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INFECTION AND IMMUNITY
Centre Infection and Immunity of Lille [CIIL]

CELLULAR MICROBIOLOGY OF INFECTIOUS PATHOGENS
Inserm U 1019 - CNRS UMR 8204
Institut Pasteur de Lille
Université Lille Nord de France
affiliated to IFR 142
Responsable
Frank LAFONT
00 33 3 20 87 11 36

 
 
Publications
2007 | 2008 | 2009 | 2010
PhD
Group members
BERTOUT Julie | CICZORA Yann | WAREIN Joëlle
Postdoctoral : POMEL Sébastien | SMAALI Nassima

 

RESEARCH REPORT
Frank Lafont inaugurated his team at the Pasteur Institute of Lille the 26th of September, 2005. The laboratory was initially equipped by grants form the French Minister for Research and Higher Education (Chaire d’Excellence) and from the charity foundation “Fondation pour la Recherche Médicale”. In 2005 and 2007, grants from the National Research agency were obtained as well as an ARCI program from the Région Nord Pas-de-Calais in 2008 and in 2006 the team obtained the “Fondation pour la Recherche Médicale” designation. The team has introduced several new techniques on the Lille Pasteur campus like atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF) microscopy.
During his post-doctoral training at the European Molecular Biology Laboratory in Heidelberg in Kai Simon’s team F. Lafont worked on the polarity, dynamics and compartmentalization of membrane microdomains. He became interested in the hijacking of lipid rafts by bacterial pathogens during his work as a junior lecturer in G van der Goot’s laboratory at the University of Geneva. In particular, he demonstrated that Shigella flexneri subverted rafts for the entry step and that the type three secretion system of S. flexneri interacts directly with raft molecular components. Also, while in Switzerland, he initiated an original research program on the use of AFM to investigate the biophysical properties of the plasma membrane in living cells with colleagues at the Swiss Federal Institute of Technology at Lausanne. This was the basis of a new line of research based on an interdisciplinary approach combining bacterial genetics, biochemistry, cellular and molecular biology, imaging and biophysics.
The working hypothesis of the team is that the mechanical properties of the plasma membrane influence the dynamics of the signaling cascade triggered by the adhesion of pathogens on cell membranes. One main original idea is that there is a basic component common to many adhesion and invasion phenomena that is hindered by specific interactions (that may rely on high and/or low affinities). By adhering to the cell surface, pathogens define specialized membrane domains. How does this confinement influence the cell signaling response? What is the molecular basis of the mechanical changes? How long is the signal maintained and how is it regulated? Which molecular components from the host and from the pathogen can be targeted for designing new therapeutic tools interfering with this early signaling response activated at the interface level? These are the questions the team addresses by using several pathogen models that have evolved different adhesion and invasion strategies. The team develops a translational project in which several pathogens are used as experimental models in order to analyze the similar and specific molecular mechanisms involved. The main point herein is that investigations are focused at the interface between the pathogen and the host cell membranes.

1. The membrane-associated signaling pathway during the first steps of Shigella flexneri invasion
We are interested in identifying signaling molecules that could be recruited to the host cell membranes (at the cell surface and vacuolar membrane levels) during the early steps of Shigella host cell invasion. As a first approach, we characterized the ubiquitinylation pathway. We reasoned that many signaling molecules are targets for ubiquitinylation. This led us to discriminate between mono-, multi- and polyubiquitinylated proteins. We obtained data for a massive ubiquitinylation occuring on vacuolar membranes after disruption that drives a sustained signaling activation until the membranes are trapped by the autophagy pathway. On these membranes, we identified molecular components of important signaling pathways leading to an inflammatory response (sequestosome, inflammasome) and to cell death (pyroptosis inducer caspase-1). This study addressed the original issue of the signaling associated with the remnant membranes left after the escape of the bacterium to the cytoplasm. We unveiled an underestimated signaling regulation at the vacuolar membrane that will open new avenues for a better understanding of how the cell response is orchestrated by intracellular invading bacteria like Shigella and Listeria.
In the course of this study we made use of an observation obtained in Sansonetti’s laboratory at the Pasteur Institute in Paris that shows the recruitment of galectin-3 as soon as the vacuolar membrane breaks. We were able to monitor the recruitment of Gal-3 by videoTIRF and deciphered the recruitment mechanism. We provided evidence that the glycosylated residues of the surface host molecules that were exposed at the cell surface, or were intraluminal in the vacuole, and that became accessible in the cytoplasm after vacuolar membrane disruption, recruit the cytoplasmic galectin-3.

