The Rip1 protease of Mycobacterium tuberculosis controls the SigD regulon

Abstract

Regulated intramembrane proteolysis of membrane-embedded substrates by site-2 proteases (S2Ps) is a widespread mechanism of transmembrane signal transduction in bacteria and bacterial pathogens. We previously demonstrated that the Mycobacterium tuberculosis S2P Rip1 is required for full virulence in the mouse model of infection. Rip1 controls transcription in part through proteolysis of three transmembrane anti-sigma factors, anti-SigK, -L, and -M, but there are also Rip1-dependent, SigKLM-independent pathways. To determine the contribution of the sigma factors K, L, and M to the Δrip1 attenuation phenotype, we constructed an M. tuberculosis ΔsigKΔ sigL ΔsigM mutant and found that this strain fails to recapitulate the marked attenuation of Δrip1 in mice. In a search for additional pathways controlled by Rip1, we demonstrated that the SigD regulon is positively regulated by the Rip1 pathway. Rip1 cleavage of transmembrane anti-SigD is required for expression of SigD target genes. In the absence of Rip1, proteolytic maturation of RsdA is impaired. These findings identify RsdA/SigD as a fourth arm of the branched pathway controlled by Rip1 in M. tuberculosis.

FIG 1

ΔsigKLM deletion does not phenocopy Δrip1 in mouse infection. (A) Confirmation of the M. tuberculosis ΔsigKLM strain. Southern blot analysis of M. tuberculosis chromosomal DNA was used to confirm the genotype of a ΔsigKLM triple mutant. When probed with the 3′ flanking region of sigM, EcoRI-digested genomic DNA produces a 12.2-kb band in ΔsigL (ΔL) cells, whereas the ΔsigLM (ΔLM) strain has a 5.2-kb band representing the sigM deletion. When probed with the 5′ flanking region of sigK, PstI-digested genomic DNA produces a 4.8-kb band in the ΔLM strain, indicating a wild-type sigK allele, whereas the ΔsigKLM (ΔKLM) triple mutant strain has a 551-bp band, representing the ΔsigK allele. (B) Phenotype of the Δrip1 strain in mice. Recovered CFU from lungs of mice infected via aerosol with wild-type M. tuberculosis and the Δrip1 strain are depicted. Error bars represent standard deviations and when not visible are within the symbol. (C) Recovered CFU from lungs of mice infected via aerosol with wild-type M. tuberculosis and the ΔsigKLM mutant. (D) Recovered CFU from spleens of mice infected via aerosol with wild-type M. tuberculosis and the ΔsigKLM mutant.

FIG 2

Topology of anti-sigma factors D and G in the mycobacterial membrane. (A) The amino acid sequence of anti-sigma factor G was analyzed for potential transmembrane domains using the TMpred server. The red line indicates the threshold for significance. (B) β-Gal or PhoA was fused to the C terminus of anti-sigma factor G (G), anti-sigma factor D (D), or anti-sigma factor L (L), and the plasmids encoding these fusions were transformed into M. smegmatis along with a vector control (vect) or a positive control (+) for lacZ (consisting of unfused lacZ) or phoA (an antigen85-phoA fusion [26]). The left side shows three replicates of each strain with β-galactosidase fusions cultured on medium containing X-Gal, and the right side shows three replicates of M. smegmatis with each PhoA fusion cultured on medium containing BCIP.

FIG 3

Allelic exchange of rsdA and sigD in M. tuberculosis. (A and B) Targeted allelic exchange strategy for replacement of sigD (A) or rsdA (B) with a hygromycin resistance cassette flanked by loxP sites. (C) Southern blot analysis of M. tuberculosis chromosomal DNA was used to confirm the genotype of ΔsigD and ΔsigD Δrip1 strains. Hybridization of the 3′ flanking region of SigD to SmaI-fragmented genomic DNA produces a 941-bp hybridization product in the wild type, whereas the ΔsigD mutant produces a 2,972-bp hybridization product. (D) When probed with the 5′ flanking region of rsdA, BamHI-digested chromosomal DNA produces a 1,148-bp hybridization product, whereas a ΔrsdA strain produces a 7,288-bp hybridization product.

FIG 4

Activation of SigD-dependent transcription requires Rip1 cleavage of RsdA. Quantitative real-time PCR was used to measure the mRNA levels of rpfC (A) or rv1815c (B) in the strains as indicated. Strains were grown in 7H9 medium to log phase for RNA collection. Relative gene expression was normalized to sigA (housekeeping) gene expression. Significance is indicated by horizontal bars connecting specific pairs of measurements, with single asterisks indicating a P value of <0.01 and double asterisks indicating a P value of <0.001.

FIG 5

Rip1 is required for the proteolytic processing of RsdA. (A) Experimental schematic for degradation of RsdA with an N-terminal MBP tag in the mycobacterial membrane. Shown are the possible species of MBP-RsdA after proteolysis by site-1 (S1P) and site-2 (i.e., Rip1) proteases. Lysates from the M. smegmatis wild type (WT) and ΔMSMEG_2579 mutant (ΔMsrip1) with plasmids encoding MBP-RsmA (B), MBP-RsdA(C), and MBP-RsgA (D) were analyzed by immunoblotting with antibodies recognizing MBP or RNAPβ as a loading control. (E) Immunoblots for MBP (top) or RNAPβ (bottom) of MBP-RsdA expressed in biologic triplicates of WT or Δrip1 M. tuberculosis.