Mycobacteriophage–antibiotic therapy promotes enhanced clearance of drug-resistant Mycobacterium abscessus

International audienceABSTRACT Infection by multidrug-resistant Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF) patients, leaving clinicians with few therapeutic options. A compassionate study showed the clinical improvement of a CF patient with a disseminated M. abscessus (GD01) infection, following injection of a phage cocktail, including phage Muddy. Broadening the use of phage therapy in patients as a potential antibacterial alternative necessitates the development of biological models to improve the reliability and successful prediction of phage therapy in the clinic. Herein, we demonstrate that Muddy very efficiently lyses GD01 in vitro, an effect substantially increased with standard drugs. Remarkably, this cooperative activity was retained in an M. abscessus model of infection in CFTR-depleted zebrafish, associated with a striking increase in larval survival and reduction in pathological signs. The activity of Muddy was lost in macrophage-ablated larvae, suggesting that successful phage therapy relies on functional innate immunity. CFTR-depleted zebrafish represent a practical model to rapidly assess phage treatment efficacy against M. abscessus isolates, allowing the identification of drug combinations accompanying phage therapy and treatment prediction in patients. This article has an associated First Person interview with the first author of the paper

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

International audienceMycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRS Mabs and LeuRS Mtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis

Prion replication occurs in endogenous adult neural stem cells and alters their neuronal fate: involvement of endogenous neural stem cells in prion diseases.

International audiencePrion diseases are irreversible progressive neurodegenerative diseases, leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits, vacuolisation, astrocytosis, neuronal degeneration, and by cognitive, behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation, but also from the stimulation of endogenous neural stem cells (NSC) or by the combination of both approaches. However, the development of such strategies requires a detailed knowledge of the pathology, particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade, several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS) and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However, the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly, this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease

Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium abscessus

International audiencePeptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors

Exploring Macrophage-Dependent Wound Regeneration During Mycobacterial Infection in Zebrafish

International audienceThe molecular and cellular mechanisms associated with tissue degradation orregeneration in an infectious context are poorly defined. Herein, we explored the role ofmacrophages in orchestrating either tissue regeneration or degradation in zebrafishembryos pre-infected with the fish pathogen Mycobacterium marinum. Zebrafish wereinoculated with different infectious doses of M. marinum prior to fin resection. While mildinfection accelerated fin regeneration, moderate or severe infection delayed this processby reducing blastemal cell proliferation and impeding tissue morphogenesis. This wascorrelated with impaired macrophage recruitment at the wound of the larvae receivinghigh infectious doses. Macrophage activation characterized, in part, by a high expressionlevel of tnfa was exacerbated in severely infected fish during the early phase of theregeneration process, leading to macrophage necrosis and their complete absence inthe later phase. Our results demonstrate how a mycobacterial infection influences themacrophage response and tissue regenerative processes

NSC were infected before their isolation.

<p><b>A</b>. Schematic presentation of the experiment. Hippocampus and Lateral Ventricles were isolated from actin-GFP mice. An equivalent amount (weight) of non infected or infected tissue (in which cells have been frozen and then heat inactivated) was added in the same tube. The cells obtained were all positive for the GFP marker. <b>B</b>. PrP^Sc Western blot using Saf-Mix anti-PrP antibody: PK digested cell lysates of NSC cells derived from the lateral ventricle (1) and the hippocampus (2) of actin-GFP mice isolated in the presence of ME7, or from the lateral ventricle (3) and the hippocampus (4) of actin-GFP mice isolated in the presence of non infected brain samples after 2 subpassages. (5) PrP^Sc control from ME7 infected brain.</p

PrP^Sc is present in NSC and neuroblasts areas.

<p><b>A</b>. PrP^Sc immunostaining (brown+arrow) using Saf84 anti-PrP antibody in the lateral wall (SVZ) and the surrounding of the lateral ventricle (LV). <b>B</b>. Schematic representation of the LV localisation in the mouse brain. <b>C</b>. Nestin immunostaining (brown+arrow) in the SVZ. <b>D and G</b>. Double-immunostaining of PrP^Sc (Blue) and Nestin (Brown) in the surrounding of the LV. <b>E</b>. Doublecortin (DCX) immunostaining (brown+arrow) showing neuroblasts exiting the SVZ of the lateral ventricle. <b>F and H</b> Double-immunostaining of PrP^Sc (Blue) and DCX (Brown) in the surrounding of the LV. Scale bar 20 µm.</p

Infection of NSC during differentiation.

<p>Western blots showing the generation of PrP^Sc: (A) in hippocampus derived NSC, (B) in lateral ventricle derived NSC, (C) in KOPrP LV derived NSC, during the differentiation process and after incubation with ME7 brain homogenate at the time of the differentiation induction. All the samples were treated with PK, Saf Mix cocktail of anti-PrP antibodies was used to detect proteinase K resistant PrP^Sc. Cells were harvested at 2, 4, 6, 8, 11 and 14 dpi, after brain homogenate removal. III: Positive control ME7, MW : Molecular Weight (in kDa). Lanes without numbers were left empty on purpose.</p

Neural precursor cells isolated from infected mice accumulate PrP^Sc.

<p><b>A</b>. Immunofluorescence analysis of the nestin marker in proliferative NSC derived from the hippocampus (H) or lateral ventricle (LV) of mock or ME7 infected mice. (red: nestin, Blue: Hoechst nuclei coloration, scale bar 5 µm). Most of the cells are positive for the nestin NSC marker in proliferation conditions. <b>B</b>. PrP^Sc Immunofluorescence in NSC cells derived from the lateral ventricle and the hippocampus of mock or ME7 infected mice (red: PrP^Sc immunofluorescence using saf61 anti-PrP antibodies, Blue: Hoechst nuclei coloration, scale bar 5 µm). <b>C</b>. PrP^Sc Western blot using Saf-Mix anti-PrP antibody: PK digested cell lysates of NSC cells derived from the lateral ventricle (1) and the hippocampus (2) of non infected mice or the lateral ventricle (3) and the hippocampus (4) of ME7 infected mice after 2 subpassages. (5) PrP^Sc control from ME7 infected brain. Lanes without numbers were left empty on purpose. <b>D</b>. PrP^Sc Western blot using Saf-Mix anti-PrP antibody: PK digested cell lysates of NSC cells derived from the lateral ventricle (1) and the hippocampus (2) of non infected mice or the lateral ventricle (3) and the hippocampus (4) of ME7 infected mice after 15 subpassages. (5) PrP^Sc control from ME7 infected brain. Lanes without numbers were left empty on purpose.</p

Impairment of the neuronal differentiation of NSC derived from prion infected mice.

<p>Immunofluorescence analysis of nestin, DCX, BetaIII-Tubulin and GFAP markers in differentiated NSC derived from the hippocampus (H) or Lateral ventricle (LV) of ME7 infected or non infected mice. (<b>A</b>. red: nestin; green: DCX, <b>B</b>. green: GFAP, red: BetaIII-Tubulin, blue: Hoechst nuclei coloration, scale bar 5 µm). <b>C</b>. Quantification of DCX and beta-III-Tubulin positive cells using ImageJ software. After differentiation induction, NSC gave rise to significantly more DCX positive cells in non infected conditions when compared with ME7 infected conditions (p<0.05 and p<0.01 by Mann-Whitney test). The number of neurons also differed significantly (p<0.01 by Mann-Whitney test) between non infected and infected NSC derived cells in differentiation conditions. Inversely astrocyte proportions were higher in the ME7 infected context (p<0.05 by Mann-Whitney test). Data represent means +/− SEM from one experiment performed in triplicate. Similar results were obtained in the three independent experiments.</p