Team Frank LAFONT

Cellular Microbiology of Infectious Pathogens

According to the WHO 2005 world report, infectious diseases constitute the second leading cause for human mortality. New emerging pathogens and threat of outbreaks responsible of epidemic and pandemic leading to high rate of morbidity and mortality reinforce the research effort devoted to infectious agents. Also, the possibility of using pathogens for bioterrorism actions implies the development of new efficient therapeutic strategies.

We are interested in understanding the early steps of infection at the molecular and cellular levels. Our research focuses on the mechanisms hijacked by pathogens to interact and possibly invade the host cells. Pathogens have evolved many strategies to subvert molecular machineries of the host to escape immune response and manage a successful invasion.

We want to develop an interdisciplinary approach combining bacterial genetics, microbiology, cell and molecular biology, biochemistry and biophysics to investigate how pathogens interact with the host cell surface, how the cell responds to this attack and how pathogens subvert this response. The aim is to identify new therapeutic targets and to design new tools in order to inhibit infection.

We have been interested for many years in the plasma membrane organization and especially in the identification of molecular machineries associated with specific membrane sub-micron domains (Fiedler et al. 1995; Lafont et al, 1998, 1999; Verkade et al 2000; Plant et al, 2000; Lecat et al., 2000). These domains, so-called lipid rafts (Simons and van Meer, 1988; Simons and Ikonen, 1997), have attracted growing interest as they have been proposed to play an important role in several diseases such as neurodegenerative disorders (Alzheimer diseases, Prion, Parkinson, …), lipid storage diseases (Niewmann-Pick disease), lipid metabolism diseases (diabetes, atherosclerosis), asthma, allergic B and T cell responses (Simons and Ehehalt, 2002). Viruses, parasites, bacteria and bacterial toxins are also using these domains (Lafont et al, 2004; Lafont and van der Goot, 2005).

We are studying at the nanoscale resolution the host-pathogen interactions using a near-field approach. We have characterized by atomic force microscopy (AFM) with functionalized cantilevers on living cells the distribution and interaction forces of bacterial toxins and adhesins with their cellular receptors (Roduit et al., 2008; Dupres et al., 2010; Mostowy et al., 2011).  We moreover developed a method to analyze the biophysical features of the plasma membrane engaged in these interactions (Roduit et al., 2008; Mostowy et al., 2011). This led us to launch research towards stiffness tomography of living cells (Roduit et al., 2009). We now challenge this possibility by coupling AFM to super high-resolution biophotonic measurements.

Although mainly extracellular, Yersinia pseudotuberculosis can in vitro and in vivo invade macrophages. We have unveiled how Y. pseudotuberculosis hijacks the autophagy pathway, blocking the maturation of autophagosomes in order to replicate within intracellular niches (Moreau et al., 2010). We have shown that after S. flexneri entry, the membrane remnants (identified by the recruitment of the galectin-3 (Paz et al., 2009)) left after the bacteria have escaped to the cytoplasm are targeted to autophagy (Dupont et al., 2009a). In the same study, we moreover demonstrated the implication of ubiquitylation and adaptors such as P62. We also could analyze the participation of autophagy in the cell response investigating cell death (necrosis and pyroptosis pathways). We hypothesized that targeting to destruction the membrane-associated signaling triggered upon bacteria entry participates in shutting down the cellular defense response (Dupont et al., 2009b). This has launched research on how ubiquitylation, adaptors and autophagy participate in the orchestration of the cellular response to infection by modulating both the fate of the pathogen and that of the host cell (Dupont et al., 2010; Ligeon et al., 2011).

Our working hypothesis is that a part of the cell signaling response is triggered upon adhesion of the pathogen independently of the specific interaction mediated by interaction between high affinity binding sites. This part of the signaling could be partially due to the biophysical mechanical properties of the membrane. Upon adhesion some signaling molecules could be recruited at the level of the membrane that senses the stress due to the adhesion of the bug. We want to understand this phenomenon. To reach this long-term goal, we decided to investigate several pathogen infection models with microorganisms interacting and entering into the host cell using different mechanisms. We want to decipher the signaling complexes that are recruited at the membrane level to analyze similarities and differences. One of our approaches is based on studying the elastic properties of the interacting membranes and simultaneously the dynamics of signaling molecules recruited upon pathogen binding. Another approach is to analyze by high-content and high-throughput microscopy screening signaling molecules involved in the early steps of infection focusing our research to membrane-associated proteins. This is a very exciting topic both in term of techniques to develop, technological barriers to overcome, concepts to challenge, and mechanisms to elucidate. We think that this approach will bring insights to open new avenues for the design of new drugs. This drives our motivation. We hope that by surfing on our pages you will share our enthusiasm!

