Many bacteria use chemical signals to coordinate group behaviour. A signal that suppresses virulence has been identified in the bacterium that causes cholera, and could be a new therapeutic target.
Like people, many bacteria do things in groups that they don't do on their own. These communal activities can be spectacular; the marine bacterium Vibrio fischeri, for example, produces bioluminescence in the light organs of deep-sea fish. But bacterial group behaviour can also be deadly — many bacteria become virulent only when they reach a certain local concentration. Such coordinated actions require bacteria to 'talk' to each other by sending chemical signals, a process known as quorum sensing. Although some of the molecules involved are known, it is likely that there are many more.
It's been known for a few years that Vibrio cholerae, the bacterium that causes cholera in humans, is 'bilingual' — that is, it uses two distinct signalling molecules1 to suppress its virulence2. One of these signals is a molecule that many species of bacteria use for quorum sensing3, but the identity of the second signal has remained a mystery. In this issue, Higgins et al.4 report the structure of this second signal. Their discovery represents a new structural class of quorum-sensing signal that may be exclusive to Vibrio bacteria, making it a possible lead for drug discovery.
In general, quorum sensing is straightforward: bacteria release signals into the surrounding environment; if the signals reach a critical concentration, they are detected by bacteria in the vicinity and this stimulates a response. In V. cholerae, quorum sensing proceeds through two parallel systems1, either of which is sufficient to independently initiate a response. The first of these involves the AI-2 molecule (Fig. 1), a signal used by many species of bacteria. AI-2 is detected by the sensory proteins LuxP and LuxQ, which are associated with the bacterium's cell membrane.
Although the identity of the signal in the second system was unknown, the enzyme responsible for producing the signal had been identified as the CqsA protein. The second signal is thought to be detected by a putative membrane-associated sensor called CqsS. Both systems in V. cholerae funnel information into the same signalling cascade (through the transducing proteins LuxU and LuxO) so that an analogous functional response is produced in each case: LuxO is inactivated, resulting in increased activity of HapR, a negative regulator that represses expression of virulence genes.
In their detective work identifying the unknown quorum-sensing signal of V. cholerae, Higgins et al.4 took their cue from the known biochemistry. They introduced the cqsA gene into Escherichia coli, creating a recombinant strain that produces much more signal than the parent V. cholerae species. By extracting the culture fluids of the E. coli, the authors obtained a mixture of compounds that they separated into its constituent parts. They next tested the purified compounds on a strain of V. cholerae that had been engineered to emit light in response to the unknown signal. Certain fractions of the mixture were 10,000 times more active than controls.
Using a combination of spectroscopic techniques, Higgins et al. then identified the active compound as 3-hydroxytridecan-4-one (Fig. 1). They confirmed this by preparing the compound chemically, and testing the synthetic version in their activity assay. The authors were finally able to obtain sufficient material from cultures of natural V. cholerae for analysis, and so to prove conclusively that 3-hydroxytridecan-4-one is the second signal for this bacterium.
So why does V. cholerae adopt a 'belt and braces' approach to quorum sensing, using two parallel systems when one would be sufficient? There are many potential explanations, one of which relates to the distinct chemical properties of the two signals. These properties might influence the stability or rates of diffusion of the signals, perhaps making one molecule superior to the other for quorum sensing in a particular environment. Higgins et al.4 suggest that, because V. cholerae encounters environments that are rich in other AI-2-producing bacteria (such as the large intestine), the AI-2 system might be used for interspecies signalling. Conversely, they propose that the specificity of the Cqs signal for the Vibrio genus makes it ideal for quorum sensing with other bacteria of the same species. To test this hypothesis, it will be necessary to show that one system dominates, depending on the environmental context.
The authors also suggest that the Cqs signal could be exploited therapeutically to dampen V. cholerae virulence. Quorum-sensing systems that trigger virulence have already been targets for therapies against several species, such as Pseudomonas aeruginosa — an opportunistic pathogen that causes infections in people with compromised immunity. Usually the goal is to block quorum sensing with a small-molecule inhibitor5. This is a difficult task, because such inhibitors must be specific, stable, easily deliverable to the infection site and able to out-compete the natural quorum-sensing signal for the target receptor.
But quorum sensing in V. cholerae may be unique. Unlike in many other species, in which quorum sensing induces virulence, in V. cholerae the response shuts virulence down, allowing the bacteria to escape the host and re-enter the environment. This means that the signals themselves could be used as therapeutics. The high specificity of 3-hydroxytridecan-4-one for V. cholerae certainly makes it an excellent candidate for drug development. In fact, in a proof-of-concept experiment, Higgins and colleagues4 show that their synthetic version of the signal does indeed terminate production of known virulence factors in V. cholerae. But this idea raises possible public-health issues: the activation of quorum sensing in V. cholerae also induces active movement of the bacterium, potentially mobilizing the pathogen and encouraging the spread of infection from one person to another.
For several years, the repertoire of bacterial quorum-sensing signal molecules and receptors was thought to be rather limited and restricted to a few species. But recent studies have revealed an array of different signals, suggesting that we have only just scratched the surface of possible mechanisms. As new signals are identified and their use by bacteria is assessed, the list of quorum-sensing organisms will undoubtedly grow. We may eventually reach a point at which bacteria that do not engage in quorum sensing are regarded as the exception, rather than the norm. The challenge now is not only to identify new systems, but also to make sense of why an organism would use one type of system over another.
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Bioorganic & Medicinal Chemistry (2008)