Project description:A longitudinal multi-omics analysis was carried out over a 26-hour small-scale fermentation of B. pertussis. Fermentations were performed in batch mode and under culture conditions intended to mimic industrial processes.
Project description:B. pertussis Tohama I was grown in iron-depleted or iron-replete media and sampled at several time points to assess global gene expression
Project description:Copper is both essential and toxic to living beings, which therefore tightly control its intracellular concentration. At the host-pathogen interface, copper is used by phagocytic cells to kill invading microorganisms. We investigated copper homeostasis in the whooping cough agent Bordetella pertussis, which lives in the human respiratory mucosa and has no environmental reservoir. B. pertussis has considerably streamlined copper homeostasis mechanisms relative to other Gram-negative bacteria. Its single remaining defense line against copper intoxication consists in a metallochaperone diverted for copper passivation and two enzymes involved in peroxide detoxification, which together fight two stresses encountered in phagolysosomes. The three proteins are encoded by an original, composite operon assembled in an environmental ancestor and which is under sensitive control by copper. Interestingly, this system appears to play a role in persistent infection in the nasal cavity of B. pertussis-infected mice. Combining responses to co-occurring stresses in a tailored operon reveals a new strategy adopted by a host-restricted pathogen to optimize survival at minimal energy expenditure.
Project description:In this study we analyzed and compared total proteome and secretome of the wild-type and JN1 mutant strains of Bordetella pertussis. The single-nucleotide transversion in the 5’-UTR of the rplN gene of JN1 mutant led to the increased transcription of the whole operon encoding ribosomal proteins and of the adjoining rpoA gene. These events led to the downregulation and decreased secretion of virulence factors on the background ofgenerally deregulated expression of B. pertussis genome. To get deeper inside in the molecular mechanisms of the observed genome deregulation we then performed the immunoprecipitation of RpoA and compared its binding partners in wild-type and JN1 mutant strains. Nano Reversed phase column (EASY-Spray column, 50 cm x 75 µm ID, PepMap C18, 2µm particles, 100Å pore size) was used for LC/MS analysis. Mobile phase buffer A was composed of water and 0.1% formic acid. Mobile phase B was composed of acetonitrile and 0.1% formic acid. Samples were loaded onto the trap column (Acclaim PepMap300, C18, 5µm, 300Å wide Pore, 300 µm x 5 mm) at a flow rate of 15 μl/min. Loading buffer was composed of water, 2% acetonitrile and 0.1% trifluoroacetic acid. Peptides were eluted with gradient of B from 4% to 35% over 60 min at a flow rate of 300 nl/min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT- qIT, Thermo). Survey scans of peptide precursors from 350 to 1400 m/z were performed at 120K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1.5 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS 2 ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with the charge state 2–6 were sampled for MS 2. The dynamic exclusion duration was set to 45 s with a 10ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2s cycles (76). The data were analyzed and quantified with label-free quantification (LFQ) algorithms in MaxQuant v1.6.3.3 (77) and the Andromeda search engine(78). The false discovery rate (FDR) parameter was set to 1 % for both proteins and peptides. The enzyme specificity of trypsin was set as C-terminal to Arg and Lys. Carbamidomethylation was set as the fixed modification, while N-terminal protein acetylation and methionine oxidation were variable modifications. Maximal number of missed cleavages was set to 2. All hits identified in searches as contaminants were filtered out. The data were searched against Bordetella pertussis reference proteome database (strain Tohama I / ATCC BAA-589 / NCTC 13251).
