| Metadata |
| datasetIdentifier | PASS00685 |
| datasetType | SWATH |
| submitter | Olga Schubert <olga.schubert@gmail.com> |
| submitter_organization | ETH Zurich |
| lab_head_full_name | Ruedi Aebersold |
| lab_head_email | aebersold@imsb.biol.ethz.ch |
| lab_head_organization | ETH Zurich |
| lab_head_country | Switzerland |
| datasetTag | MtbMboComparison |
| datasetTitle | Comparative 'omics analyses differentiate Mycobacterium tuberculosis and Mycobacterium bovis and reveal distinct macrophage responses to infection with the human and bovine tubercle bacilli |
| publicReleaseDate | 2018-02-27 00:00:00 |
| finalizedDate | 2018-02-27 14:02:17 |
| summary | Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in a range of mammals, including humans. A key feature of MTBC pathogens is their high degree of genetic identity, yet distinct host tropism. Notably, while Mycobacterium bovis is highly virulent and pathogenic for cattle, the human pathogen M. tuberculosis is attenuated in cattle. Previous research also suggests that host preference amongst MTBC members has a basis in host innate immune responses. To explore MTBC host tropism, we present in-depth profiling of the MTBC reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv at both the global transcriptional and translational level via RNA-sequencing and SWATH mass spectrometry. Furthermore, a bovine alveolar macrophage infection time course model was employed to investigate the shared and divergent host transcriptomic response to infection with M.tuberculosis H37Rv or M. bovis AF2122/97. Significant differential expression of virulence-associated pathways between the two bacilli was revealed, including the ESX-1 secretion system. A divergent transcriptional response was observed between M. tuberculosis H37Rv and M. bovis AF2122/97 infection of bovine alveolar macrophages, in particular cytosolic DNA-sensing pathways at 48 hours post-infection, and highlights a distinct engagement of M. bovis with the bovine innate immune system. The work presented here therefore provides a basis for the identification of host innate immune mechanisms subverted by virulent host-adapted mycobacteria to promote their survival during the early stages of infection. |
| contributors | Kerri M. Malone, Kévin Rue-Albrecht, David A. Magee, Kevin Conlon, Olga T. Schubert, Nicolas C. Nalpas, John A. Browne, Alicia Smyth, Eamonn Gormley, Ruedi Aebersold, David E. MacHugh, Stephen V. Gordon |
| publication | Malone et al., Comparative 'omics analyses differentiate Mycobacterium tuberculosis and Mycobacterium bovis and reveal distinct macrophage responses to infection with the human and bovine tubercle bacilli, Microbial Genomics, 2018, in press |
| growth | Exponentially grown mycobacterial liquid cultures were established in Sauton’s basal media +0.025% tyloxapol. For mid to late-log phase culture, mycobacterial cells were grown to an optical density (OD600nm) of 0.6 – 0.8 at 37C prior to harvest. Six M. bovis AF2122/97 and six M. tuberculosis H37Rv replicates were prepared. |
| treatment | No treatment. |
| extraction | Mycobacterial cells were harvested by centrifugation at 2,500 x g for 10 min and the pellet was re-suspended in 1 ml LB buffer (0.1 M ammonium bicarbonate buffer, 8 M urea, 0.1% RapiGEST SF (Waters, UK)). The suspension was transferred to a 2-ml screw cap tube and the cells were lysed by bead-beating for 30 sec at maximum setting using 3 mm glass beads (Sigma) and a MagNaLyser instrument (Roche). The lysate supernatant was harvested by centrifugation at 12,000x g for 10 min and transferred to a clean 1-ml tube. The remaining pellet was re-suspended in LB buffer and the bead beating cycle was repeated twice more. Protein lysate samples were stored at -80°C. Protein samples were removed from -80°C storage and thawed on ice. Total protein content was measured using the Qubit Protein Assay kit according to manufacturer’s guidelines and protein concentrations were adjusted to 0.5 mg/ml. |
| separation | No separation. |
| digestion | Protein disulphide bonds were reduced by addition of 0.2 M Tris(2-carboxyethyl)phosphine (TCEP) and the resulting free cysteine residues were alkylated by addition of 0.4 M iodoacetamide (IAA). Extracted protein samples were diluted with 0.1 M ammonium bicarbonate buffer to reach a urea concentration of < 2 M and then digested with 1:50 enzyme/substrate ratio of sequencing grade modified trypsin (Promega). 50% trifluoroaceticacid (TFA) was added to lower the pH to 2 in order to stop the tryptic digest and to precipitate the RapiGEST. Water-immiscible degradation products of RapiGEST were pelleted by centrifugation at 12,000 x g for 10 min. The cleared peptide solution was desalted with C18 reversed-phase columns (SepWPak Vac C18, Waters). The columns were pre-conditioned 2-3 times with acetonitrile and equilibrated 3 times with Buffer A (2% acetonitrile, 0.1% trifluoroacetic acid in H2O) prior to sample loading. The flow-through was re-loaded onto the column and the column was then washed 3 times with Buffer A. The peptides were eluted from the column using Buffer B (50% acetonitrile, 0.1% trifluoroacetic acid in H2O) and the elution step was repeated. The eluate was dried under vacuum using a rotary evaporator at 45C. Dried peptide pellets were re-suspended in MS buffer (2% acetonitrile, 0.1% trifluoroacetic acid in ultra pure H2O) to a concentration of 1 ug/ul, sonicated in a water bath for 3 min and supernatant was harvested by centrifugation at 12,000 x g for 10 min. |
| acquisition | 1 ug of each peptide sample was measured in SWATH mode on a TripleTOF 5600 mass spectrometer using data-independent acquisition settings essentially as described earlier (Schubert et al., Cell Host & Microbe 2015). |
| informatics | SWATH data was analysed using an automated pipeline and the software OpenSWATH with the M. tuberculosis H37Rv SWATH assay library essentially as described earlier (Schubert et al., Cell Host & Microbe 2015). |
| instruments | AB SCIEX TripleTOF 5600+ |
| species | Mycobacterium tuberculosis, Mycobacterium bovis |
| massModifications | C+57.021464 |
