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).

Project description:Background Bordetella pertussis is a Gram-negative bacterium that infects the human respiratory tract and causes pertussis or whooping cough. The disease has resurged in many countries including Finland where the whole-cell pertussis vaccine has been used for more than 50 years. Antigenic divergence has been observed between vaccine strains and clinical isolates in Finland. To better understand genome evolution in B. pertussis circulating in the immunized population, we developed an oligonucleotide-based microarray for comparative genomic analysis of Finnish strains isolated during the period of 50 years. Methodology/Principal Findings The microarray consisted of 3,582 oligonucleotides (70-mer) and covered 94% of the genome of Tohama I, the strain of which the genome has been sequenced [21]. Twenty isolates from 1953 to 2004 were studied together with two Finnish vaccine strains and two international reference strains. The isolates were selected according to their characteristics, e.g. the year and place of isolation and pulsed-field gel electrophoresis profiles. Genomic DNA of the tested strains, along with reference DNA of Tohama I strain, was labelled and hybridized. The absence of genes as established with microarrays, was confirmed by PCR. Compared to the Tohama I strain, Finnish isolates lost 7 (8.6 kb) to 49 (55.3 kb) genes, clustered in one to four distinct loci. The number of lost genes increased with time, and one third of lost genes had functions related to ion transport, metabolism, or energy production and conversion. All four loci of lost genes were flanked by the insertion sequence element IS481. Conclusion/Significance Our results showed that the progressive gene loss occurred in Finnish B. pertussis strains isolated during a period of 50 years and confirmed that B. pertussis is dynamic and is continuously evolving, suggesting that the bacterium may use gene loss as one strategy to adapt to highly immunized populations. Keywords: comparetive genomic hybridisation

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Bordetella pertussis strain:Tohama I-derived mutant

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