Cell to Cell Spread of Intracellular Pathogens
Dr. Southwick’s Lab
Investigating Host-Pathogen Interactions
Cell to Cell spread of Intracellular pathogens
L. monocytogenes and S. flexnerii are food borne pathogens and possess the ability to cross the intestinal epithelial barrier. L. monocytogenes surface proteins (e.g. Internalin A, Internalin B) interact with host cell surface receptors (e.g. E-cadherin, Met Receptor) to force entry of bacteria into epithelial and other non-phagocytic cells. Similarly, S.flexnerii enters epithelial cells using its Type III Secretion System (TTSS). Inside the cell, they break open the phagocytic vacuoles and rapidly multiply in the nutrient rich cytosol. Both bacteria then exploit host actin cytoskeleton to travel through the cytosol to reach cell membrane, where they extend filopodia-like structures to reach and subsequently enter the adjacent host cells. This mode of cell-to-cell spread protects the bacteria from humoral immune surveillance.
Initiation of actin polymerization begins with the activation of host Arp2/3 complex by L. monocytogenes ActA (which directly interacts with Arp2/3) or S. flexnerii IcsA (which acts through N-Wasp). This process is fairly well understood. In-vitro experiments suggest that only host Arp2/3 (actin nucleator), ADF/Cofilin (actin recycling) and CapZ (capping protein) are sufficient for actin based motility of these bacteria. The in-vitro experiments are extremely helpful in deciphering the mechanism of action of various proteins in actin polymerization, but may not reveal complex regulatory mechanisms that exist in a cell. We are investigating how intracellular pathogens manipulate the host cell physiology during cell-to-cell spread. We have shown that L. monocytogens requires PI3 Kinase to initiate and sustain actin based motility inside the cells whereas PI3 Kinase is not required for in-vitro motility. Similarly, cell-to-cell spread of S. flexnerii is facilitated by host Myosin 10 (http://youtu.be/siGbFSoOghQ). These findings suggest that other host factors may also be involved in the intracellular motility of these bacteria.
Listeria moving through PtK2 cell by actin-based motility.
Green = actin filament stain, Red = Listeria stain.
Note the long actin filament tails behind the moving bacteria.
Effect of Anthrax toxins on the Immune System
- In collaboration with the CDC we are investigating the effects of the anthrax toxins on the immune system. Lethal and Edema toxin are able to enter the cytoplasm of cells when combined with protective antigen, and have been shown to affect neutrophil chemotaxis and phagocytosis. By paralyzing the innate immune system anthrax is able to rapidly invade the host and cause fatal disease.
- We examined the effects of anthrax lethal toxin (LT) on human neutrophil chemotaxis, a process that requires actin filament assembly and discovered that exposure to LT markedly impairs PMN actin assembly, and reductions in actin filament content are accompanied by a profound paralysis of PMN chemotaxis. Our findings provide a previously unappreciated mechanism for LT virulence, and emphasize a central role for p38 MAP kinase-mediated phosphorylation of Hsp27 in actin-based motility and innate immunity. We propose that Hsp27 facilitates actin-based motility through a phosphorylation cycle that shuttles actin monomers to regions of new actin filament assembly. Anthrax edema toxin (ET) also significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize.
- Although hemorrhage is a prominent clinical manifestation of systemic anthrax, we found that anthrax lethal toxin fails to cleave its target, mitogen-activated protein kinase 1, and anthrax edema toxin fails to increase intracellular cyclic adenosine monophosphate. The toxins failed to attach to platelets, as platelets exhibit highly reduced expression of toxin receptors tumor endothelial marker 8 (TEM8) and capillary morphogenesis gene 2 (CMG2), as well as coreceptor low density lipoprotein receptor-related protein 6 (LRP6). Our studies suggest that the hemorrhagic clinical manifestations of systemic anthrax are unlikely to be caused by the direct binding and entry of anthrax toxins into human platelets.
- Listeria moving through PtK2 cell by actin-based motility. Green = actin filament stain, Red = Listeria stain. Note the long actin filament tails behind the moving bacteria.