2. The role of the septin cytoskeleton in Listeria monocytogenes invasion
The detailed mechanism allowing Listeria monocytogenes to invade the host cells awaits comprehensive characterization. Listeria uses the zipper mode of entry at variance with the triggered mode used by Shigella. However, both pathogens end up in the cytoplasm where they use an actin-based motility mechanism to invade the neighboring cells. Moreover, contrary to Shigella that binds loosely to the host cell surface, Listeria binds tightly to two receptors. Hence, the Listeria model offers a complementary experimental model to investigate the pathogen-host membrane interface. We are interested in the molecular basis of the adhesion of Listeria to the host cell surface and on the subsequent signaling response at the interface. We decided first to examine the mechanical features underlying the adhesion step. Listeria’s membrane receptors are anchored to the subcortical network of actin. In this context we wanted to investigate the role played by the septin family. The function of septins remains unclear, although they are involved in the regulation of the cytoskeleton and in the membrane-cytoskeleton relationships. We have shown in collabration with the P Cossart’s Team at the Pasteur Insitute in Paris, using atomic force microscopy (AFM), that depletion of septin family members leads to different cell shapes, either bigger or smaller than those of control cells, especially along the z-axis. Interestingly, invasion of Listeria monocytogenes into cells depleted for septins is differently affected depending on the family member targeted and is correlated with the cell shape. Also using AFM, we have shown that the interaction between one Listeria adhesin, InlB, and its receptor, Met, is also affected and correlated with the cell shape. By targeting some members of the septin family it is possible to modify the interaction forces between InlB and its receptor. As our measurements were performed on living cells, the interaction forces recorded take into account both the molecular interaction and the elasticity of the cell. Hence, our approach gave a hint concerning the anchorage of the receptor on the cytoskeleton and on the mechanical properties of the membrane engaged in adhesion to the microorganism. Altogether, these data led to the working hypothesis that septins can regulate the subcortical network on the cytoskeleton onto which the receptor is anchored. As such, this work using an original approach in the field gives a clue into the physiological function of septins.

3. The internalization pathway of Yersinia pseudo tuberculosis in host cells
Although Yersinia is described as being bound at the cell surface, where it can replicate, there are reports, both in vivo and in vitro, arguing for Yersinia internalization into macrophages. This pathogen, like Listeria, uses the zipper mode of invasion but remains intravesicular. With Yersinia, we are interested in identifying the molecular signaling machineries that are recruited at the host cell membrane level while the bacteria are internalized and replicating (interestingly bacteria remain closely associated with the vacuolar membrane as observed by electron microscopy). However, the membrane trafficking of the microorganism remained unclear and we first aimed at deciphering the entry pathway. We showed in collaboration with M. Simonet’s Team at the Institut Pasteur de Lille that Y. pseudotuberculosis hijacked membrane lipid rafts during its entry into the host cells. Then, the pathogen subverted the autophagy pathway as shown using specific markers and functional approaches based on the expression of dominant inhibitory mutants of this pathway. Moreover, we could establish a link between activation of autophagy and inhibition of apoptosis, which allows successful bacterial invasion. However, cell death is observed at later stage of infection and we could demonstrate that the binding of the bacterium to the cell surface triggered activation of pyroptosis that ultimately led to cell death and bacterial release.
We also have investigated the role of the microbiota flora in protection against Y. pseudotuberculosis and have shown that MyD88- and TLR2-dependent sensing is instrumental in eliciting degranulation of Paneth cells.

