Phosphate starvation: a novel signal that triggers ESX-5 secretion in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis uses the Type VII ESX secretion systems to transport proteins across its complex cell wall. ESX-5 has been implicated in M. tuberculosis virulence, but the regulatory mechanisms controlling ESX-5 secretion were unknown. Here we uncover a link between ESX-5 and the Pst/SenX3-RegX3 system that controls gene expression in response to phosphate availability. The DNA-binding response regulator RegX3 is normally activated by phosphate limitation. Deletion of pstA1, which encodes a Pst phosphate uptake system component, causes constitutive activation of RegX3. A ΔpstA1 mutant exhibited RegX3-dependent overexpression of esx-5 genes and hyper-secretion of the ESX-5 substrates EsxN and PPE41 when the bacteria were grown in phosphate-rich medium. In wild-type M. tuberculosis, phosphate limitation activated esx-5 transcription and secretion of both EsxN and PPE41, and this response required RegX3. Electrophoretic mobility shift assays revealed that RegX3 binds directly to a promoter within the esx-5 locus. Remarkably, phosphate limitation also induced secretion of EsxB, an effector of the virulence-associated ESX-1 secretion system, though this induction was RegX3 independent. Our work demonstrates that the Pst/SenX3-RegX3 system directly regulates ESX-5 secretion at the transcriptional level in response to phosphate availability and defines phosphate limitation as an environmental signal that activates ESX-5 secretion.

Figure 1

Overexpression of esx-5 genes in the ΔpstA1 mutant is RegX3-dependent. A. Schematic representation of the esx-5 locus. Genes encoding ESX-5 conserved components are in black, known ESX-5 substrates are in white, and a known ESX-5 cytoplasmic chaperone is in light gray. Genes with no confirmed function in ESX-5 secretion are in dark gray. The * indicates a gene with a known frame-shift mutation. B. Quantitative RT-PCR analysis of esx-5 transcription. Wild-type M. tuberculosis Erdman (WT), ΔpstA1, ΔregX3, ΔpstA1ΔregX3, ΔregX3 pNDregX3 and ΔpstA1ΔregX3 pNDregX3 were cultured in P_i-rich 7H9 medium to mid-exponential phase and RNA was extracted. Abundance of the eccB₅, esxM, esxN, espG₅, eccD₅, mycP₅ and ppe41 transcripts relative to sigA was determined by quantitative RT-PCR. Data shown are the means +/- standard deviations of three independent experiments. Asterisks indicate a statistically significant difference in transcript abundance compared to the WT control (P < 0.05).

Figure 2

Hyper-secretion of ESX-5 substrates by the ΔpstA1 mutant requires RegX3. Wild-type M. tuberculosis Erdman (WT), ΔpstA1, ΔregX3, ΔpstA1ΔregX3, ΔregX3 pNDregX3 and ΔpstA1ΔregX3 pNDregX3 were cultured in Sauton's complete medium without Tween-80 for 5 days. 10 μg of cell lysate (CL) and 4 μg of culture filtrate (CF) proteins were subjected to SDS-PAGE and Western blot analysis. Antibodies used are indicated. Anti-GroEL2 was used as both a loading control for the CL fraction and a cell lysis control in the CF fraction. Anti-ModD was used as a loading control for the CF fraction. Results shown are from a single experiment and are representative of 3 independent experiments.

Figure 3

Induction of ESX-5 genes by phosphate limitation requires RegX3. Wild-type M. tuberculosis Erdman (WT), ΔregX3, and ΔregX3 pNDregX3 were cultured in P_i-free 7H9 medium for 96 hours. RNA was extracted at 0, 24, 48, 72 and 96 hours. Abundance of the esxN, espG₅, eccD₅, eccB₅, ppe41, modD, esxB, and espA transcripts relative to 16S rRNA was determined by quantitative RT-PCR. Data shown are the means +/− standard deviations of three independent experiments. Asterisks indicate statistically significant differences in transcript abundance between WT and ΔregX3 or ΔregX3 pNDregX3 and ΔregX3: * P < 0.05; ** P < 0.005

Figure 4

Phosphate limitation induces ESX-5 and ESX-1 protein secretion. Wild-type M. tuberculosis Erdman was grown for 5 days in Sauton's complete medium without Tween-80 (control) or P_i-free Sauton's medium without Tween-80 to which 250, 25, or 2.5 μM KH₂PO₄ was added exogenously. 10 μg of cell lysate (CL) and 4 μg or 8 μg (as indiciated) of culture filtrate (CF) proteins were subjected to SDS-PAGE and Western blot analysis. Antibodies used are indicated. Anti-GroEL2 was used as both a loading control for the CL fraction and a cell lysis control in the CF fraction. Anti-ModD was used as a loading control for the CF fraction. Results shown are from a single experiment and are representative of 2 independent experiments.

