and ?makE showed reduced survival inside EFVs compared to the wild type. Our findings reveal new insights into the grazing resistance mechanisms of V. cholerae, identify factors associated with the survival of V. cholerae within EFVs and more broadly, highlight the connection between antiprotozoal and virulence factors displayed by pathogenic bacteria. Overall design: Intracellular Vibrio cholerae bacteria recovered from Tetrahymena pyriformis and from EFVs were fixed in 4% formaldehyde at 4 degc overnight and then the MicroSPLiT protocol was perfromed to individalluy tage the mRNA of the bacteria