 

References :

Fiedler K, Lafont F, Parton RG, Simons K. Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane. J Cell Biol. (1995) 128:1043-53.

Lafont F, Lecat S, Verkade P, Simons K. Annexin XIIIb associates with lipid microdomains to function in apical delivery. J Cell Biol. (1998) 142:1413-27.

Lafont F, Verkade P, Galli T, Wimmer C, Louvard D, Simons K. Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells. Proc Natl Acad Sci U S A. (1999) 96:3734-8.

Lafont F, Tran Van Nhieu G, Hanada K, Sansonetti P, van der Goot FG. Initial steps of Shigella infection depend on the cholesterol/sphingolipid raft-mediated CD44-IpaB interaction. EMBO J. (2002) 21:4449-57.

Lafont F, Abrami L, van der Goot FG. Bacterial subversion of lipid rafts. Curr Opin Microbiol. (2004) 7:4-10.

Lafont F, van der Goot FG. Bacterial invasion via lipid rafts. Cell Microbiol. (2005) 7:613-20.

Lecat S, Verkade P, Thiele C, Fiedler K, Simons K, Lafont F. Different properties of two isoforms of annexin XIII in MDCK cells. J Cell Sci. (2000) 113:2607-18.

Plant PJ, Lafont F, Lecat S, Verkade P, Simons K, Rotin D. Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb. J Cell Biol. (2000) 149:1473-84.

Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin Invest. (2002) 110:597-603.

Simons K, Ikonen E. Functional rafts in cell membranes. Nature. (1997) 387:569-72.

Simons K, van Meer G. Lipid sorting in epithelial cells. Biochemistry. (1988) 27:6197-202.

Verkade P, Harder T, Lafont F, Simons K. Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells. J Cell Biol. (2000) 148:727-39.

Yersin A., Hirling H., Kasas S., Roduit C., Kulangara K., Dietler G., Lafont F., Catsicas S., Steiner P. Elastic properties of the cell surface and trafficking of single AMPA receptors in living primary hippocampal neurons. Biophys. J. (2007) 92, 4482-4489.

Roduit C., van der Goot F.G., De Los Rios P., Yersin A., Steiner P., Dietler G., Catsicas S., Lafont F. * and Kasas S.*.  Elastic membrane heterogeneity of living cells revealed by stiff nanoscale membrane domains. Biophys. J. (2008) 94, 1521-1532.

Dupres V., Verbelen C., Raze D., Lafont F. and Dufrêne Y.F. Force spectroscopy of the interaction between mycobacterial adhesions and heparan sulphate proteoglycan receptors ChemPhysChem (2009) 10:1672-1675.

Dupont N, Lafont F. How autophagy regulates the host cell signaling associated with the postpartum bacteria cocoon experienced as a danger signal.Autophagy. (2009) 5:1222-3.

Roduit C., Dietler G., Sekatski S., Catsicas S., Lafont F. and Kasas S.. Stiffness tomography by atomic force microscopy. Biophys. J. (2009) 97:674-677.

Dupont N, Lacas-Gervais S, Bertout J, Paz I, Freche B, Van Nhieu GT, van der Goot FG, Sansonetti PJ, Lafont F. Shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy. Cell Host Microbe. (2009) 6:137-49.

Paz I, Sachse M, Dupont N, Cederfur C, Enninga J, Leffler H, Poirier F, Prevost MC, Lafont F, Sansonetti P. Galectin-3, a marker for vacuole lysis by invasive pathogens. Cell Microbiol. (2010) 12 :530-544.

Moreau K, Lacas-Gervais S, Fujita N, Sebbane F, Yoshimori T, Simonet M, Lafont F. Autophagosomes can support Yersinia pseudotuberculosis replication in macrophages. Cell Microbiol. (2010) 12 :1108-1123.

Dupont N., Temime N. and Lafont F How ubiquitination and autophagy participate in the regulation of the cell response to bacterial infection Biology of the Cell (2010) 102:621-634.

Mostowy S, Janel S, Forestier C, Roduit C, Kasas S, Pizarro-Cerda J, Cossart P, Lafont F A Role for Septins in the Interaction between the Listeria monocytogenes Invasion Protein InlB and the Met Receptor. Biophys. J. (2011) 100 :149-159.

Ligeon L-A., Temime-Smaali N., and Lafont F. Ubiquitylation and autophagy in the control of bacterial infections and related inflammatory responses (2011) Cell. Microbiol. In press

 

For more information (complete list of research projects, publications, people) please go to: http://www.cmip.cnrs.fr/

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