Kulkarni has continued to develop both bioinformatics tools and computational models. “Looking at small RNAs in the CsrA pathway with our bioinformatics tool (CSRNA_FIND) allowed us allowed us to predict several previously undiscovered small RNAs,” says Kulkarni.Since most of his work is theoretical, his team often relies on collaborators to validate their predictions. “This has worked out well,” he says. “Our predictions have been validated with a high degree of success.” He notes that the sRNAs they predicted in Legionella pneumophila have been validated by several laboratories recently, something he finds gratifying. “It aids research efforts in multiple labs worldwide,” he says.
CsrA (carbon storage regulator) is a global, RNA-binding regulatory protein. Both quorum sensing and the CsrA pathways control virulence and colonization in several species of human bacterial pathogens. They are also involved in biofilm formation in a variety of bacterial species, including biofilm colonization infections by Pseudomonas aeruginosa in the airways of patients with cystic fibrosis. Information about the potential targets that mediate biofilm formation in P. aeruginosa might eventually help patients with cystic fibrosis; this provides motivation for Kulkarni's group. They are developing a bioinformatic tool to predict target genes regulated by CsrA in multiple bacterial species, and collaborating to see if it can be validated. “If we identify targets, it should help with therapeutic targets,” he says. Based on genomes currently sequenced, CsrA is present in more than 100 species of bacteria, so Kulkarni suspects that the tools they develop will likely be applicable to multiple species. “This will open up several new avenues of research.” Kulkarni says that his collaborators are expanding what they know about quorum sensing by examining downstream targets and pathways, and by developing and analyzing stochastic computational models for regulation by sRNAs.Lab Studies
Ann M. Stevens, an associate professor of Biological Sciences at the Virginia Polytechnic Institute and State University, has collaborated with Kulkarni, among others. She has been studying the effects of intercellular interaction among bacteria on virulence, biofilm formation, and bioluminescence. Stevens’ interest is in quorum sensing in V. fischeri and V. parahaemolyticus, as well as in the corn (maize) pathogen Pantoea stewartii, which is injected into the corn plant by its symbiont, the corn flea beetle (Chaetocnema pulicaria). The production of P. stewartii's polysaccharide capsule is controlled by quorum sensing, which causes the bacteria to clog the xylem of the plant. This leads to streaks of chlorosis (yellowing), necrosis, and wilting. It is the higher cell density that results in de-repression of capsule production. “It's not the number-one corn pathogen,” explains Stevens, “but corn is an important commodity.” Understanding the quorum sensing response in this system could allow enhancement and modulation or control of this response.
Stevens studies signaling in several species, but she says understanding quorum sensing in one system doesn't always lead to the same understanding in another. Although increased cell density in Vibrio populations leads to upregulation of gene expression (e.g., luminescence), increased cell density in P. stewartii populations leads to downregulation of genes. Her group just received a grant to study the regulation of pathogenesis by quorum sensing in P. stewartii. “By looking at quorum sensing in P. stewartii, we have the capacity to address questions not asked before, including how receptors respond to signals and how the cells respond at low as well as high cell densities,” Stevens says. Along with standard molecular biology methods, she believes that a multi-disciplinary approach coupling experiments with modeling will provide answers on how quorum sensing and cell signaling can be controlled.
Stevens also collaborates with Andre Levchenko, assistant professor at the Institute for Computational Medicine at Johns Hopkins University (Baltimore, MD), on the use of microfluidics chambers that enable visualization of single cells. Stevens notes that most people believe quorum sensing systems involve one positive feedback loop that reinforces a signal when it is turned on. However, her work with Levchencko suggested that there are two interlocked feedback loops in V. fischeri that regulate luminescence: both the amount of signal and the amount of receptor are critical for a robust response.
“Historically, people have thought bacteria were entities only concerned with their [own] propagation,” says Stevens. But she says that idea is changing as it becomes clear that most species can measure how many similar cells are near them in their environment, as well as how many other types of bacteria are present. These bacteria often respond to multiple extracellular signals through different receptors on the bacterial surface, creating complex signal transduction systems. Researchers are now realizing that bacterial signaling systems are more complicated than previously thought, she says.
Possible medicinal and environmental applications of quorum sensing, such as interfering with biofilm formation, are still being explored, although significant developments are still needed, according to Stevens. “My work is basic in nature, but I hope there will be practical applications down the road.”