A novel copper-responsive regulon in Mycobacterium tuberculosis

Abstract

In this work we describe the identification of a copper-inducible regulon in Mycobacterium tuberculosis (Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five-locus regulon, which includes a gene that encodes the copper-protective metallothionein MymT, was highly induced in wild-type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper-resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for regulated in copper repressor. Intriguingly, several of the RicR-regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper-responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host.

Fig. 1. Identification of a regulatory element in five loci repressed in proteasome degradation-defective Mtb

(A) Alignment of the lpqS, Rv2963, mymT ricR, and orfAB promoters. The putative transcriptional start sites as determined by 5’RACE are indicated by double-underlines, and the presumed start codons are at the 3’ end of the sequence in capitals and in bold. The palindromic sequences are in bold and indicated by arrows. (Below) Genomic organization around each palindrome (gray boxes). Genes that were down-regulated in the pafA and mpa strains are in black arrows. (B) qRTPCR revealed that ricR orfA and mymT transcripts were decreased in pafA and mpa mutant strains when compared to WT Mtb. Transcript levels were normalized to rpoB (RNA polymerase β-subunit) for each gene in each condition or strain tested. Fold changes relative to WT Mtb are plotted on the y-axis. Experiments are representative of three biological replicates, each performed in triplicate. dlaT (dihydrolipoamide acyltransferase, Rv2215) was used as a negative control. Transcript levels of lpqS, Rv2963, mymT ricR, and orfAB were significantly lower in the pafA and mpa mutants (Student’s t-test, p < 0.05). (C) Complementation of the mpa mutation resulted in restoration of near WT levels of lpqS, Rv2963, mymT ricR, and orfA transcripts. Strains used for complementation were MHD18 (WT+pMV306, empty vector), MHD22 (mpa+pMV306), and MHD23 (mpa+pMV-mpa). The decreased levels in the mpa mutant were statistically significantly different from the WT and complemented strains. (D) Conservation of lpqS, Rv2963, mymT, orfAB and Rv0190 in other Mycobacteria. Mka = M. kansasii, Mma = M. marinum; Mul = M. ulcerans; Mav = M. avium, Mle = M. leprae, Mka = M. kansansii, Mgi = M. gilvum, Msm = M. smegmatis. The asterisk (*) signifies the homologue did not have the palindromic sequence found upstream of Mtb lpqS.

Fig. 2. Genes repressed in pafA and mpa mutants are copper inducible

(A) Induction of genes in response to 500 µM CuSO₄ for four hours. The genes repressed in the proteasome degradation-defective strains (in black) were all significantly induced in WT Mtb (H37Rv) in response to copper as shown by qRTPCR. csoR and ctpV are known copper-inducible genes that were used as positive controls. dlaT served as a negative control. (B) In WT Mtb, lpqS, Rv2963, mymT, ricR, and orfA were significantly induced by 500 µM CuSO₄, 10-fold more iron (0.05% w/v ferric ammonium citrate) and 500 µM ZnSO₄ when compared to untreated samples grown in Sauton’s minimal media. ricR transcripts appear to have the lowest response to the metals tested. Data are representative of two biological replicates, each performed in triplicate.

Fig. 3. RicR is a copper-responsive regulator

(A) Alignment of RicR (Rv0190/MT0200) homologues in other Mycobacteria along with MtbCsoR, and BsuCsoR. Gene identification: Mbo, Mb0196; Mma, MMAR_0433; Mul, MUL_1083; Mav, MAV_4988; Mgi, Mflv_0484; Msm, MSMEG_0230; Mle, ML2609. In black is conserved copper coordinating amino acids [cysteine (Cys) 38, histidine 63, Cys67] based on the crystal structure of MtbCsoR (Liu et al., 2007). The asterisk (*) indicates amino acids predicted to be important for DNA interactions in MtbCsoR (Ma et al., 2009a, Liu et al., 2007). Secondary structure prediction (alpha helices 1–3) was adapted from (Liu et al., 2007). Increasing blue intensity indicates increasing conservation of the highlighted amino acids. (B) Growth curves comparing the WT, ricR transposon mutant, and ricR-complemented strains in 7H9+ADN media (6 µM CuSO₄) and Sauton’s minimal media (trace copper). OD₅₈₀ was measured daily. These experiments represent three independent experiments, each done in duplicate. (C) lpqS, Rv2963, orfA, and mymT are highly expressed in the ricR mutant, comparable to levels in copper-induced WT bacteria. Cultures were grown in Sauton’s minimal media until to OD₅₈₀ of ~0.6, after which RNA was harvested for analysis. For copper treatment, cells were treated for four hours with 500 µM CuSO₄ prior to RNA harvest. ricR transcript was not detected (ND) in the ricR mutant due to disruption by the transposon insertion. qRTPCR was performed as described in the Experimental Procedures and Fig. 1. This is representative of two experiments, each done in triplicate.

Fig. 4. Complementation of the ricR mutation

(A) Immunoblot analysis shows that RicR levels were partially restored in the ricR-complemented strain grown in Sauton’s minimal media. Non-specific bands recognized by the RicR polyclonal antibodies served as loading controls. Data are representative of three biological replicates. (B) lpqS transcript levels at early stationary phase were highly expressed in the ricR mutant compared to WT Mtb and were restored to WT levels in the ricR-complemented strain. Bacteria were grown in Sauton’s minimal media for these experiments and this is representative of two biological replicates performed in triplicate. (C) Immunoblot analysis showed MymT levels were markedly increased in the ricR mutant and were restored to undetectable levels in the complemented strain grown in Sauton’s minimal media. Cross-reactive proteins served as the loading control.

Fig. 5. RicR binds the lpqS promoter

(A) DNA affinity chromatography revealed RicR associates with the lpqS promoter (lpqSp). Clarified lysates of E. coli expressing untagged ricR were incubated with biotinylated DNA (see Experimental Procedures for details). RicR-DNA interaction was disrupted upon palindrome mutagenesis (sequences indicated below). RicR did not bind the csoR promoter (csoRp) or intragenic DNA (pks12). (B) Copper disrupted the RicR-DNA interaction in a dose-dependent manner.

Fig. 6. The ricR mutant is hyper-resistant to copper toxicity

Copper sensitivity assay comparing the ability of WT, ricR and ricR-complemented Mtb strains to survive in 500 µM copper sulfate. The bottom panel shows colony-forming units (CFU) per ml from the same experiment. Data are representative of at least two experiments, each done in triplicate. CFU after 10 days in untreated versus copper-treated ricR strain were not statistically significantly different (Student’s t-test, p > 0.05), while the WT and complemented strains had significantly fewer CFU between copper-treated and untreated cultures.

Fig. 7. Model for a dual copper response in Mtb

Two parallel pathways appear to respond to copper in Mtb. (Top half) Copper induces the release of CsoR from its own promoter, resulting in the expression of ctpV, a predicted P-type ATPase metal transporter. (Bottom half) Copper also de-represses RicR from its own promoter as well as four other promoters. MymT binds up to six Cu(I) ions and may play a role in the sequestration or transport of excess copper. Rv2963 and LpqS are predicted to be membrane associated proteins and may play a role in copper export.