Bacteriophages and other mobile genetic elements in microbial host adaptation, physiopathology and potential therapy

La présente thèse comprend trois chapitres qui étudient la manière dont les éléments génétiques mobiles (EGMs), y compris les bactériophages (phages), peuvent être impliqués dans (i) l’adaptation des bactéries à différents mammifères, (ii) le parasitage des bactéries (par les phages) tout en préservant l’hôte bactérien, et (iii) le détournement des phages à des fins thérapeutiques (phagothérapie) pour traiter les infections bactériennes. Le premier chapitre s’intéresse au récent saut d’espèces de la souche humaine de Staphylococcus aureus du Complexe Clonal 8 (CC8) aux bovins, chez qui elle est responsable de mastites. Nous démontrons que le premier événement responsable de ce saut est l’acquisition d’une cassette chromosomique staphylococcique appelée SCCbov. SCCbov est un EGM codant pour une nouvelle protéine de surface appelée « Adherence-Like Bovine protein » (ADLB) impliquée dans la pathogenèse des mastites. En effet, les souches de CC8 bovines envahissent et tuent les cellules mammaires bovines en culture. Au contraire, l’excision de SCCbov ou l’inactivation d’ADLB diminuent la virulence dans ces conditions. Le second événement du saut est la perte d’un phage inséré dans le chromosome (ou prophage) qui interrompt le gène de la lipase. La restauration de l’activité lipase est susceptible de faciliter la croissance des staphylocoques dans le milieu riche en lipides du lait. Le troisième événement est la perte d’un prophage interrompant le gène de la -hémolysine. La restauration de la -hémolysine promeut la colonisation épithéliale. Enfin, nous avons construit des souches isogéniques de CC8 comportant différentes combinaisons de ces EGMs, permettant d’étudier le saut d’espèce dans une approche « d’évolution inverse ». En conclusion, c’est ce trafic d’EGMs qui est à l’origine du saut des CC8 humains aux bovins. Le second chapitre étudie la relation structure-fonction et la régulation de l’activité de la lysine du phage PlySK1249 de Streptococcus dysgalactiae. Les phages produisent des lysines en fin de réplication pour lyser les bactéries hôtes et libérer leur progéniture. Cependant, libérer les lysines dans l’environnement risque de lyser des bactéries voisines qui portent le même phage, ou des bactéries constituant de nouvelles proies. PlySK1249 a une structure multi- modulaire intrigante, constituée d’un domaine central de liaison à la paroi bactérienne, encadré par un domaine amidase (bactériolytique) et domaine endopeptidase (CHAP; non- bactériolytique). Nous démontrons que cette multi-modularité remplit trois fonctions. (A) la combinaison des deux domaines enzymatiques et du domaine de liaison est hautement synergique en termes de dégradation de la paroi et de lyse bactérienne. (B) le domaine de liaison prévient la diffusion de la lysine en la gardant liée aux débris de paroi cellulaire après la lyse bactérienne. (C) en présence de paroi bactérienne PlySK1249 est progressivement clivée et par une protéase de la paroi bactérienne (encore non-identifiée), qui désamorce son activité bactéricide. En résumé, la structure multi-modulaire de PlySK1249 implique une régulation inter-domaines sophistiquée, permettant de circonscrire la lyse à la cellule infectée afin de ne pas compromettre l’intégrité des cellules avoisinantes. Le troisième chapitre est une preuve de concept de la phagothérapie, utilisant un cocktail de 12 phages anti-Pseudomonas aeruginosa et un modèle d’endocardite expérimentale (EE) chez le rat. Les phages se sont révélés hautement bactéricides in vitro (perte de >6 logs CFU/ml en 6 h), mais sélectionnaient des pseudomonas résistants. Les phages étaient aussi bactéricides in vivo (perte de >2 logs CFU/g de végétations en 6 h), mais ne sélectionnaient pas de résistance chez les animaux. L’absence de résistance in vivo était due au coût d’adaptation chez l’animal, car elle affectait la synthèse des pili et du LPS, deux facteurs de virulence critiques de pseudomonas. Enfin, la combinaison de phages et d’antibiotiques (dans ce cas la ciprofloxacine) s’est révélée hautement synergique, prévenant la résistance in vitro, et démontrant une fréquence de stérilisation des végétations cardiaques (>50% en 6 h) sans précédent dans l‘EE à P. aeruginosa. En conclusion, la phagothérapie combinée ou non aux antibiotiques ouvre des