The bacterium that causes cholera, Vibrio cholerae, can grow both aquatically and in a human host. To survive, the bacterium must be able to live in very distinct conditions: imagine how different the temperature, available nutrients, and neighboring microbes are between these two niches. To adapt, V. cholerae must turn on and off the genes appropriate for its current environment. How does the bacterial cell know what its current environment is and which genes it needs?
This is the question being addressed by Dr. Jim Bina and graduate student Vanessa Ante. Their research focuses on the questions: what are the environmental cues, and how do these cues affect gene expression in V. cholerae? Ante’s recently published findings in the Journal of Bacteriology demonstrate that a component of the human intestine, bile salt, acts as one of the environmental cues to activate expression of virulence-related gene, leuO.
Bile is secreted by the liver into the small intestine, and contains a number of antimicrobial factors. The major component is bile acids, such as deoxycholate, cholate, and chenodeoxycholate. These detergent-like molecules are important for digestion and can also disrupt the microbial membrane – but Ante’s work shows they can be an alert to V. cholerae. As V. cholerae is ingested from contaminated water, it passes into the gastrointestinal tract. “Because we have bile salts present in the small intestines, these bile salts act as a signaling molecule, with ToxR responding and regulating LeuO in response,” explains Ante. ToxR and LeuO are two cholera proteins that play an important role in detecting environmental cues.
Bina adds: “It's been known for a long time that ToxR, a membrane-associated regulator, has a domain in the periplasm of the organism that is thought to be a sensing domain.” After sensing environmental molecules, the ToxR sends a signal to the DNA binding domain, which can then regulate the target genes. ToxR signaling has several arms: regulating expression of virulence factors, such as cholera toxin, and regulating expression of porin proteins that are important for bile resistance (the latter is not pictured in the image at right).
“What Vanessa has shown is that in addition to porin protein, in the presence of bile, ToxR also turns on LeuO. LeuO goes on and regulates a number of downstream effects and also contributes to bile resistance, suggesting that the regulatory cascade being controlled by ToxR to adapt to the environment of the intestine is broader than just these porin proteins,” explains Bina. “What this paper is showing is that one of the environmental signals turns out to be bile acids – at least in the test tube,” he continues, and that ToxR “is necessary for sensing bile acids and then turning on the downstream effects,” including bile resistance.
The research adds to the known targets of ToxR signaling. Further, it shows that LeuO activation doesn’t require an intermediary protein. “ToxR can regulate a number of genes, and the major one is ToxT, which then goes on and regulates virulence factor production. We show that ToxR is able to directly bind to LeuO without ToxT being involved,” says Ante. This research shows “the role of ToxR, at least in adaptation to the environment in the intestine, is broader than what was previously appreciated,” says Bina.
The Bina lab had previously shown that ToxR regulates LeuO when cyclic dipeptides are present. When cyclic dipeptides, which are produced by a number of bacteria, are the activating molecule, LeuO activation occurs with simultaneous decrease of virulence factor genes. However, bile salt activation of LeuO did not have a similar decrease of virulence factor genes. Why not?
Bina speculates: “The way we’re thinking about this is ToxR is sensing where the bug is in the intestine – whether or not the organism is at the epithelial surface or at the lumen.” These locations may not seem very far apart to us, but this is the equivalent of about 15 miles away. “In the lumen, the bug would be exposed to bile acids perhaps not present at the epithelial surface because the mucus provides a diffusion barrier. In this way, we think what’s happening is ToxR is sensing the environment and regulating LeuO, which is then regulating downstream genes that are important for adaptation.”
Input: environmental cue. Output: proper gene expression. As the above toxin regulation schematic suggests, the circuit is rarely linear. Understanding these complex circuits allows researchers to understand how bacteria sense and adapt to their current environment. In the case of pathogens like V. cholerae, these circuits can also identify potential drug targets, if a given drug throws a wrench in the circuitry gears. Researchers have previously described small molecule inhibitors of ToxT transcription – just the sort of wrench to stop V. cholerae from adjusting to its environment and producing toxin. But before the circuit can be interrupted, it must first be fully understood. The story of bile salt stimulation of ToxR-LeuO moves us one step closer to that goal.
-- Julie Wolf