Type IV Pili, Microbes and Disease Microbes have evolved numerous mechanisms to adapt to, and co-exist with, their human host. Our goal is to define these mechanisms in molecular terms, and to understand how they influence the genesis of disease. We focus on two bacterial pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, and the role their Type IV pili play in infection. Using cell biology, biochemistry and biophysics approaches we elucidate the bacteria-host interactions driven by Type IV pili, and the biological consequences that result from them. Read about:Â Type IV pili, in briefType IV pili are filamentous surface structures expressed by many pathogenic and free-living bacteria. The image at the top of the screen shows bundles of Tfp fibers extending from a microcolony of N. gonorrhoeae cells. The pilus fiber is a dynamic structure that undergoes cycles of extension, substrate tethering and retraction. Multiple pili engaging in this process power twitching motility (crawling over surfaces). Pilus retraction also enables the bacterium to take up exogenous DNA (a form of genetic exchange), engage in biofilm formation and quorum sensing, and, in the case of some pathogens, attach to host cells. In the case of Neisseriae, pilus retraction also transduces signals into the infected cell. top Measuring pilus retraction forces
With our collaborators M. Sheetz and N. Biais (Columbia U), we are characterizing the biophysical parameters of pilus retraction. By means of laser tweezers we measured filament retraction forces of 50-100 picoNewtons (pN) (1). Pilus filaments form bundles. Using hydrogel micropillars we measured bundle retraction forces that reach 1 nanoNewton (2). (To put the latter forces into perspective, a human exerting a proportionate force can move an object 10,000 times his/her body weight.) Â Â Title: Laser tweezers analysis of pilus retraction. In this videorecording, a small bead (in the laser trap) can be seen to move towards the diplococcus adhered to the large bead. The small bead subsequently relaxes back into the laser trap then moves again towards the large bead. The forward movements of the small bead are due to retraction of the pilus that is attached to the small bead. See refs (1,2) for details. top Type IV pili establish a physical and chemical dialogue with epithelial cellsDuring infection, the force of pilus retraction activates signaling cascades (3,4) and alters gene expression (4) in the infected cell. These events affect the cell in many ways. They stimulate the cell to produce its own chemical that further enhances bacterial aggregation into microcolonies (3). These enlarging microcolonies pull on the cell with higher forces, presumably forming and reenforcing a feedback loop. Thus begins a two-way, multi-dimensional communication system between bacteria and epithelial cell.
Retraction force induces a massive remodeling of the host cell cortex at the site of infection: microvilli beneath the microcolony are extended and distorted; membrane receptors and cytoskeleton components cluster beneath the microcolony in structures called cortical plaques (3,5). Our working hypothesis is that the plaque functions as a signaling center that controls many early host cell responses to infection. We have only begun to delve into the signals sent through these plaques.  Legend: Fluorescence microscopy image of N. meningitidis microcolonies (yellow) attached to human epithelial cells (blue). See (5) for details. Legend: Scanning Electron Microscopy image of N. gonorrhoeae microcolonies (pseudocolored in blue) attached to human epithelial cells. Microvilli at the edges of microcolonies are elongated and deformed by interactions with the bacteria; some microvilli can be seen to wrap around the bacteria (6). Force-induced signaling has yet another outcome. Neisseria induces an apoptotic cell death response in the infected cell. Pilus retraction force overrides this response – through the activation of signal cascades that interfere with apoptosis pathways (7). It is counterintuitive that a pathogen would evolve a mechanism that lowers virulence. However, in the case of a human-specific pathogen such as Neisseria, it is not in the best interest of the pathogen to damage the host, as this would lower its chances of transmission and ultimately survival. Legend: Videomicroscopy of human epithelial cells expressing actin-GFP infected with wt N. gonorrhoeae strain MS11 (5a) or MS11pilT (5b). The pilT mutant produces pili that are not retractible. The mutant adheres to cells but is nonmotile. See (6) for details. top Type IV pilus signaling through CD46CD46 is an immunoregulatory protein on the membrane of nucleated human cells; it also acts as a signaling intermediate during Neisseria infection. Bacterial attachment