promesses nouvelles pour le traitement des infections bactériennes systémiques sévères. -- The present thesis comes in three chapters studying how mobile genetic elements (MGEs), including bacterial viruses called bacteriophages (phages), may be involved in (i) adaptation of bacteria to different mammalian hosts, (ii) parasiting bacteria (in the case of prophages) while preserving their bacterial hosts, and (iii) new therapeutic applications. The first chapter studied the recent human-to-bovine host jump of typical human Staphylococcus aureus Clonal Complex 8 (CC8) to cowherds, where it causes invasive mastitis. We show that the first event driving the human-to-bovine jump was the acquisition, by human CC8 strains, of a new “staphylococcal cassette chromosome” called SCCbov. SCCbov is a new MGE encoding for a new bacterial surface protein called Adherence-Like Bovine protein (or ADLB), which is implicated in mastitis pathogenesis. Indeed, while parent bovine CC8 readily invaded mammary cells lines, then escaped endosomes, and eventually lysed cultured cells, excision of SCCbov or knocking out ADLB decreased invasiveness and mammary cell death. We then show that the second event of the host-jump was the loss of a phage inserted in the bacterial chromosome (called a prophage), which interrupted the lipase- encoding gene. The loss of this prophage restored S. aureus lipase activity, which is likely to facilitate staphylococcal growth in the lipid-rich milk milieu. The third event was the loss of a β- hemolysin-interrupting prophage, restoring β-hemolysin activity, which was shown to promote epithelial colonization. Finally, we constructed isogenic bovine CC8 strains carrying these MGEs in various combinations, which will help determine the host-jump dynamics in a “reverse evolution” approach. Thus, MGE trafficking was the driving force behind the adaptation of human CC8 staphylococci to cows. The second chapter dissected the molecular structure-function activity and regulation of phage lysin PlySK1249 from Streptococcus dysgalactiae. Phages produce lysins at the end of their replication cycle to lyse host bacteria and release their progeny. However, uncontrolled diffusion of lysins in the surrounding might also lyse neighboring bacteria carrying sibling phages or potential new bacterial hosts. PlySK1249 has an intriguing multimodular structure with a central cell wall-binding domain (CWBD) bracketed by a bacteriolytic amidase domain and a non-bacteriolytic endopeptidase (CHAP) domain. We show that this multi-modularity serves a triple purpose. On the one hand, combining the two enzymatic and the CWBD domains led to a synergistic activity in cell wall degradation and bacterial lysis. On other hand, the CWBD prevented lysin diffusion after bacterial lysis by keeping lysin attached to cell wall debris. Finally, we found that in the presence (but not in the absence) of cell wall protein extract, PlySK1249 was subject to proteolytic cleavage by an as yet unknown bacterial wall protease, further introducing posttranscriptional modification in order to control its lytic activity. Thus, the multimodular structure of PlySK1249 implicates sophisticated inter-domain interactions promoting synergistic – yet cell-restricted – lysis, in order to release the phage progeny without jeopardizing neighboring host cells. The third chapter was a proof of concept study of phage therapy, using a cocktail of 12 phages directed against Pseudomonas aeruginosa and a model of P. aeruginosa experimental endocarditis (EE). Phages were highly bactericidal in vitro (loss of >6 logs CFU/ml in 6 h), but selected for phage resistance. Phages were also bactericidal in vivo (loss of >2 logs CFU/g of vegetations in 6 h), but did not select for resistance in animals. The absence of in vivo resistance selection was due to the fitness cost of resistance, which affected synthesis of bacterial pilus or LPS, two P. aeruginosa virulence factors that are critical for in vivo infection. Finally, combining phages with antibiotics (namely ciprofloxacin) was highly synergistic, prevented resistance selection in vitro, and cured notoriously difficult-to-treat EE due to P. aeruginosa at an unprecedented rate, healing >50% of the animals within 6 h of therapy. Thus, thoughtful utilization of phage therapy combined or not with antibiotics might become an alternative to treat severe systemic bacterial infections

