Increased iron utilization and oxidative stress tolerance in a Vibrio cholerae flrA mutant confers resistance to amoeba predation.

The flagellar transcriptional regulator flrA initiates the regulatory cascade of flagellum synthesis in Vibrio cholerae . Previously, we observed an increase in sub-populations of V. cholerae carrying mutations in the flrA gene during long-term co-adaptation with the amoeba host Acanthamoeba castellanii . The flrA mutants exhibit increased growth and intracellular survival in the amoeba host but the molecular mechanisms were unknown. Using an in-frame deletion of flrA, here we show that the absence of flrA increases pathogen growth and induces a distinct V. cholerae transcriptomic signature during amoeba predation. Transcriptome analysis of a flrA mutant in A. castellanii revealed that several genes involved in iron acquisition and amino acid biosynthesis are highly up-regulated compared to the wild-type strain. Furthermore, we show that iron availability is crucial for the survival of V. cholerae in amoeba. We also report that V. cholerae KatB and KatG catalases confer an increased tolerance to oxidative stress. We conclude that the increased survival of the flrA mutant in amoeba is due to a combination of factors, including resistance to oxidative stress and an increased capacity to utilize essential nutrients such as iron and amino acids. Together, the results presented here detail how a bacterial pathogen increases resistance to protozoan predation, thereby allowing for increased survival in the environment. IMPORTANCE Persistence of V. cholerae in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face predation pressure by heterotrophic protists such as the free-living amoeba A. castellanii . This study explores how a mutant of V. cholerae adapts to acquire essential nutrients and survive predation. Here, we observed that up-regulation of iron acquisition genes and genes regulating resistance to oxidative stress enhances pathogen fitness. Our data show that V. cholerae can defend predation to overcome nutrient limitation and oxidative stress, resulting in an enhanced survival inside the protozoan hosts.