Edward E. Penhoet Distinguished Chair in Global Public Health and Infectious Diseases and 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, circumvent autophagy, avoid cell death pathways 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). LLO is expressed in a phagosome and triggers autophagy, but the bacteria are able to escape from autophagy. Upon entry into the cytosol, LLO is still expression, but has a number of fail-safe mechanisms that prevent its toxicity in the host cytosol and thereby compartmentalize its activity to acidic vacuoles. For example, during infection LLO is ubiquitylated and has two fates; one it is degraded by the proteasome while a second pool is sequestered into aggregates that are subject to autophagy. 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 small signaling molecule, c-di-AMP, through bacterial multidrug efflux pumps that activates a host cytosolic protein called STING leading to the transcription of type I interferon and co-regulated genes. 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. Also, we are using genetics and biochemistry to deduce the role played by c-di-AMP for bacterial growth. Most recently, we've identified a set of bacterial factors that respond to redox stress and are specifically necessary for growth in macrophages. Bacterial and host derived glutathione are required to activate bacterial virulence gene expression.
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. Surprisingly, in the context of L. monocytogenes immunity, the STING pathway has a negative impact on development of adaptive immunity. These studies have implications for the rational design of vaccines. Indeed, L. monocytogenes is being developed in the private sector as a vector-based vaccine for both cancer immunotherapy and infectious diseases applications.
McKay SL and Portnoy DA. Ribosome hibernation facilitates tolerance of stationary-phase bacteria to aminoglycosides. In press, Antimicrobial Agents and Chemotherapy. (2015).
Whiteley AT, Pollock AJ, Portnoy DA. The PAMP c-di-AMP is essential for Listeria growth in macrophages and rich but not minimal media, due to a toxic increase in (p)ppGpp. Cell Host Microbe. 2015 Jun 10;17(6):788-98. (2015).
Kellenberger CA, Chen C, Whiteley AT, Portnoy DA, Hammond MC. RNA-based fluorescent biosensors for live cell imaging of second messenger cyclic di-AMP. J Am Chem Soc. 2015 May 27;137(20):6432-5. (2015).
Mitchell G, Ge L, Huang Q, Chen C, Kianian S, Roberts M, Schekman R, Portnoy DA. Avoidance of autophagy mediated by PlcA or ActA is required for Listeria monocytogenes growth in macrophages. Infect Immun. 2015 May;83(5):2175-84. (2015).
Fu J, Kanne DB, Leong M, Hix Glickman L, McWhirter SM, Lemmens E, Mechette K, Leong JJ, Lauer P, Liu W, Sivick KE, Zeng Q, Soares KC, Zheng L, Portnoy DA, Woodward JJ, Pardoll DM, Dubensky Jr TW, Kim Y. STING agonist formulated cancer vaccines can cure established tumor resistant to PD-1 blockade. Sci Transl Med. 2015 Apr 15;7(283):283ra52. (2015).
Siegrist MS, Aditham A, Espaillat A, Cameron T, Whiteside S, Cava F, Portnoy DA, Bertozzi CR. Host actin polymerization tunes the cell division cycle of an intracellular pathogen. Cell Rep. 2015 Apr 28;11(4):499-507. (2015).
Reniere ML, Whiteley AT, Hamilton KL, John SM, Lauer P, Brennan RG, Portnoy DA. Glutathione activates virulence gene expression of an intracellular pathogen. Article. Nature. 2015 Jan 8;517(7533):170-3. (2015).
Durack J, Burke TP, Portnoy DA. A prl mutation in secY suppresses secretion and virulence defects of Listeria monocytogenes secA2 mutants. J. Bacteriol. 2015 Mar;197(5):932-42. (2015).
Kline B, McKay S, Tang W, Portnoy DA. The Listeria monocytogenes hibernation-promoting factor (HPF) is required for the formation of 100S ribosomes, optimal fitness, and pathogenesis. J Bacteriol. 2015 Feb 1;197(3):581-91. (2015).
Burke TP, Loukitcheva A, Zemansky J, Wheeler R, Boneca IG, Portnoy DA. Listeria monocytogenes is resistant to lysozyme by the regulation, not acquisition, of cell wall modifying enzymes. J. Bacteriol. 2014 Nov;196(21):3756-67. (2014).
Archer, K.A., Durack, J., and Portnoy, D.A. STING-dependent Type I IFN production inhibits cell-mediated immunity to Listeria monocytogenes. PLoS Pathog. 2014 Jan;10(1):e1003861. (2014).
Witte C.E., Whiteley A.T., Burke T.P., Sauer, J.D., Portnoy, D.A., Woodward J.J. Cyclic di-AMP is critical for Listeria monocytogenes growth, cell wall homeostasis, and establishment of infection. mBio. 4(3). Doi:pii:e00282-13. 1128/mBio.00282-13. (2013).
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).
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).
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).
Photo credit: Mark Hanson at Mark Joseph Studios.
Last Updated 2015-02-03