Professor of Biochemistry, Biophysics and Structural Biology*
*And Affiliate, Division of Immunology & Pathogenesis
The overall goal of our research is to understand the molecular and cellular basis of microbial pathogenesis and the mechanisms used by the host to defend against infection. Specifically, the lab is focused on the interaction of the facultative intracellular bacterial pathogen Listeria monocytogenes and its mammalian host. This fascinating microorganism is able to enter cells, escape from a phagosome and grow rapidly in the cytosol. By exploiting a host system of actin-based motility, the bacteria move through the cytosol to the cell membrane and into pseudopod-like projections (listeriopods) that are ingested by neighboring cells. This mechanism allows pathogens to spread from one cell to another without ever leaving the host cytoplasm thereby avoiding the immune response.
Escape from a vacuole and cell to cell spread of L. monocytogenes. The stippled material depicts F-actin.
Cell biology of infection. The primary L. monocytogenes determinant responsible for lysis of host cell vacuoles is the pore-forming cytolysin, listeriolysin O (LLO). We will continue to focus on the control of LLO synthesis and secretion, and its mechanism of action. The ultimate goal is to relate structural and biochemical information to its precise mechanism of action in both tissue culture and in mice. We are also characterizing a number of fail-safe mechanisms that prevent LLO toxicity in the host cytosol and thereby compartmentalize its activity to acidic vacuoles. Interestingly, mutants that fail to properly compartmentalize LLO activity are cytotoxic to infected host cells and attenuated for virulence in mice.
Innate immunity to infection. Murine listeriosis is an outstanding model to study basic aspects of innate and acquired cell-mediated immunity. Using bacterial mutants blocked at various stages in the infection process, we are elucidating pathways of host cell gene expression in response to microbial infection. Our studies clearly document the presence of a vacuolar and cytosolic pathway of innate immune recognition. Most recently, we identified that bacteria secrete a c-di-AMP that activates a host protein called STING leading to the transcription of interferon. We are currently investigating the role of this pathway during infection and immunity.
Bacterial determinants that control pathogenesis. We continue to use genetic screens and genomic approaches to identify and characterize bacterial determinants required for pathogenesis. Among the bacterial factors we are currently studying include enzymes that synthesize, degrade, and export c-di-AMP during bacterial growth both in culture and in cells. Most recently, we've identified a set of bacterial factors that respond to redox stress and are specifically necessary for growth in macrophages.
Acquired immunity to infection and vaccine development. Mice that survive a challenge with sublethal doses of virulent L. monocytogenes acquire antigen-specific cell-mediated immunity that renders the mice resistant to subsequent challenge. Importantly, killed bacteria or bacterial mutants unable to access the host cell cytosol fail to induce immunity, while mutants that enter the cytosol, but fail to spread from cell to cell retain their capacity to induce immunity. We are interested on both the bacterial and host factors that contribute to immunity. These studies have implications for the rational design of vaccines. Indeed, L. monocytogenes is being developed as a vector-based vaccine for both cancer immunotherapy and infectious diseases applications. We are currently working on L. monocytogenes-based vaccines expressing Mycobacterium tuberculosis antigens as potential tuberculosis vaccines.
Manzanillo, P.S., Shiloh, M.U., Portnoy, D.A., Cox, J.S. Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages. Cell Host Microbe. 11:469-480. (2012).
Melton-Witt, J.A., McKay, S.L., Portnoy, D.A. Development of single-gene, signature-tag-based approach in combination with alanine mutagenesis to identify listeriolysin O residues critical for the in vivo survival of Listeria monocytogenes. Infect Immun. 80(6):2221-30. Epub 2012 Mar 26. (2012).
Witte, C.E., Archer, K.A., Rae, C.S., Sauer, J.D., Woodward, J.J., and Portnoy, D.A. Innate immune pathways triggered by Listeria monocytogenes and their role in the induction of cell-mediated immunity. Review. Advances in Immunology. 113: 135-156. (2012).
Sauer, JD., Peryere, S., Archer, K.A., Hanson, B., Lauer, P., Portnoy, D.A.Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and fail to induce protective immunity. Proc. Natl. Acad. Sci. USA. 108(30):12419-24. (2011).
Woodward, J.J., A.T. Iavarone, Portnoy, D.A. c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response. Science. 328: 1703-1705. (2010).
Sauer, John-Demian, C.E. Witte, J. Zemansky, B. Hanson, P. Lauer, and Portnoy, D.A. Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol. Cell Host Microbe. 7(5):412-9. (2010).
Bahjat, K.S., N. Meyer-Morse, E.E. Lemmens, J.A. Shugart, T.W. Dubensky, Jr., D.G. Brockstedt, and Portnoy, D.A. Suppression of cell-mediated immunity following recognition of phagosome-confined bacteria. PLoS Pathog. 5(9):e1000568. (2009).
Zemansky, J., B. Kline, J.J. Woodward, J. H. Leber, H, Marquis, and Portnoy, D.A. Development of a mariner-based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-proline isomerase, PrsA2, that contribute to its hemolytic phenotype. J Bacteriol. 191(12):3950-64. (2009).
Crimmins, G. T., A. A. Herskovits, K. Rehder, K.E. Sivick, P. Lauer, T. W. Dubensky, and Portnoy, D.A. Listeria monocytogenes multi-drug efflux resistance transporters activate a cytosolic surveillance pathway of innate immunity. Proc Natl Acad Sci U S A. 105(29):10191-6. (2008).
Leber, J. H., G. T. Crimmins, S. Raghavan, N. P. Meyer-Morse, J. S. Cox, and D. A. Portnoy. Distinct TLR- and NLR-mediated transcriptional responses to an intracellular pathogen. PloS Pathogens. 4(1):e6 (2008).
Last Updated 2012-08-16