Bacteria are unicellular organisms, but it has become obvious in recent years that, like cells in a multicellular tissue, bacteria can synchronize their gene expression and engage in group behaviors. Bacterial group behaviors include the formation of biofilms, which are multicellular structures of bacteria encased in an extracellular matrix; production of bioluminescence, as in many marine bacteria; or the synchronized production of virulence factors during chronic infections of human pathogens. These traits are essential for hostile, as well as beneficial, relationships between different species of bacteria and between bacteria and their hosts.
The main molecular mechanism involved in the regulation of these multicellular behaviors is a process called quorum sensing. This mechanism allows bacteria to count their cell numbers and regulate gene expression accordingly. Bacteria do so by producing small signaling molecules—autoinducers that are secreted and sensed by themselves and their neighbors. Because quorum sensing regulates virulence in important human pathogens, therapies to quench quorum sensing are already being explored as alternative methods to traditional antibiotics for controlling bacterial infections.
Most researchers studying quorum sensing focus on systems that involve species-specific signals, which are important for communication within bacteria of the same species (intraspecies communication). In nature, however, most ecosystems are composed of many different species of bacteria; therefore, I am interested in studying communication between different bacterial species (interspecies communication).
One quorum-sensing signal, autoinducer-2 (AI-2), is produced by many bacteria widely distributed through the kingdom Bacteria. During my postdoctoral studies with Bonnie Bassler (HHMI, Princeton University), I cocultured different bacterial species together and showed that they can use AI-2 to regulate each other's gene expression. Thus I demonstrated that AI-2 can foster interspecies communication. My hypothesis is that interspecies quorum sensing is crucial for species interactions in multispecies communities and that its manipulation can lead to new therapeutic avenues.
To understand interspecies signaling and its role in natural ecosystems, we use an integrative approach that investigates interspecies quorum sensing across many scales, spanning the molecules, receptors, biochemical pathways, and signal transduction networks. We developed many tools to manipulate and probe interspecies signaling in vitro, and we have achieved considerable advances in the field. We now propose to apply the same approaches in vivo in the microbiota of the mammalian gut, a natural multispecies environment of great importance to human health. My expectation is that we will be able to establish strategies to tailor gut microbiota to improve health conditions.
AI-2: A Signal That Fosters Interspecies Communication in Bacteria
My recent research has focused on the mechanisms of bacterial interspecies signaling mediated by AI-2, a signal produced and detected by a wide variety of different bacterial species. I characterized the molecular players involved in the signal transduction pathway in the model bacterium Escherichia coli. This work provided the tools to develop the first laboratory system to study AI-2 signaling in multispecies consortia. With this setup, I demonstrated that different bacterial species can use AI-2 to communicate, supporting the initial hypothesis that AI-2 is a universal signal for bacterial communication.
Meanwhile, we and others have shown that AI-2 regulates a wide diversity of bacterial behaviors, such as virulence, biofilm formation, and antibiotic production. However, despite the large number of studies identifying phenotypes regulated by AI-2, the molecular mechanisms involved in AI-2 recognition and signal transduction are still poorly understood. Identification of such mechanisms has been one of the main interests of my research. Two receptors for AI-2 have been identified: the LuxP receptor, which was identified by members of the laboratories of Bonnie Bassler and Frederick Hughson (Princeton University), and the LsrB receptor, which we identified in collaboration with Stephen Miller (presently at Swarthmore College). The LuxP receptor is exclusively found in the vibrios, and we wonder if LsrB, which was identified first in Salmonella typhimurium, could be present in other organisms. We used a combination of structural, genetic, and bioinformatics approaches to identify functional LsrB AI-2 receptors in bacteria with sequenced genomes. With this study we showed that the presence of LsrB goes beyond enteric bacteria; this allowed us to establish a general method to identify new functional AI-2 receptors, which we validated experimentally.
Clues for the function of the LsrB AI-2 receptor came from my initial work in E. coli. This bacterium has an unusual profile of AI-2 production and accumulation. At early stages of growth, E. coli produce and secrete AI-2, until AI-2 reaches a certain critical concentration; at this point a mechanism dependent on LsrB that internalizes AI-2 is turned on, removing the signal from the environment.Using mixed-species cocultures, I demonstrated that consumption of AI-2 by enteric bacteria can be used to interfere with AI-2–mediated communication in other species, including the ability of human pathogens to regulate virulence. With this work I realized that we can use E. coli to manipulate AI-2 levels in environments colonized by this bacterium. We have constructed mutants that produce AI-2 and keep it at high levels (thus promoting communication) and others that efficiently remove and destroy the signal (thus silencing communication). Our plan is to use E. coli as a tool to control AI-2–dependent communication in vivo and to determine the role of AI-2 in natural environments.
Identifying the Bacterial Chemical Lexicon
Every year, new quorum-sensing signals are being discovered; thus knowledge of the chemical molecules that foster bacteria communication is in its early beginnings. To understand interspecies quorum sensing, we aim to identify and characterize new signals. There is substantial evidence of additional signals mediating interspecies quorum sensing in important pathogens, but the molecules remain unidentified. We are conducting genetic screens in these pathogens to identify genes involved in the biosynthesis of these signals. By combining the results obtained in these screens with biochemical approaches we expect to identify novel signals that should be important for virulence control in multispecies ecosystems.
Silencing and Promoting Interspecies Communication in the Gut Microbiota
The mammalian gut is an amazing ecosystem: it contains far more bacterial cells than the human cells we have in our body and more than 1,000 different bacterial species that have to coexist in the gut and interact with each other and with the host. The molecular mechanisms involved in these interactions remain basically unknown. My hypothesis is that interspecies communication plays a major role in colonization and homeostasis of gut microbiota. In this work we will establish a method to manipulate AI-2 interspecies signaling in the gut of mice, using the different E. coli mutants in the mechanism for AI-2 accumulation/removal mentioned above. Once we identify new signals, we will use a similar approach for other systems. Our goals are to identify the microbiota species that are favored when promoting or silencing interspecies communication and to determine the consequences of manipulating interspecies communication on the protective properties of gut microbiota against pathogens and inflammatory agents.
I expect this work will bring us closer to implementing strategies to tailor microbiota composition and to using the protective function of microbiota to develop new treatments to solve pathogenic infections, inflammatory diseases, and diseases related to nutritional imbalances, such as obesity and diabetes.
Grants from the Portuguese Foundation for Science and Technology provided partial support for this research
As of January 17, 2012