Interaction of bacterial pathogens with host cells
Our general interest is in the interaction of bacterial pathogens with mammalian cells, interactions that promote colonization and provide insight into central functions of eukaryotic cells. These interactions also trigger immune responses that can enhance tissue damage and/or result in clearance of the pathogen.
Enterohemorrhagic Escherichia coli
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is important intestinal pathogen that, like several gram negative pathogens, utilizes a specialized secretion system to inject bacterial “effectors” into mammalian cells. These effectors directly target eukaryotic signaling pathways or cellular functions to promote the infectious process. EHEC is able to stimulate robust actin polymerization in the host cell, leading to a large actin ‘pedestal’ directly beneath the bound bacterium (Fig 1).
Figure 1. Enterohemorrhagic E. coli O157:H7 (blue) induces filamentous actin pedestals (red) upon binding mammalian cells. EspFU, a bacterial protein (yellow) that is translocated into the host cell, is localized at the tip of the pedestal.
We have identified bacterial effectors that are required for pedestal formation, and shown that one of these, termed EspFU, activates the eukaryotic actin nucleation promoting factor WASP by mimicking and thus displacing a WASP helix critical for control of WASP activity (Fig 2). Thus, EspFU provides insight into fundamental mechanisms of actin assembly in mammalian cells.
Figure 2. The nucleation promoting factor WASP was previously shown to be retained in an inactive conformation by an interaction between an amphipathic helix in the WCA domain (red) and the WASP GBD (GTPase Binding Domain; blue; see Kim et al., Nature 404, 151-8 (2000)). The EHEC effector EspFU (green) harbors a helix that binds to the same hydrophobic grove in the WASP GBD, thus activating WASP to signal actin assembly (Cheng et al, 2008 ).
An equally important goal is to understand how interactions between these bacteria and the intestinal epithelium promote colonization and disease. EHEC localized in the intestine can produce the potent toxin Shigatoxin that penetrates the epithelial barrier to cause serious systemic disease. We have been developing a murine model for Shigatoxin-mediated disease, and are utilizing this model to test whether actin rearrangement triggered by EHEC promotes epithelial translocation of Shigatoxin and the promotion of systemic disease.
Lyme disease and relapsing fever bacteria of the genus Borreliae are tick-borne spirochetes (Fig 3) that cause multisystemic infection. We investigate the spirochetal factors that promote colonization, and components of the host immune response that contribute to spirochetal clearance. A confounding factor in the analysis of spirochetal adhesion is the presence of many potentially redundant adhesion pathways. An approach that my laboratory has pioneered is the conversion of an otherwise nonadherent (and noninfectious) B. burgdorferi strain into a highly adherent strain by the ectopic expression of a single spirochetal adhesin, permitting a straightforward analysis of a given adhesion pathway in the absence of confounding pathways. Then, to assess the role of specific adhesion pathways in vivo, we infect animals with strains that are either deficient in a single adhesion pathway or deficient in all but a single adhesion pathway. Our goal is a comprehensive understanding of the mechanism and function of the multiple bacterial binding pathways of this invasive pathogen.
Figure 3. Borrelia hermsii, the relapsing fever spirochete (orange) attaches to red blood cells (green) during mammalian infection (Benoit, et al, 2010; photo by Annette Moter )
Lyme disease and relapsing fever spirochetes not only establish systemic infection, but also often are capable of resisting immune clearance. We are utilizing highly tractable murine infection models to elucidate mechanisms of host defense, including a novel mechanism of T cell-independent immunologic memory.
Invasive pneumococcal disease caused by Streptococcus pneumoniae is responsible for over 1.6 million deaths per year worldwide. Lung infection, one of the most common forms of invasive pneumococcal disease, is associated with a robust influx of polymorphonuclear cells (PMNs) into alveolar spaces. This acute inflammatory response, due to its nonspecific mode of action, causes significant tissue damage and contributes to pneumococcal disease. We have found that the lipid-based chemoattractant hepoxilin A3 (HXA3), an arachidonic acid metabolite generated via the 12-lipoxygenase (12-LOX) pathway, is a critical mediator of S. pneumoniae-induced PMN migration. Blocking the production of HXA3 virtually eliminated PMN migration in response to S. pneumoniae infection in cultured monolayers and significantly diminished pulmonary inflammation after experimental infection of mice (Figure 4). Notably, mice unable to produce HXA3 were completely protected from an otherwise lethal dose of S. pneumoniae, indicating that during this infection the acute inflammatory response is detrimental to the host.
Figure 4. Mice that had been mock-treated or treated with a 12-LOS inhibitor were infected intratracheally with Streptococcus pneumoniae, and lungs were H&E stained after 2 days of infection.
Intranasal colonization by S. pneumoniae is very common in humans and may represent a natural immunization by which most individuals are protected from serious pneumococcal infections. We found that nasopharyngeal colonization with S. pneumoniae results in subsequent protection from lung infections. We have developed evidence that T cells contribute to a protective colonization-induced antibody response by radio-resistant, differentiated plasma cells. A detailed understanding of this response may lead to strategies for prevention of invasive S. pneumoniae infection.