Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.

Effects of antibacterial agents and drugs monitored by atomic force microscopy

Sandor Kasas, Giovanni Longo

Originally invented for topographic imaging, atomic force microscopy (AFM) has evolved into a multifunctional biological toolkit, enabling to measure structural and functional details of cells and molecules. Its versatility and the large scope of information it can yield make it an invaluable tool in any biologically oriented laboratory, where researchers need to perform characterizations of living samples as well as single molecules in quasi-physiological conditions and with nanoscale resolution. In the last 20years, AFM has revolutionized the characterization of microbial cells by allowing a better understanding of their cell wall and of the mechanism of action of drugs and by becoming itself a powerful diagnostic tool to study bacteria. Indeed, AFM is much more than a high-resolution microscopy technique. It can reconstruct force maps that can be used to explore the nanomechanical properties of microorganisms and probe at the same time the morphological and mechanical modifications induced by external stimuli. Furthermore it can be used to map chemical species or specific receptors with nanometric resolution directly on the membranes of living organisms. In summary, AFM offers new capabilities and a more in-depth insight in the structure and mechanics of biological specimens with an unrivaled spatial and force resolution. Its application to the study of bacteria is extremely significant since it has already delivered important information on the metabolism of these small microorganisms and, through new and exciting technical developments, will shed more light on the real-time interaction of antimicrobial agents and bacteria. For further resources related to this article, please visit the . Conflict of interest: The authors have declared no conflicts of interest for this article.

Wiley-Blackwell2014

The role of the inflammasome in cellular responses to toxins and bacterial effectors

Françoise Gisou van der Goot Grunberg

Invading pathogens are recognized by mammalian cells through dedicated receptors found either at the cell surface or in the cytoplasm. These receptors, like the trans-membrane Toll-like Receptors (TLR) or the cytosolic Nod-like Receptors (NLR), initiate innate immunity after recognition of molecular patterns found in bacteria or viruses, such as LPS, flagellin, or double-stranded RNA. Recognition of molecules produced only by a specific pathogen, such as a viral envelop protein or a bacterial adhesin does not appear to occur. Bacterial protein toxins, however, might compose an intermediate class. Considering the diversity of toxins in terms of structure, it is unlikely that cells respond to them via specific molecular recognition. It rather appears that different classes of toxins trigger cellular changes that are sensed by the cells as danger signals, such as changes in cellular ion composition after membrane perforation by pore-forming toxins or type III secretion systems. The signaling pathways triggered through toxin-induced cell alterations will likely play a role in modulating host responses to virulent bacteria. We will here describe the few studied cases in which detection of the toxin by the host cell was addressed. The review will include not only toxins but also bacteria effectors secreted by the bacterium in to the host cell cytoplasm.

Springer-Verlag2007

From polymers to gene regulation

Aleksandre Japaridze

DNA molecule is the fundamental component of every living organism, since it encodes the hereditary information. Although the genetic code of almost 200 species has been sequenced, the direct connection between gene sequence, DNA structure and biological function remains poorly understood. The aim of this thesis was to investigate the connection between DNA function and structure. Throughout the thesis we will discuss experiments testing the link between different physical properties of DNA with its biological function. For this purpose we used atomic force microscopy (AFM), a high resolution imaging technique. The first chapter is devoted to explain the basic concepts of AFM technique, as well as the composition and structure of DNA molecules. We also describe some of the polymer physics models of DNA, defining basic quantities like the persistence and the critical exponent. In chapter 2 we discuss experiments, in which we studied changes in the physical properties of DNA molecules, when bound to the substrate surface with various methods. We show that, when DNA is strongly bound to the surface, it retains its physiological B-form. On contrary, when weakly bound, a conformational B-to-A-form transition takes place. We also discuss a novel experimental method, combining nanometer sized PDMS slits with AFM, for studying DNA in geometrical confinement. When DNA is weakly confined, DNA persistence length increases, molecules elongate and adopt more anisotropic shapes. Under stronger confinement, DNA hairpins form. In chapter 3 we study binding of staining dyes, proteins and RNA polymerase (RNAP) to DNA molecules. First we show that DNA physical properties are significantly affected when stained with dyes. Depending on the dye binding mode, either the contour length, or the persistence length or both simultaneously change. We move further and investigate the role of protein amino acids and DNA sequence in the formation of nucleoprotein complexes. On the example of EspR protein, a key virulence regulator in Mycobacterium tuberculosis (MTB), we show that single amino acid mutations, lead to lower DNA binding affinity. These mutations also impair the formation of higher order protein-DNA complexes, silencing the MTB virulence. Afterwards, on the examples of RNAP and H-NS protein, we show the importance of DNA sequence for the binding and higher order oligomerization. By introducing DNA mutations disrupting the helical phasing between RNAP binding sites in the fis promoter sequence, we significantly lower the RNAP binding affinity. The helical phasing of the binding sites somehow coordinates the cooperative binding of RNAP to the promoter. Finally on the model of H-NS protein, we show how different spatial arrangements of strong binding sites for an architectural protein in the DNA, determine the final 3D structure of the assembled nucleoprotein complex. The final chapter, is fully devoted to the study of a completely new type of DNA organisation, which we call Hyperplectonemes. Hyperplectonemes are very ordered DNA structures, formed by large supercoiled molecules, in the presence of attractive DNA-DNA potential. First, we describe their structure and its dependence on various environmental factors, than their binding to nucleoid associated proteins. We argue that this emerging DNA organisation is the basic structure of the bacterial chromatin, which is further modulated by numerous DNA binding and condensing proteins in vivo.

EPFL2015

From polymers to gene regulation

Aleksandre Japaridze

EPFL2015