Research on Improving the Quality and Safety of Patient Care
- In 2010 and 2011 Dr. Southwick served as an Advanced Leadership Fellow at the Harvard Business School. He learned how to apply business principles to health care and studied how to effectively lead and manage teams as well as how to organize people to bring about meaningful change. In addition to these skills he learned the latest approaches for preventing medical errors. Following his fellowship, Dr. Southwick has developed and implemented a multidisciplinary rounding program based on Athletic Principles that was recently published in Academic Medicine. The program significantly shortened patient length of stay as well as reduced hospital readmissions. This program had the added benefits of ishortening work rounds by 16 minutes and improving physician, nurse, and student satisfaction. He also has published a book outlining his recommendations for curing our dysfunctional healthcare delivery system entitled: Critically Ill A 5-point Plan to Cure Healthcare Delivery March 2014 2nd edition. And will be teaching an 8-week open online course through the UF branch of Coursera beginning September 1, 2014 entitled: Fixing Healthcare Delivery.
- Bacillus anthracis’ Lethal Toxin Induces Broad Transcriptional Responses in Human Peripheral Monocytes. Chauncey KM, Lopez MC, Sidhu G, Szarowicz SE, Baker HV, Quinn C, Southwick FS. BMC Immunol. 2012 Jul 2;13(1):33.
- Anthrax lethal and edema toxins fail to directly impair human platelet function. Chauncey KM, Szarowicz SE, Sidhu GS, During RL, Southwick FS. J Infect Dis. 2012 Feb 1;205(3):453-7.
- Brucella, voles, and emerging pathogens. Morris JG Jr, Southwick FS. J Infect Dis. 2010 Jul 1;202(1):1-2.
- Commentary: “Who was caring for Mary?” revisited: a call for all academic physicians caring for patients to focus on systems and quality improvement. Southwick FS, Spear SJ. Acad Med. 2009 Dec;84(12):1648-50.
- Bacillus anthracis edema toxin impairs neutrophil actin-based motility. Szarowicz SE, During RL, Li W, Quinn CP, Tang WJ, Southwick FS. Infect Immun. 2009 Jun;77(6):2455-64.
- Theodore E. Woodward Award: spare me the PowerPoint and bring back the medical textbook. Southwick FS. Trans Am Clin Climatol Assoc. 2007;118:115-22.
- Anthrax lethal toxin paralyzes actin-based motility by blocking Hsp27 phosphorylation. During RL, Gibson BG, Li W, Bishai EA, Sidhu GS, Landry J, Southwick FS. EMBO J. 2007 May 2;26(9):2240-50.
- A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing. Zhang Y, Vorobiev SM, Gibson BG, Hao B, Sidhu GS, Mishra VS, Yarmola EG, Bubb MR, Almo SC, Southwick FS. EMBO J. 2006 Oct 4;25(19):4458-67.
- Phosphoinositide 3-kinase is required for intracellular Listeria monocytogenes actin-based motility and filopod formation. Sidhu G, Li W, Laryngakis N, Bishai E, Balla T, Southwick F. J Biol Chem. 2005 Mar 25;280(12):11379-86.
- Anthrax lethal toxin paralyzes neutrophil actin-based motility. During RL, Li W, Hao B, Koenig JM, Stephens DS, Quinn CP, Southwick FS. J Infect Dis. 2005 Sep 1;192(5):837-45.
- CapG(-/-) mice have specific host defense defects that render them more susceptible than CapG(+/+) mice to Listeria monocytogenes infection but not to Salmonella enterica serovar Typhimurium infection. Parikh SS, Litherland SA, Clare-Salzler MJ, Li W, Gulig PA, Southwick FS. Infect Immun. 2003 Nov;71(11):6582-90.
- Chauncey, KM, MC.Lopez, G Sidhu1, S E Szarowicz, HV Baker,, C Quinn and FS Southwick. Bacillus anthracis Lethal Toxin Induces Broad Transcriptional Responses in Human Peripheral Monocytes. BMC Immunology 13: 33-58, 2012
- Bharwani, AM., Harris, GC, and Southwick, FS. A Business School Perspective on Medical Interprofessional Rounds: Transforming Rounding Groups into Rounding Teams. Acad. Med. 87: 1768-1771, 2012
- Bishai, EA, Sidhu, GS, Li, W, Dhillon, J, Bohil, AB, Cheney, RE, Hartwig, JH and Southwick, FS. Myosin-X facilitates Shigella-induced membrane protrusions and cell-to-cell spread. Cellular Micro. 15:353-67, 2013
- Southwick, F, Lewis, M, Treloar, D, Cherabuddi, K, Radhakrishnan, N, Leverence,, R, Han, X. and Cottler, L Applying Athletic Principles to Medical Rounds to Improve Teaching and Patient Care. Acad. Med. 89: July 2014