Identification and characterization of population heterogeneity of Vibrio cholerae in vivo

Abstract

Vibrio cholerae is a facultative pathogen that is found naturally in aquatic environments around the world. Upon ingestion of contaminated water or food, V. cholerae can colonize the human small intestine and induce profuse secretory diarrhea known as rice water stool (RWS). During the course of the infection V. cholerae undergoes through various transcriptional changes to adapt to the shifting environment. In the early stage of infection, bacteria rapidly replicate and increase expression of key virulence factors. The middle stage of infection is characterized by upregulation of the chemotaxis and flagellar genes. This allows V. cholerae to exit the luminal fluid of the small intestine. Finally in the late stage of infection, chemotaxis becomes repressed as V. cholerae is shed in the RWS. Studies have shown that V. cholerae exiting the mammalian host can enter into a hyperinfectious state. These hyperinfectious bacteria have been implicated to be major contributors in household transmission of the disease. Factors that promote hyperinfectivity are still largely unknown.Other studies have demonstrated that host‐passed bacteria upregulate important late genes that prepare V. cholerae transition into the aquatic environment. For example, several of these late genes are involved in metabolism and are shown to be important for V. cholerae survival in pond water. One such gene is glpK, which encodes for a glycerol kinase and plays an important role in glycerol metabolism. By quantitative real time PCR, previous work has shown that mice‐passaged V. cholerae had an increased expression of glpK by 10‐ to 14‐fold. However, using recombination‐based in vivo expression technology (RIVET), our lab demonstrated that only 27% of the bacterial population exiting the host induces glpk expression. This suggests that glpK is highly expressed during the late stage of infection but only in a quarter of the population. These results led to the idea that the population of V. cholerae leaving the host may be heterogeneous, in which one subset of the population undergoes preparation for survival in the aquatic environment while the other remains hyperinfectious and therefore, can be more fit for rapid transmission to the next host. To examine this heterogeneous population in vivo, we generated a chromosomal GFP‐transcriptional fusion to glpK in the wave three Haiti V. cholerae strain. Using this strain we infected infant rabbits, which serve as an animal model for cholera in humans, and showed that only a subset of the host‐passed vibrios express GFP or glpK. This is consistent with previous RIVET studies. We will use fluorescence‐activated cell sorting (FACS) to separate the two populations and perform RNA‐seq to study their transcriptomes. Analysis of the subpopulation will provide further understanding of the mechanism of transmission and dissemination of V. cholerae.