Construction of Mbv MPB70 mutants and secretome analysis.

(A) Genetic arrangement Mbv wild-type (WT) and Mbv mpb70 knock-out (Δmpb70) strains. (B) Genetic organisation of dipZ-mpb70 genes and primer locations (C) Characterisation of Mbv mutants by PCR. Deletion of mpb70 and maintenance of dipZ in Mbv Δmpb70 and complemented compared to WT. dipZ is amplified in all the strains. mpb70 (244 bp) was amplified in the WT strain but not in Δmpb70 and complemented strains. The PCR with a forward primer in dipZ and reverse primer in mpb70 gave the expected amplification of a 370 bp product for WT, but was absent in Δmpb70. Its absence from the complemented Δmpb70/mpb70 (Compl) strain, confirms that the location of the mpb70 gene, carried by the replicative plasmid pEW70c2, is distal to the wild type chromosomal location. (D) Western immunoblot for detection of MPB70 in the supernatant of the Mbv WT, Δmpb70 and complemented (Mbv-Compl) strains. Each sample was analysed in duplicate. A 23 kDa band corresponding to MPB70 was detected for the WT and complemented strain but not for Δmpb70 strain. (E) Graphical map of the plasmid pGM221-1 used to generate the strain Mtb overexpressing MPT70 (Mtb-MPT70+). The Mtb-H37Rv gene mpt70 is expressed under the control of hsp60 promotor. The clonality of the Mtb-MPT70+ strain was sustained by the Kanamycin resistance cassette carried by the plasmid pGM221-1. (F) Western immunoblot for detection of MPB70 and its homologous MPT70 in the supernatant of the Mbv WT, Δmpb70, Mbv-Compl, Mtb WT and Mtb-MPT70+ strains. A 23 kDa band corresponding to MPB70 was detected for Mbv WT, Mbv-Compl and MTB-MPT70+ strains but not for Mbv Δmpb70 and Mtb WT strains. (G) Principal component analysis of secretome samples. Distinct clusters of sample groups are observed that indicate high reproducibility between replicates and major protein expression differences between the samples analysed. (H) Unsupervised hierarchical clustering of secretome samples. Distinct clusters of sample groups indicate high reproducibility of replicates and distinct protein expression differences between the samples. Two major clusters are observed with uninfected controls clustering separately from mycobacterial-infected samples. Protein groups with lower abundance (e.g., lower LFQ intensity) are in purple and protein groups with higher abundance are in orange.

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