Phage diversity, genomics and phylogeny

Recent advances in viral metagenomics have enabled the rapid discovery of an unprecedented catalogue of phages in numerous environments, from the human gut to the deep ocean. Although these advances have expanded our understanding of phage genomic diversity, they also revealed that we have only scratched the surface in the discovery of novel viruses. Yet, despite the remarkable diversity of phages at the nucleotide sequence level, the structural proteins that form viral particles show strong similarities and conservation. Phages are uniquely interconnected from an evolutionary perspective and undergo multiple events of genetic exchange in response to the selective pressure of their hosts, which drives their diversity. In this Review, we explore phage diversity at the structural, genomic and community levels as well as the complex evolutionary relationships between phages, moulded by the mosaicity of their genomes

Aspirin plus ticlopidine prevented experimental endocarditis due to Enterococcus faecalis and Streptococcus gallolyticus

Enterococcus faecalis and Streptococcus gallolyticus cause infective endocarditis (IE), which can originate from the continuous release or translocation of low bacterial numbers into the bloodstream. In this context, IE cannot be prevented with antibiotics. We previously demonstrated that aspirin plus ticlopidine protected rats from IE due to S. gordonii and Staphylococcus aureus. Here we showed that aspirin plus ticlopidine significantly reduced vegetation weight and protected 73 and 64% rats (P < 0.005) from IE due to E. faecalis and S. gallolyticus, respectively. These results further support the potential use of aspirin plus ticlopidine for a global prevention of IE in high-risk patient

Local simulations of the magnetized Kelvin-Helmholtz instability in neutron-star mergers

Context. Global MHD simulations show Kelvin-Helmholtz (KH) instabilities at the contact surface of two merging neutron stars. That region has been identified as the site of efficient amplification of magnetic fields. However, these global simulations, due to numerical limitations, were unable to determine the saturation level of the field strength, and thus the possible back-reaction of the magnetic field onto the flow. Aims. We investigate the amplification of initially weak fields in KH unstable shear flows, and the back-reaction of the field onto the flow. Methods. We use a high-resolution ideal MHD code to perform 2D and 3D local simulations of shear flows. Results. In 2D, the magnetic field is amplified in less than 0.01ms until it reaches locally equipartition with the kinetic energy. Subsequently, it saturates due to resistive instabilities that disrupt the KH vortex and decelerate the shear flow on a secular time scale. We determine scaling laws of the field amplification with the initial field strength and the grid resolution. In 3D, this hydromagnetic mechanism may be dominated by purely hydrodynamic instabilities limiting the amplification. We find maximum magnetic fields of 10^16 G locally, and r.m.s. maxima within the box of 10^15 G. However, such strong fields exist only for a short period. In the saturated state, the magnetic field is mainly oriented parallel to the shear flow for strong initial fields, while weaker initial fields tend to lead to a more balanced distribution of the field energy. In all models the flow shows small-scale features. The magnetic field is at most in equipartition with the decaying shear flow. (abridged)Comment: 26 pages, 22 figures (figure quality reduced); accepted for publication in Astronomy & Astrophysic

Neutron-Rich Freeze-Out in Viscously Spreading Accretion Disks Formed from Compact Object Mergers

Accretion disks with masses ~0.001-0.1 Msun form during the merger of neutron star (NS)-NS and black hole-NS binaries. Initially, such hyper-accreting disks cool efficiently by neutrino emission and their composition is driven neutron-rich by pair captures under degenerate conditions. However, as the disk viscously spreads and its temperature drops, cooling becomes inefficient and the disk becomes advective. Analytic arguments and numerical simulations suggest that once this occurs, powerful winds likely drive away most of the disk's remaining mass. We calculate the thermal evolution and nuclear composition of viscously spreading accretion disks formed from compact object mergers using one-dimensional height-integrated simulations. We show that freeze-out from weak equilibrium necessarily accompanies the disk's late-time transition to an advective state. As a result, hyper-accreting disks generically freeze out neutron-rich (with electron fraction Ye ~ 0.2-0.4), and their late-time outflows robustly synthesize rare neutron-rich isotopes. Using the measured abundances of these isotopes in our solar system, we constrain the compact object merger rate in the Milky Way to be < 1e-5 (M_d,0/0.1 Msun)^(-1) per year, where M_d,0 is the average initial mass of the accretion disk. Thus, either the NS-NS merger rate is at the low end of current estimates or the average disk mass produced during a typical merger is << 0.1 Msun. We also show that if most short duration gamma-ray bursts (GRBs) are produced by compact object mergers, their beaming fraction must exceed f_b ~ 0.13(M_d,0/0.1 Msun), corresponding to a jet half-opening angle > 30(M_d,0/0.1 Msun)^(1/2) degrees. This is consistent with other evidence that short duration GRB outflows are less collimated than those produced in long duration GRBs.Comment: 12 pages, 9 figures, 1 table; accepted to MNRAS; minor changes to text and figure

Common Genetic Variants Contribute to Risk of Transposition of the Great Arteries.

RATIONALE: Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. OBJECTIVE: We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. METHODS AND RESULTS: We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10 CONCLUSIONS: This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 nea

Frank Oechslin & Co., everything in potted plants.

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Aspirin plus ticlopidine prevented experimental endocarditis due to Enterococcus faecalis and Streptococcus gallolyticus

Enterococcus faecalis and Streptococcus gallolyticus cause infective endocarditis (IE), which can originate from the continuous release or translocation of low bacterial numbers into the bloodstream. In this context, IE cannot be prevented with antibiotics. We previously demonstrated that aspirin plus ticlopidine protected rats from IE due to S. gordonii and Staphylococcus aureus. Here we showed that aspirin plus ticlopidine significantly reduced vegetation weight and protected 73 and 64% rats (P < 0.005) from IE due to E. faecalis and S. gallolyticus, respectively. These results further support the potential use of aspirin plus ticlopidine for a global prevention of IE in high-risk patients.status: publishe