Bortezomib treatment increases transcription of clpP1P2, clgR, and acr2 genes in M. bovis BCG. (A) Bortezomib dose-dependent increase of RFP expression under the control of P-clpP1P2, P-clgR, and P-acr2 promoters after 24 h of bortezomib treatment. RFU, relative fluorescence units. Primers and plasmid construction procedures using the integrative plasmid pMV306 (18) to generate the respective reporter strains are listed in Table S1 in the supplemental material. OD600 was measured during the course of the experiment and was found to increase a maximum of 2-fold in the drug-free samples and less in the drug-containing samples. (B) Bortezomib-dependent increase of clpP1P2, clgR, and acr2 mRNA. Transcript levels were measured after 16 h of bortezomib treatment. Primer sequences (16, 19) can be found in Table S2 in the supplemental material. Relative expression (quantification cycle [ΔΔCq]) was calculated as described previously (20) by using 16S RNA as the reference. BZ, bortezomib. CIP, ciprofloxacin. MIC90, drug concentration that inhibited growth of the bacteria by 90%. MIC90 of BZ, 12.5 µM. MIC90 of CIP, 1.6 µM. Data in panels A and B are represented as means ± standard deviations from two biological and four technical replicates.
ClgR is a substrate of mycobacterial Clp, and its accumulation is toxic for M. bovis BCG. (A) Schematics of ClgR-RFP fusion proteins, their transformability, and colony color of M. bovis BCG transformants. Red, RFP; gray, ClgR. “Recovery” indicates whether transformants with the respective constructs could be obtained. “Color” indicates the color of the M. bovis BCG colonies. “GGSG” indicates the peptide sequence used as a short linker inserted between RFP and ClgR. Primers used and plasmid construction procedures employing the episomal plasmid pMV262 (18) to generate the respective strains are listed in Table S3 in the supplemental material. All ClgR-RFP fusion proteins as well as the ClgR nonfusion proteins were overexpressed in M. bovis BCG under the control of the constitutive P-hsp60 promoter (17). Note that the transformation efficiencies for the overexpression constructs for which colonies could be recovered were in the range of 5 × 104/µg DNA and were similar for all constructs. Colony sizes were also similar with the exception of RFP-ClgR, ClgR-RFP-SsrA, and ClgR-RFP-ClgR(C9) transformants, which displayed somewhat smaller colony sizes. For overexpression constructs for which no transformants were obtained, the plates were incubated and observed for 2 months. (B) Fluorescence measurements of M. bovis BCG cultures carrying various ClgR-RFP fusion constructs shown in panel A without and with 24-h bortezomib treatment. (C) Effect of increasing bortezomib concentrations on fluorescence and growth of M. bovis BCG cultures expressing the RFP–full-length ClgR fusion protein (RFP-ClgR [A]). RFU, relative fluorescence units. The bacteria were grown in 96-well plates for 5 days as described in the text with a starting OD600 of 0.05. Turbidity and fluorescence measurements were taken after day 5 with an Infinite M200 Pro plate reader (Tecan). Data shown in panels B and C represent means ± standard deviations from two biological and four technical replicates.