Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Molecular mechanisms of horizontal gene transfer in pathogen Helicobacter pylori

Helicobacter pylori est une bactérie à Gram-négatif qui colonise la muqueuse de l’estomac humain. Elle se distingue des autres bactéries par un nombre de gènes très limité et de nombreuses particularités physiologiques et biochimiques. Elle provoque des infections associées à différentes maladies gastro-duodénales (ulcères et cancers). Depuis quelques années, une recrudescence de multi-résistances aux antibiotiques est observée. La transformation naturelle est l’un des processus clés qui les propage. Il s’agit d’un mécanisme de transfert horizontal de gènes qui permet aux bactéries de s’adapter à leur environnement, en internalisant des fragments d’ADN exogène à travers leur membrane, puis en les intégrant dans le chromosome par recombinaison homologue. Mes travaux ont visé à étudier de façon structurale et fonctionnelle trois protéines d’H. pylori décrites comme étant essentielles dans le processus de transformation naturelle: NucT, DprA et ComFc. La première partie de ce travail s’est concentrée sur la nucléase périplasmique NucT, supposée être impliquée dans la transformation chez H. pylori. Cependant, la délétion de son gène a permis de démontrer qu’elle ne joue en fait qu’un rôle mineur dans ce processus. La résolution de sa structure 3D a permis de mieux comprendre sa spécificité pour les acides nucléiques simple brin. Dans la seconde partie, la protéine DprA, responsable du chargement de la recombinase RecA sur l’ADN internalisé, a été étudiée. DprA d’H. pylori n’est composée que de 2 des 3 domaines qui constituent habituellement DprA, et fixe aussi bien l’ADN double brin que l’ADN simple brin mais uniquement via son domaine RF. Malgré son homologie structurale avec le domaine WH de liaison à l’ADN, le domaine C-terminal de HpDprA n’a pas d’affinité pour l’ADN. Nous avons mis en évidence des acides aminés conservés dans ce domaine dont l’étude pourrait permettre de comprendre son rôle. Enfin, une étude structurale de la protéine ComFc dont la délétion du gène entraîne la disparition totale de la capacité de transformation d’H. pylori a été réalisée. L’obtention de sa structure 3D a permis de mettre en évidence la présence d’un domaine catalytique phosphoribosyl-transférase ainsi que d’un domaine en doigt en zinc. Ce dernier pourrait être responsable de la capacité de ComFc à fixer l’ADN. Le substrat naturel de cette enzyme reste à découvrir.L’ensemble de ce travail a permis de contribuer à une meilleure compréhension à l’échelle moléculaire du mécanisme de transformation génétique naturelle d’H. pylori. L’avancement sur ces connaissances pourrait à long terme aider à réduire la propagation des multi-résistances par l’élaboration de nouvelles thérapies.Mots-clés : H. pylori, transformation naturelle, NucT, DprA, ComFc, interaction protéine-ADNHelicobacter pylori is a Gram-negative bacterium that colonizes the mucus of the human stomach. It is distinguished from other bacteria by a limited number of genes and many physiological and biochemical characteristics. It causes infections associated with various gastro-duodenal diseases (ulcers and gastric cancers). In recent years, an increase in multi-resistance to antibiotics has been observed. Natural transformation is one of the key processes that spreads these multi-resistances. It is a horizontal gene transfer mechanism that allows bacteria to adapt to their environment by internalizing exogenous DNA fragments through their membrane and then integrating them into the chromosome by homologous recombination. My work aimed to study in a structural and functional approach three proteins of H. pylori described as essential in the natural transformation process: NucT, DprA and ComFc. The first part of this work focused on periplasmic nuclease, NucT, which is supposed to be involved in transformation in H. pylori. However, the deletion of its gene has shown that it actually plays only a minor role in this process. The resolution of its 3D structure has led to a better understanding of its specificity for single-stranded nucleic acids. In the second part, the protein DprA, responsible for loading RecA recombinase onto internalized DNA, was studied. HpDprA is composed of only 2 of the 3 domains that usually constitute DprA, and binds both double-stranded and single-stranded DNA but only via its RF domain. Despite its structural homology with the WH DNA binding domain, the C-terminal domain of HpDprA has no affinity for DNA. We have identified conserved amino acids in this domain that could be studied to understand its role. Finally, a structural study of ComFc, whose deletion of the gene leads to the total disruption of the transformation capacity of H. pylori, has been carried out. The acquisition of its 3D structure has highlighted the presence of a phosphoribosyl transferase catalytic domain as well as a zinc finger domain. The latter could be responsible for capacity of ComFc to bind DNA. The natural substrate of this enzyme remains to be discovered.All this work has contributed to a better knowledge at the molecular level of the natural genetic transformation mechanism of H. pylori. Advancing this knowledge could in the long term help to reduce the spread of multiresistance through the development of new therapies.Keywords: Helicobacter pylori, natural transformation, NucT, DprA, ComFc, protein-DNA interactio