to host cells via Type IV pili stimulates tyrosine-phosphorylation of one of the two CD46 isoforms, and reroutes CD46 to the cortical plaque (7,8). We recently discovered that infection stimulates Presenilin/γ-Secretase to process CD46, releasing the cytosolic tail. Furthermore, Presenilin cleavage of CD46 is enhanced by pilus retraction. Presenilin/γ-Secretase is a protease complex that regulates many membrane proteins through proteolytic processing. The Presenilin-released cytosolic tails of many of these proteins have signaling and/or transcriptional activity. Our working hypothesis is that the Presenilin-released CD46 tails participate in signaling in the cytosol and/or transactivate transcription in the nucleus.  Legend: This cartoon illustrates steps involved in Presenilin/γ-Secretase cleavage of a substrate, and lists the functions of some of the released tails. First, the substrate is cleaved by a matrix metalloprotease, then by Presenilin/γ-Secretase. The released tail traffics to various parts of the cells, depending on the substrate, where it influences cell function. top Commensalism, Co-adaptation, Co-evolutionMany bacteria belonging to the Neisseria genus colonize the human body without causing disease. The relationship between bacteria and man is considered commensalistic as the host provides a niche for the bacteria and the bacteria do no harm to the host. The two pathogenic Neisseria species, N. gonorrhoeae and N. meningitidis, can also colonize the human body for lengths of time without causing disease. They are establishing a “carrier state†within the host that is reminiscent of commensalism. We have begun to study Neisseria commensalism. We believe that understanding the basis of commensalism will help us understand how the pathogens cause disease. top Lab Members and Collaborator top Publications and Other Reading MaterialSelected So Lab Publications Merz, A.J., So, M. and Sheetz, M. (2000) M. Pilus retraction powers bacterial motility Nature 407:98-101. Biais, N., Ladoux, B., Higashi, D.L., So, M. and Sheetz, M. (2008) Cooperative retraction of bundled Type IV pili enables nanonewton force generation. PLoS Biol. 6:e87. Lee, S., Higashi, D.L., Snyder, A., Merz, A.J., Potter, L. and So, M. (2005) PilT is required for PI[3,4,5]P3-mediated crosstalk between Neisseria gonorrhoeae and epithelial cells. Cell Micro. 7:1271-1284. Howie, H.L., Glogauer, M. and So, M. (2005) N. gonorrhoeae Type IV pilus stimulates mechanosensitive pathways and cytoprotection through a pilT-dependent mechanism. PLoS Biol. 4:e100. Merz, A.J., Enns, C.A. and So, M. (1999) Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells. Mol. Microbiol. 32:1316-1332. Higashi, D.L., Lee, S.W., Snyder, A. Weyand, N.J., Bakke, A. and So, M. (2007) Dynamics of Neisseria gonorrhoeae Attachment: Microcolony Development, Cortical Plaque Formation and Cytoprotection, Infect. Immun. 75:4743-4753. Howie, H., Shiflett and So, M. (2008) ERK mediated downregulation of Bim and Bad during infection with N. gonorrhoeae. Infect Immun. 76:2715-2721. Lee, S.W., Bonnah, R. A., Milgram, S.L., Atkinson, J. and So, M. (2001) CD46 is tyrosine phosphorylated at tyrosine 354 upon infection of epithelial cells by N. gonorrhoeae. J. Cell Biol. 156:951. Weyand, N., Lee, S.W., Higashi, D.L., Cawley, D., Yoshihara, P. and So, M. (2006) Monoclonal antibody detection of CD46 clustering beneath Neisseria gonorrhoeae microcolonies. Infect Immun. 74:2428-2435.
Type IV Pili
Mattick, J.S. (2002) Type IV pili and twitching motility. Annu. Rev. Microbiol. 56:289-314. Merz, A. and So, M. (2000) Interactions of pathogenic Neisseriae with epithelial cell membranes. Ann. Rev. Cell Devel. Biol.16:423-457. Merz, A.J. and Forest, K.T. (2002) Bacterial surface motility: slime trails, grappling hooks and nozzles. Curr. Biol. 12:R297-303. Forest, K.T. and Tainer, J.A. (2007) Type-4 pilus structure: outside to inside and top to bottom-a view. Gene 192:165-169. Craig, L., Volkmann, N., Arvai, A.S., Pique, M.E., Yeager, M., Egelman, E.H. and Tainer, J.A. (2006) Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol. Cell. 23:651-662. Measuring Biological Forces Moffitt, J.R. et al. (2008) Recent Advances in Optical Tweezers. Annu. Rev. Biochem. 77:205-228. Tanase, M., Biais, N. and Sheetz, M. (2007) Magnetic Tweezers in Cell Biology. In: Methods in Cell Biology. Elsevier, NY. pp. 473-493. CD46 Gill, D.B. and Atkinson, J.P. CD46 in Neisseria pathogenesis. Trends in Mol. Med. 10:459-465. Astier, A. (2008) T-cell regulation by CD46 and its relevance in multiple schlerosis. Immunology. 124:149-154. Marie, J.C. et al. (2002) Linking innate and acquired immunity: divergent roles of CD46 cytoplasmic domains in T cell-induced inflammation. 3:659-666
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