Bacterial type IV secretion systems: a tool for DNA transfer into mammalian cells

New Biotechnology · Volume 25S · September 2009

Bacterial type IV secretion systems: a tool for DNA transfer into mammalian cells E. Fernández-González 1,∗ , H.D. de Paz 1 , C. Dehio 2 , F.J. Sangari 1 , M. Llosa 1 1

Departamento Biología Molecular (Universidad de Cantabria) & IBBTEC (UCCSIC-IDICAN), Santander, Spain 2 Division of Molecular Microbiology, Biozentrum of the University of Basel, Basel, Switzerland

Type IV secretion systems (T4SS) are versatile machineries for the selective secretion of macromolecules that are involved in different biological processes such as pathogenicity (pT4SS) or horizontal DNA transfer by conjugation (cT4SS). Human intracellular pathogens, such a Bartonella henselae and Brucella suis, use a pT4SS to deliver effector proteins into the cytoplasm of eukaryotic cells. To recruit its specific substrate each T4SS has a coupling protein (CP). We have previously shown that CP can interact with heterologous T4SS. We investigated if a pT4SS could be used to deliver DNA (the substrate of the cT4SS) into its target mammalian cells. To test if the pT4SS of Bartonella and Brucella are able to recruit the cT4SS substrate of the conjugative plasmid R388, we constructed a plasmid coding for the R388 components required for substrate recruitment and a GFP cassette that is expressed only in the eukaryotic cells. Mammalian cells were infected with bacteria harbouring the plasmid. DNA transfer from intracellular bacteria is detected by GFP expression using flow cytometry. In the case of Bartonella about 1% of cells were GFP positive, indicating DNA mobilization from the bacteria into the human cells. The transfer was dependent on the T4SS of the bacteria and the CP of the plasmid, confirming that we have achieved the recruitment of a heterologous DNA substrate by a pT4SS. This system will be an interesting tool for the introduction of any DNA molecule into specific cellular types, since each pathogen containing a T4SS has a different target tissue. This has prompted us to test DNA transfer through pT4SS of other human pathogens. Experiments with Brucella are underway. doi:10.1016/j.nbt.2009.06.086

Size-controlled and stealth silica-based nano-spheres as potential drug carriers E. Papini 1,∗ , I.M. Rio Echevarria 2 , F. Selvestrel 2 , F. Mancin 2 , E. Reddi 3 , D. Segat 3 , R. Tavano 1 1

Dept. Biomedical Sciences and CRIBI, University of Padova, Padova, Italy Department of Chemical Sciences, University of Padova, Italy 3 Dept. of Biology, University of Padova, Italy 2

Silica nanoparticles provide a quasi-ideal scaffold for the realization of medical nanodevices: they are cheap and easy to prepare, water soluble, relatively inert from the chemical point of view. In addition they can be easily functionalized (or doped) either at the surface or in the interior. These remarkable qualities make silica nanoparticles excellent candidates for a prominent place in the next generation of drug carriers and nanotherapeutic agents. However, nanostructures may be potentially dangerous for living organisms and even biocompatible silica nanoparticles need

ABSTRACTS

to be effectively modified to minimize and possibly ablate the interaction with immune cells and macrophages within the body. Biocompatibility and low clearance-rate (conventionally referred to as stealth property) of nanoparticles of various nature can be classically achieved by PEG coating. Here we discuss the preparation of silica nanoparticles by ammonia catalyzed polymerization of vinyltriethoxysilane (ORMOSIL ORganically MOdified SILica) in the presence of surfactants. By including a trialkcoxysilane modified PEG in the reaction we obtain a thick and dense PEG coating of the nanoparticles, which are produced in the desired size (20—100 nm) by a one-pot procedure. Bare or PEG-modified nanoparticles were not toxic to endothelial and blood cells. Finally, we evaluated the stealth properties of fluorescent-labeled nanoparticles using primary macrophages differentiated in vitro from human monocytes, as a model of the Reticular Endothelial System particle-capturing phagocytes. Flowcytofluorimetry and confocal fluorescence microscopy analysis demonstrated that, while the non-PEGylated silica nanoparticles were efficiently endocytosed in acidic phagosomes, their PEG-coated versions eluded macrophage association and internalization. PEGylated ORMOSIL nanoparticles proved to be a promising candidate for the design of nano-drugs and diagnostic tools. Further studies will be performed to monitor their full biocompatibility and to verify the possibility of specific ligand-based cell targeting. doi:10.1016/j.nbt.2009.06.087

Poster 1.5.05 DNA interaction studies of a salen-type Schiff base ligand (N,N -bis{5-[(triphenylphosphonium)methyl]salicylidine}-o-phenylenediamine using circular dichroism and fluorescence techniques N. Shahabadi ∗ , S. Kashanian, F. Darabi Razi University, Kermanshah, Islamic Republic of Iran

Compounds with the structure of C N (azomethine group) are known as Schiff bases, which are usually synthesized from the condensation of primary amines and active carbonyl groups. Schiff bases are important class of compounds in medicinal and pharmaceutical field. They show biological applications including antibacterial, antifungal and antitumor activity. We has been synthesized and characterized the salen-type Schiff base ligand (N,N -bis{5-[(triphenylphosphonium)-methyl]salicylidine}-ophenylenediamine (SF) by spectroscopic (NMR, FT-IR and UV—vis) techniques. Then the binding of this Schiff base ligand to doublestrand DNA, in 10 mM HEPES buffer at neutral pH, has been investigated by fluorescence spectroscopy and circular dichroism. In fluorescence experiments, the emission intensity of SF grow to around 3.7 times larger than that in the absence of DNA at a [DNA]/[SF] ratio of 2 and followed by a red shift of 12 nm. This marked increase in emission intensity also agree with these observed for other intercalators. In addition, detectable changes in the CD spectrum of CT-DNA in the presence of SF indicated deep conformational changes of the DNA double helix following the www.elsevier.com/locate/nbt S35