The C-terminal domain of HpDprA is a DNA-binding Winged Helix domain that does not bind double-stranded DNA

International audienceDprA, a ubiquitous multidomain DNA-binding protein, plays a crucial role during natural transformation in bacteria. Here, we carried out the structural analysis of DprA from the human pathogen Helicobacter pylori by combining data issued from the 1.8 Å resolution X-ray structure of the Pfam02481 domain dimer (RF), the NMR structure of the carboxy-terminal domain (CTD) and the low-resolution structure of the full-length DprA dimer obtained in solution by SAXS. In particular, we sought a molecular function for the CTD, a domain that we show here is essential for transformation in H. pylori. Albeit its structural homology to winged helix DNA binding motifs, we confirmed that the isolated CTD does not interact with ssDNA nor with dsDNA. The key R52 and K137 residues of RF are crucial for these two interactions. Search for sequences harboring homology to either HpDprA or Rhodopseudomonas palustris DprA CTDs led to the identification of conserved patches in the two CTD. Our structural study revealed the similarity of the structures adopted by these residues in RpDprA CTD and HpDprA CTD. This argues for a conserved, but yet to be defined, CTD function, distinct from DNA binding. This article is protected by copyright. All rights reserved

Alpha repeat proteins (αRep) as expression and crystallization helpers

International audienceWe have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize

ComFC mediates transport and handling of single-stranded DNA during natural transformation

International audienceAbstract The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes and virulence factors across bacteria. However, its role remains largely unknown. Here, we show that Helicobacter pylori ComFC is involved in DNA transport through the cell membrane, and is required for the handling of the single-stranded DNA once it is delivered into the cytoplasm. The crystal structure of ComFC includes a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for the protein’s in vivo activity. Furthermore, we show that ComFC is a membrane-associated protein with affinity for single-stranded DNA. Our results suggest that ComFC provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery

ComF is a key mediator in single-stranded DNA transport and handling during natural transformation

Natural transformation plays a major role in the spreading of antibiotic resistances and virulence factors. Whilst bacterial species display specificities in the molecular machineries allowing transforming DNA capture and integration into their genome, the ComF(C) protein is essential for natural transformation in all Gram- positive and - negative species studied. Despite this, its role remains largely unknown. Here, we show that Helicobacter pylori ComF is not only involved in DNA transport through the cell membrane, but it also required for the handling of the ssDNA once it is delivered into the cytoplasm. ComF crystal structure revealed the presence of a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for its in vivo activity. ComF is a membrane-associated protein with affinity for single-stranded DNA. Collectively, our results suggest that ComF provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery

Dimer of NucT and comparative analysis with PLD family nucleases.

<p><b>(A)</b> Ribbon diagrams of the dimers of NucT, Nuc, Zuc, hBJ-TSA-9 and hFAM83B in two orientations (profile and on top). One monomer is in green and the other on grey. The helices and loops which line the groove of the catalytic pockets are in orange. The extra loops of NucT, hBJ-TSA-9 and hFAM83B are indicated by a pink arrow. <b>(B)</b> Electrostatic surfaces of the top of the dimers. The basic electropositive residues are in blue and the acidic electronegative ones are in red.</p

Data collection and structure refinement statistics.

<p>Data collection and structure refinement statistics.</p

Nuclease NucT prefers single-stranded nucleic acids.

<p><b>(A)</b> Schematic representation of the different 5’ radiolabelled DNA substrates (S.) used. See also the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189049#pone.0189049.s003" target="_blank">S1 Table</a>. Asterisks indicate the radiolabelled 5’ end of the XV98 oligonucleotide common to the substrates used in <b>B</b> and <b>C</b>. <b>(B to D)</b> Nuclease activity tests. NucT at the indicated concentrations was incubated for 30 min at 37°C with the indicated substrate. Quantification of three independent cleavage experiments on SI (open squares) and SII (black squares) is reported as a graph in the B right panel. In <b>D</b> NucT activity was tested on the 24-mer single-stranded osf344 DNA oligonucleotide (ssDNA 24nt) or a 25-mer RNA Hxv82 (RNA 25nt), both radiolabelled in 5’. The samples were run on denaturing PAGE as described in Materials and methods.</p