Characterization of c-di-AMP signaling in the oral commensal and opportunistic pathogen S. mitis.
Streptococcus mitis is a commensal colonizer of the oral cavity that occasionally causes serious opportunistic infections. Our data indicate that the cyclic di-adenosine monophosphate signaling system influences the transition of S. mitis from a commensal to a pathogenic lifestyle. This project will identify and characterize effector proteins putatively involved in this process.
The human microbiota consists of an enormous number of symbiotic microorganisms that normally live on or within the human body without causing disease. This is achieved by a delicate balance in host-microbe interactions. Under certain conditions, such as a weakened host-immune system, typically commensal microorganisms can transition into a pathogenic lifestyle. Streptococcus mitis is one of the first colonizers of the oral cavity and normally lead a commensal lifestyle in humans from infancy to senescence. However, S. mitis can also cause serious infections such as bacteremia in neutropenic patients, and infective endocarditis. Our preliminary data from an animal experiment revealed that neutropenia is required for S. mitis to cause severe disease in mice. Interestingly, we noticed that S. mitis mutants with an aberrant intracellular signaling system mediated by the second messenger cyclic di-adenosine monophosphate (c-di-AMP) were cleared significantly quicker from internal organs such as the heart and spleen of neutropenic mice (preliminary data). This indicates that a functional c-di-AMP signaling system is required for bacterial survival and/or persistence, even in neutropenic individuals.
The intracellular concentration of c-di-AMP is regulated by specific diadenylate cyclases that produce c-di-AMP and phosphodiesterases that degrade c-di-AMP via phosphadenylyl-adenosine (pApA) to AMP. Several c-di-AMP specific receptors have been identified in different bacteria, including transcriptional regulators, cation transporters and riboswitches, but due to the diverse structures of the identified receptors, the full complement of effectors is not yet known in any organism and completely unknown in S. mitis. The potential regulatory role of pApA has not been characterized in detail in any organism. Our in silico analysis of the genome of S. mitis NCTC12261 identified one gene encoding a putative di-adenylate cyclase (DacA) and two genes encoding putative phosphodiesterases Pde1 and Pde2 that degrade c-di-AMP via pApA to AMP. We have created knock-out mutants of each of these genes separately, and in combination, and used them to characterize the role of c-di-AMP signaling in a number of phenotypes relevant for colonization and survival in the oral cavity. Our results show that the c-di-AMP signaling system is involved in regulation of growth, biofilm formation, resistance to DNA damage, colony morphology, and acid stress.
Although we have established that c-di-AMP is an important second messenger in S. mitis and have characterized the proteins involved in controlling the concentration of this signaling molecule we do not know the effectors of c-di-AMP signaling (i.e. the target molecules that bind to c-di-AMP and carry out a function in response to the signal). Identification of these effectors is important since they can be suitable targets for drug development. Based on published research on c-di-AMP signaling in different bacteria, we have performed bioinformatics analyses of the S. mitis (NCTC12261) genome. We identified eight genes encoding putative c-di-AMP binding proteins. In addition, our published and preliminary data have identified one previously unknown putative c-di-AMP effector protein in S. mitis. The role and function of these putative c-di-AMP effector proteins will be characterized in vitro and in vivo.
This project will generate novel knowledge about the important c-di-AMP signaling system in an opportunistic pathogen and provide information on the effectors of this signaling pathway. There is an evident invention potential since interfering with this regulatory system constitutes an attractive target for drug development.
How does c-di-AMP signaling regulate colonization, persistence and virulence of S. mitis and thereby influence the transition from a commensal to a pathogenic life style?
Mål og metode
- The main objective of this project is to characterize c-di-AMP binding proteins that function as effectors regulating processes involved in colonization, persistence and virulence of S. mitis.
- Identify putative c-di-AMP effectors involved in regulating c-di-AMP mediated phenotypes.
- Determine the role of the effectors in regulating c-di-AMP mediated phenotypes of S. mitis
- Demonstrate the c-di-AMP binding potential of the effector proteins in vitro.
