MECHANISMS OF SIGNAL TRANSDUCTION IN BRUCELLA

<b>Project Methods</b><br> Methods: a general overviewWe will use many traditional molecular genetic, biochemical, and bioinformatic approaches to understanding signal transduction mechanisms in Brucella spp, which are outlined in detail in our proposal. However, we have also developed unique molecular methods for this project, which will permit us to advance investigations of Brucella physiology and infection biology in new directions (outlined below).Generating Brucella mutantsBriefly, traditional bioinformatic approaches easily identify sensory proteins (e.g. sensor histidine kinases) in bacterial genomes, and traditional molecular genetic tools allow for facile deletion of the genes encoding these sensor kinases. However, it is difficult to predict substrates of these sensory transduction proteins, and how different kinases influence each other in vivo. Brucella is an excellent model system of study in which to investigate how multiple sensory histidine kinases interact to affect signaling through a single pathway that controls chronic infection. By generating mutant strains missing combinations of sensor kinases and testing the stress survival and mammalian infection phenotypes, we will advance understanding of how consortia of sensory kinases function together to control Brucella physiology and infection biology.A novel, pull-down sequencing methodUnderstanding the role of small RNAs in the regulation of gene expression and infection is also a major challenge, and we have developed an innovative biochemical pull-down sequencing method which we will utilize to define direct regulatory targets of Brucella small RNAs that are important for infection. Importantly, our group is conducting experiments in both B. abortus, a BSL3 select agent, and B. ovis, a BSL2 Brucella species. Working between these two species permits us to advance molecular/biochemical lines of experimentation in Brucella that may otherwise be impeded by restrictive USDA- and CDC-mandated BSL3 select agent sample removal protocols. Pull-down sequencing data to identify molecular targets of small RNAs will be conducted using open source sequence alignment and counting approaches in R.Efforts to advance new concepts through this research project: A novel concept emerging from this project is that Alphaproteobacteria, including Brucella, integrate multiple environmental signals via a set of unusual histidine kinases known as HWE-family kinases. This concept is more fully outlined in a recent review from our group (Fiebig, et al. 2015. Annu Rev. Microbiol.). We are advancing the novel concept that flavin-binding HWE kinases such as Brucella LovhK can function as AND logic gates, i.e. LovhK can only be activated when the flavin cofactor is oxidized AND when blue photons are present in the environment. If one generalizes this result, it suggests that certain sensor histidine kinases may integrate information about multiple physical/chemical features of a particular niche. To test this hypothesis, we will measure the kinase actibity of purified LovhK in the presence and abence of activating light signals in buffers of varying redox potentials. The proposed research activities (outlined more completely in our proposal) have the potential to expand our concept of sensor kinase function.An additional novel concept that we propose to test over this next grant period centers on a Brucella LolA/B-like protein, EipB. It is notable that a number of bacterial groups are missing the LolB protein, which is key molecular determinant of lipoprotein trafficking in many species. Our preliminary data suggest that we have discovered a new protein that serves this function in Brucella and other members of the alphaproteobacterial clade. To test this hypothesis, we will utilize EipB mutant strains and assess the lipoprotein compositon of these strains using biochemical fractionation and mass spectrometry.Efforts to advance an integrative experimental and computational approach to the study of Brucella:As outlined in the project proposal, my research team will use established and effective protocols to conduct genetic analyses of Brucella physiology/cell biology, and biochemical, biophysical, and computational analyses of environmental sensory transduction proteins. An important component of this proposal is our studies of the Brucella GSR sensor, LovhK, a photo/redox sensory LOV kinase. Though LOV proteins are encoded in over 10% of sequenced bacterial genomes, their functions remain largely undefined. The experience that my group brings to the study of LOV sensors makes us the ideal group to continue investigation of this system in Brucella. We have expertise across a range of disciplines, and the innovative combination of genetic analysis, biochemistry, and structural biology applied to this problem promises to more clearly define a stress signaling mechanism that is a key determinant of chronic brucellosis. On a broader level, our interdisciplinary approach will provide a more complete understanding of how bacterial cells perceive and adapt to physical and chemical changes in their environment.A very limited number of groups worldwide are conducting basic studies of B. abortus, a BSL3 select agent pathogen. Our studies of B. abortus and the BSL2 species, B. ovis are providing important new insight into variability in the molecular mechanism of GSR signaling across the alphaproteobacterial clade. Additionally, our animal infection work has provided us with important evidence that perturbing GSR signaling is a possible new approach to antimicrobial treatment of brucellosis.EvaluationWe will evaluate success of our research and research training efforts in two main ways: 1) through our Brucella research publications, which will be publically available (we expect to publish 1-2 papers per year over the course of this project), 2) though graduation and advancement of our research trainees to postdoctoral, research scientist, and research faculty positions, or advancement in other science-related careers (we expect to advance 2-4 trainees over tthe course of this project).