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.
Role of CapG in the Immune System
In the macrophage, control of actin dynamics is achieved in part by the participation of Cap G, a protein discovered in this laboratory. This calcium-sensitive actin filament capping protein can regulate the assembly and disasssembly of actin-cytoskeleton during macrophage movement. CapG binds the barbed or rapid growing end of actin filaments in the presence of calcium. When the calcium concentration is lowered CapG dissociates from the barbed end allowing actin assembly to again occur. PCR generated point mutations have been made in the molecule to assess the relationship between primary structure and function. We have created gain-of-function mutations that convert CapG from a capping to a capping and severing protein addition. In collaboration with Dr. Stephen Almo (Albert Einstein University) we have crystallized wild-type and the severing mutant and map their tertiary structure.
Wild-type (left) and CapG-null neutrophils crawling toward a chemotactic gradient. Note the lack of shape change in the CapG-null cells.
A Cap G knock-out mouse has been generated to clarify the role of this actin regulatory protein in cytoskeleton function. CapG null macrophages fail to ruffle in response to MCSF and demonstrate depressed phagocytosis. Reintroduction of CapG by microinjection restores the ruffling response. Presently the ability of CapG null mice to mount an immune response is being investigated. CapG is found in neurite growth cones, therefore studies on neurite outgrowth and dendrite ruffling are also being pursued.
Recent Publication
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.