Développement du typage moléculaire d'Ehrlichia ruminantium, l'agent de la cowdriose, par la technique MLVA (Multi Locus VNTR Analysis)

National audienc

Développement du typage moléculaire d'Ehrlichia ruminantium, l'agent de la cowdriose, par la technique MLVA (Multi Locus VNTR Analysis)

National audienc

Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable

A new typing technique for the Rickettsiales Ehrlichia ruminantium: Multiple-locus variable number tandem repeat analysis

International audienceEhrlichia ruminantium (ER) is a member of the order Rickettsiales transmitted by Amblyomma ticks. This obligatory intracellular bacterium is the causative agent of a fatal disease in ruminants, named heartwater. It represents a constraint on breeding development in sub-Saharan Africa and in the Caribbean. The genetic diversity of the strains of ER, which could be a limiting factor to obtain effective vaccines, needs to be better characterized. For this purpose, we developed a molecular typing technique based on the polymorphism of variable number tandem repeat (VNTR) sequences, MLVA (multiple locus VNTR analysis).Eight (out of 21) VNTR candidates were validated using 17 samples representing a panel of ER strains from different geographical origins from West, South Africa, and Caribbean areas and in ER infected ticks and goat tissues. This result demonstrated the ability of these VNTRs to type a wide range of strains. The stability of the selected VNTR markers was very good, at the time scale needed for epidemiological purposes: in particular, no difference in the VNTR profiles was observed between virulent and attenuated strains (for Gardel and Senegal strains) and between strains (Gardel and Blonde strains) isolated in the same area 19 years apart. We validated the strong discriminatory power of MLVA for ER and found a high level of polymorphism between the available strains, with 10 different profiles out of 13 ER strains.The MLVA scheme described in this study is a rapid and efficient molecular typing tool for ER, which allows rapid and direct typing of this intracellular pathogen without preliminary culture and gives reliable results that can be used for further epidemiological studies. (C) 2011 Elsevier B.V. All rights reserved

Caractérisation de la diversité génétique d'<em>Ehrlichia ruminantium</em> à l'échelle mondiale par l'approche MLVA et MLST

International audienc

Analysis strategies to detect the specific integrations mediated by phiC31-int.

<p>A) Illustration of the three mechanisms of the phiC31-int mediated integration of a NILV containing an <i>attB</i> sequence. According to the type of integration, the PCR results in three different profiles: - PCRs LTR+/<i>attB</i>− : integration type (1), specific integration. - PCRs LTR−/<i>attB</i>+: integration type (2), residual integration. - PCRs LTR+/<i>attB</i>+: integration type (3), illegitimate integration. P1/P1′ are the primers used for <i>attB</i> PCR and P2/P2′ are the primers used for LTR PCR. B) Schematic representations of the inverse PCR and the adapted inverse PCR strategies used to characterize phiC31-int integration sites.</p

DNA sequence of <i>att</i> and p<i>attP</i> sites.

<p>A) Wild type <i>attP</i> and <i>attB</i> sites. After recombination two hybrids sites are formed: <i>attL</i> and <i>attR</i>. B) Recombination between <i>attB</i> site and the human locus Xq22.1 This recombination generates a p<i>attR</i> which has been isolated by inverse PCR. Xq22.1 had been described previously as a human p<i>attP</i> by MP Calos et al., who isolated the same p<i>attR</i>.</p

Detection of recombination mediated by phiC31-int between an <i>attB</i> site contained into a NILV and a genomic <i>attP</i> site.

<p>A) Scheme of the DsRed2 PCR before and after the enzymatic restriction treatment. B) PCR DsRed2 results without restriction enzyme treatment. Lanes 1 to 3: cotransduction with CMV-Neo and CMV-PhiC31 increasing vector input of 50–150–300 ng of p24. Lanes 4 to 6: cotransduction with <i>attB</i>-CMV-Neo and CMV-PhiC31 increasing vector input of 50–150–300 ng of p24. Lane 7: <i>attB</i>-CMV-Neo. Lane 8: positive control generated by triple-transfection (CMV-phiC31-int, <i>attB</i>-CMV-Neo and CMV-<i>attP</i>-DsRed2). Lane 9: negative control without vector. Lane 10: negative control of PCR. C) PCR DsRed2 results after restriction enzyme treatment. Lanes are similar to figure B. D) Nested PCR from the product isolated from lane 6 to confirm the specificity of PCR DsRed2 amplification.</p