Biology subjects

Jamet, A.

Publications and source records attributed to Jamet, A..

5 recordsLinked to original sources

bioRxiv2024

A Toolbox for Neisseria meningitidis: Gene Editing, Complementation and Labelling

The efficient transformation of Neisseria meningitidis and Neisseria gonorrhoeae facilitates the rapid construction of bacterial mutants with insertion of antibiotic resistance cassettes. However, this strategy limits the construction of strains with multiple mutations. Recent advances in markerless strategies for Neisseria species have enabled the construction of mutants without antibiotic resistance markers. However, these innovative approaches have potential limitations related to the selection strategy or the possible occurrence of spontaneous mutations in the selection marker genes. In addition, complementation tools or labelling strategies for N. meningitidis are also lacking. In this study, we have introduced new tools for markerless mutation, genetic complementation and labelling in N. meningitidis, thus improving the research possibilities for understanding and tackling these pathogens.

microbiology↗

bioRxiv2021

The Pentose Phosphate Pathway constitutes a major metabolic hub in pathogenic Francisella

Metabolic pathways are now considered as intrinsic virulence attributes of pathogenic bacteria and hence represent potential targets for anti-bacterial strategies. Here, we addressed the role of the pentose phosphate pathway (PPP) and its connections with other metabolic pathways in the pathophysiology of Francisella novicida. The involvement of the PPP in Francisella intracellular life cycle was first demonstrated with the study of PPP inactivation mutants. Indeed, inactivation of tktA, rpiA or rpe genes, severely impaired intramacrophagic multiplication during the first 24 hours. Time-lapse video microscopy demonstrated that rpiA and rpe mutants were able to resume late intracellular bacterial multiplication. To get further insight into the links between the PPP and other metabolic networks of the bacterium, we next performed a thorough proteo-metabolomic analysis of these mutants. We show that the PPP constitutes a major bacterial metabolic hub with multiple connections with glycolysis, tricarboxylic acid cycle and other pathways, such as fatty acid degradation and sulfur metabolism. Hence, our study highlights how the PPP is instrumental to Francisella pathogenesis and growth in its intracellular niche.

microbiology↗

bioRxiv2020

ROS-dependent innate immune mechanisms control Staphylococcus aureus MRSA virulence in the Drosophila larval model

Antibiotics multi-resistant Staphylococcus aureus strains constitute a major public health concern worldwide and are responsible of both healthcare- and community-associated infections. Here we have established a robust and simple S. aureus oral infection model, using Drosophila melanogaster larva, which allowed to follow S. aureus fate at the whole organism level as well as the host immune responses. Fluorescence microscopy and Light sheet 3D imaging revealed bacterial clustering at the posterior midgut that displays neutral pH. Our study demonstrates that S. aureus infection triggers host H2O2 production through Duox enzyme, consequently empowering antimicrobial peptides production through Toll pathway activation. We also show that catalase-mediated quenching of H2O2 not only enhances S. aureus survival but also minimizes host antimicrobial response, hence reducing bacterial clearance in vivo. Finally, we confirm the versatility of this model by demonstrating the colonization and host stimulation capacities of two other bacterial pathogens: Salmonella Typhimurium and Shigella flexneri. Overall, the drosophila larva may constitute a general model to follow in vivo host innate immune responses triggered upon infection with human pathogens.

microbiology↗

bioRxiv2018

Persistent Staphylococcus aureus infections in children with chronic lung disease: a multi-omics analysis of bacterial adaptation to define novel therapeutic approaches

BackgroundChronic lung infection of cystic fibrosis (CF) patients by Staphylococcus aureus is a well-established epidemiological fact. Indeed, S. aureus is the most commonly identified pathogen in the lungs of CF patients. Strikingly the molecular mechanisms underlying S. aureus persistency are not understood.\n\nMethodsWe selected pairs of sequential S. aureus isolates from 3 patients with CF and from one patient with non-CF chronic lung disease. We used a combination of genomic, proteomic and metabolomic approaches with functional assays for in-depth characterization of S. aureus long-term persistence.\n\nResultsFor the first time, we show that late S. aureus isolates from CF patients have an increased ability for intracellular survival in CFBE-F508del cells compared to ancestral early isolates. Importantly, the increased ability to persist intracellularly was confirmed for S. aureus isolates within the own patient F508del epithelial cells. An increased ability to form biofilm was also demonstrated.\n\nFurthermore, we identified the underlying genetic modifications inducing altered protein expression profiles and notable metabolic changes. These modifications affect several metabolic pathways and virulence regulators that could constitute therapeutic targets.\n\nConclusionsOur results strongly suggest that the intracellular environment might constitute an important niche of persistence and relapse necessitating adapted antibiotic treatments.\n\nSummaryS. aureus persists for years in the lungs of patients with cystic fibrosis despite antibiotic therapies. We demonstrate that S. aureus adaptation leads to increased intracellular persistence suggesting a key role for intracellular niche during S. aureus chronic lung infection.

microbiology↗

bioRxiv2018

Phosphorylation of the Type VI secretion system sheath controls Francisella virulence

Francisella tularensis is a facultative intracellular pathogen that causes the zoonotic disease tularemia in human and animal hosts. This bacterium possesses a non-canonical type VI secretion systems (T6SS) required for phagosomal escape and access to its replicative niche in the cytosol of infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the Francisella T6SS in culture. We found that the amounts of essentially all the TSS6 proteins remained unchanged upon KCl stimulation. We therefore hypothesized that a post-translational modification might be involved in T6SS assembly. A whole cell phosphoproteomic analysis allowed us to identify a unique phosphorylation site on IglB, the TssC homologue and key component of the T6SS sheath. Importantly, the phosphorylated form of IglB was not present in the contracted sheath and 3D modeling indicated that the charge repulsion provoked by addition of a phosphogroup on tyrosine 139 was likely to weaken the stability of the sheath structure. Substitutions of the phosphorylatable residue of IglB (tyrosine 139) with alanine or with phosphomimetics prevented T6SS formation and totally impaired phagosomal escape. In contrast, the substitution with the non-phosphorylatable aromatic analog phenylalanine impaired but did not prevent phagosomal escape and cytosolic bacterial multiplication in J774-1 macrophages. Altogether these data suggest that phosphorylation of the sheath participates to T6SS disassembly. Post-translational modifications of the sheath may represent a previously unrecognized mechanism to finely modulate the dynamics of T6SS assembly-disassembly. Data are available via ProteomeXchange with identifier PXD012507. SynopsisFrancisella possesses a non-canonical T6SS that is essential for efficient phagosomal escape and access to the cytosol of infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the Francisella T6SS in culture. We found that KCl stimulation did not result in an increased production of TSS6 proteins. We therefore hypothesized that a post-translational modification might be involved in T6SS assembly. Using a global and site-specific phosphoproteomic analysis of Francisella we identified a unique phosphorylation site on IglB, the TssC homologue and a key component of the T6SS contractile sheath. We show that this site plays a critical role in T6SS biogenesis and propose that phosphorylation may represent a new mechanism affecting the dynamics of sheath formation.

microbiology↗