4. The role of knobs ultrastructure and elasticity in regulating cytoadherence
Once infected by Plasmodium falciparum, red blood cells express on their cell surface specific membrane structures connected to the underlying cytoskeleton that result from parasite protein synthesis. Some of these structures, known as knobs, are assumed to play an important role in the cytoadherence of the infected red blood cells to the vascular wall. With this experimental model, in collaboration with A. Scherf’s Team at the Pasteur Institute in Paris, we can investigate how a pathogen can modulate the mechanical properties of the host cell that in turn influence cytoadherence. We first aimed at characterizing, in a biophysical perspective, these membrane structures induced by the parasite. We have thus been able, using AFM, to characterize the ultrastructure of these knobs as a function of mutations and to correlate some phenotypic differences observed with different genotypes. The genotypes that gave the strongest differences as revealed with AFM were then used for further analysis of cell elasticity.

5. Interaction of LPS with the host cell surface
The LPS of Gram-negative bacteria is composed of a hydrophobic lipid A linked to a charged, densely compact oligosaccharidic core associated (smooth-LPS) or not (rough-LPS) with a hydrophilic O-polysaccharide chain (O-chain). LPS protects enteroinvasive bacteria against the inflammatory reaction and one of the bacterial elements that encounter the host is LPS. Interaction between LPS and the host cell plasma membrane may be responsible for stabilizing the bacterium onto the plasma membrane of the host. Using signature-tagged mutagenesis, genes invoved in the glucosylation of the tri-rhamnose-N-acetyl glucosamine tetrasaccharide O antigen of Shigella were shown to regulate the size of Shigella LPS molecule and regulate Shigella virulence. Therefore, it is important to check the influence of purified LPS in the reorganization of the host plasma membrane. Indeed, several lines of evidence suggest that Brucella LPS, by itself is an important modulator of membrane domains. Brucella possesses a peculiar LPS that we call non-classical LPS as compared with the so-called classical LPS from enterobacteria such as Escherichia coli. B. abortus lipid A possesses a diaminoglucose backbone (rather than glucosamine), and acyl groups are longer (C18–C19, C28 rather than C12 and C14) and are only linked to the core by amide bounds (rather than ester and amide bonds). In collaboration with JP. Gorvel’s Group at the CIML in Marseille, we thus wanted to characterize the interaction forces of several LPS from Brucella with the host plasma membrane of living cells. However, there is no method available for LPS coating onto the AFM tip. We have thus first established a method allowing targeting of the acyl chains of lipid A for tip functionalisation that will be then used in the field.

6. Mechanical adhesion properties of bacilli and bacteria
To better understand the microorganism - host cell interface, we investigate the adhesion properties of the microorganism in a simple experimental system, without the eukaryotic cell, in order to characterize some basic phenomena. We selected as experimental systems the adhesion of bacilli to inert surfaces, such as those used in food industries, and biofilm formation onto such substrata. This offered the possibility of investigating the mechanical features of pathogenic microorganisms (e.g. Bacilus anthracis and Listeria) and to address questions that are of high relevance for industry, i.e. contamination by adhering microorganisms. This project as part of a consortium with groups from the INRA, AFSSA, INSERM and INSA, launched during the second semester of 2008 aims at the characterization, using AFM, of the adhesion properties of spores (with B. Cereus as the experimental model for bacilli) onto metal substrata and biofilms (with Listeria as the model for contamination of fish products). Moreover, AFM will be also used to test on living cells the adhesion properties of bacilli expressing a mutant of BclA (Bacillus collagen-like protein of anthracis) that is localized in the exosporium and plays a role in bacilli adhesion. Conversely, tips functionalized with BclA will be used directly on living cells to measure interaction forces in corre lation with the local elasticity of the cell membrane.

PERSPECTIVES AND DEVELOPMENT 2010
Our project for the considered period will be to extend our analysis of the membrane-associated signalling activation triggered upon interaction of pathogens with the host cell membranes, based on our previous results and technological achievements. We previously found that membrane signalling can affect host cell survival, once infected, and the membrane traffic of different pathogens. Hence, we want to challenge our working hypothesis that upon pathogen interaction, the pathogen – host cell membrane interface defines membrane domains where recruited signalling molecules initiate pathways that regulate survival of the host and/or survival/replication of the pathogen. Our methodological strategy remains based on a multidisciplinary approach taking advantages of a new technological development related to the coupling of high-resolution biophotonic microscopy, to analyze the distribution of signalling molecules, and Atomic Force Microscopy (AFM), to examine the biophysical properties of the membrane into which the signalling molecules are recruited and activated.

 

 
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