- Characterize the function of c-di-AMP effector proteins in vitro.
- Genes encoding putative c-di-AMP effector proteins will be deleted in a background strain with increased c-di-AMP concentration (the pde1 and pde2 double mutant).
- The mutants will be analyzed in relevant c-di-AMP regulated phenotype assays with a focus on growth, biofilm formation and DNA damage and metabolism.
- Mutants of c-di-AMP binding proteins showing altered c-di-AMP mediated phenotypes will be further characterized genetically (eg. by reintroducing the gene of interest or creating additional mutations to elucidate the signaling pathway in detail).
- Genes encoding putative c-di-AMP effectors will be cloned into expression vectors.
- Putative effector proteins will be expressed, purified by affinity chromatography and size exclusion chromatography for characterization in vitro.
- C-di-AMP binding to effector proteins will be characterized by microscale electrophoresis (MST).
- The putative functions/activities of effector proteins will be characterized in vitro.
The student will receive training in all described methods and is expected to eventually be able to independently carry out the methods. The student will carry out the genetic manipulation of S. mitis and cloning of genes encoding the effector proteins into effector proteins. Following an initial functional screening of mutants, and protein expression trials the student will focus on characterizing the most promising candidate gene in more detail.
The research lab
My research group Dynamics of bacterial colonization currently consists of one lab engineer, two PhD students, one MSc student and a Life science summer research student. The research fields of the group include antibiotic resistance, biofilms, host-microbe interactions and cyclic di-nucleotide mediated intracellular signaling of bacteria. Recent publications have positioned the group as a leader in polyether ionophore resistance in bacteria and a member of the group is considered one of the pioneers of cyclic dinucleotide signaling in bacteria. The group shares a lab with the research group Epigenetic mechanisms in cancer that Professor Thomas Kuntziger leads and his group currently contains one PhD student, one lab engineer and one Life Science research student. The research program student will be part of this research environment that has extensive knowledge in biochemistry, microbiology, and cell biology and many years of experience in relevant techniques such as growth of bacteria under aerobic and anaerobic conditions, protein expression, purification and enzymatic characterization, as well as light and fluorescence microscopy and eukaryotic cell cultures. The infrastructure required for the experiments described in the project proposal is in most part available in our laboratory and complemented by equipment at the nearby located Structural biology core facility lead by Jon Dalhus that we collaborate with regarding certain methods for protein characterization.
The scientific environment
My group together with the groups of Thomas Kuntziger and Morten Enersen have weekly groups meetings, where the members of the groups present and discuss their results in English. The purpose of these meetings is to allow the students to practice speaking English, improve their presentation techniques, and become comfortable speaking in front of their peers with the ultimate goal of preparing the students for the dissertation defense. In addition to this, we have a close collaboration with Professor Ole Andreas Økstad at the Department of Pharmacy, UiO. Our groups work with similar research questions and Professor Økstad and I co-supervise two PhD-students (one in each group), and we have regular scientific seminars 2 – 4 times per year where the students present their research and we discuss their results. The Dynamics of bacterial colonization research group also collaborates with the Norwegian Veterinary Institute (Anne-Margrete Urdahl, Karin Lagesen), clinical researchers in periodontitis (Anders Verket, UiO) and endodontic infections (Pia Titterud Sunde, UiO). We also collaborate with international research groups working on antibiotic resistance (Ian Paulsen, Macquarie University, Australia and Karl Hassan, University of Newcastle, Australia), cyclic dinucleotide signaling (Ute Römling, Karolinska Institutet, Sweden), and protein chemistry (Vincent Postis, Leeds Beckett University, United Kingdom) and Preben Morth (Technical University of Denmark, Denmark). These collaborations opens up for research stays abroad at world leading laboratories within relevant fields of research. The institute of Oral Biology organizes weekly scientific seminars where students and staff present and discuss current research projects as well as biweekly literature seminars were research students present and discuss scientific literature. Taken together, the environment of our research group provides the student with an excellent setting for scientific development.