Figure 5

Secretion of the ESX-1 substrate EsxB is induced by phosphate limitation. Wild-type M. tuberculosis Erdman was cultured for 5 days in Sauton's complete medium without Tween-80 (control), or P_i-free Sauton's medium without Tween-80 to which either 2.5 μM KH₂PO₄ (P_i and K⁺ limitation), 2.5 μM NaH₂PO₄ and 4 mM KCl (P_i limitation) or 2.5 μM KCl and 4 mM NaH₂PO₄ (K⁺ limitation) was added exogenously. 10 μg of cell lysate (CL) and 4 μg of culture filtrate (CF) proteins were subjected to SDS-PAGE and Western blot analysis. Anti-GroEL2 was used as both a loading control for the CL fraction and a cell lysis control in the CF fraction. Anti-ModD was used as a loading control for the CF fraction. Results shown are from a single experiment and are representative of 3 independent experiments.

Figure 6

RegX3 is required for induction of ESX-5 protein secretion in response to phosphate limitation. Wild-type M. tuberculosis Erdman (WT), ΔregX3, and ΔregX3 pNDregX3 strains were cultured for 5 days either in Sauton's complete medium without Tween-80 (control) or in P_i-free Sauton's medium without Tween-80 to which 2.5 μM KH₂PO₄ was added exogenously. 10 μg of cell lysate (CL) and 4 μg of culture filtrate (CF) proteins were subjected to SDS-PAGE and Western blot analysis. Anti-GroEL2 was used as both a loading control for the CL fraction and a cell lysis control in the CF fraction (data not shown). Anti-ModD was used as a loading control for the CF fraction. Results shown are representative of 3 independent experiments.

Figure 7

Determining the RegX3 binding site within the esx-5 locus. A. Schematic representation of the 3’ esx-5 locus. The intergenic region between ppe27 and pe19 is enlarged. Locations of relevant primers and probes used for EMSAs are indicated. B. Quantitative RT-PCR analysis of esx-5 transcription. Wild-type M. tuberculosis Erdman (WT), ΔpstA1, ΔpstA1Δppe27-pe19, ΔpstA1Δppe27 and ΔpstA1Δpe19 were cultured in P_i-rich 7H9 medium to mid-exponential phase and RNA was extracted. Abundance of the pe19, esxN, espG₅, and eccD₅ transcripts relative to sigA was determined by quantitative RT-PCR. Data shown are the means +/- standard deviations of three experiments. Asterisks indicate statistically significant differences in transcript abundance between ΔpstA1 and ΔpstA1Δppe27-pe19 (P < 0.05). C. Quantitative RT-PCR analysis of mRNA levels of amplicons within the ppe27-pe19 intergenic region and pe19. WT, ΔpstA1 and ΔpstA1ΔregX3 strains were cultured in P_i-rich 7H9 medium to mid-exponential phase and RNA was extracted. Abundance of the amplicons relative to sigA was determined by quantitative RT-PCR. Data shown are the means +/− standard deviations of three independent experiments. Asterisks indicate statistically significant differences in amplicon abundance between ΔpstA1 and either WT or ΔpstA1ΔregX3 (P < 0.05). D. Identification of an esx-5 operon by RT-PCR. WT, ΔpstA1, and ΔpstA1ΔregX3 were cultured in P_i-rich 7H9 medium to mid-exponential phase (+P_i). WT, ΔregX3 and ΔregX3 pND−regX3 strains were cultured for 24 hours in 7H9 no P_i (−P_i). RNA was extracted, reverse transcribed to cDNA, and PCR amplified using the indicated primers. +RT and −RT denote cDNA synthesis reactions where reverse transcriptase was included or excluded, respectively. gDNA was included as a template for each primer pair as a positive control. E. EMSA analysis of binding between purified His₆-RegX3 the SenX3 probe (positive control), and Probes A and B probes. 0.5 ng of DIG-labeled probe was incubated with increasing concentrations (0-1 μg) of purified recombinant His₆-RegX3. F. EMSA analysis of RegX3 binding specificity. DIG-labeled senX3 probe (positive control) or Probe A was incubated with purified recombinant His₆-RegX3. Unlabeled competitors (specific or non-specific) or α-His₆ antibodies were added to the binding reactions as indicated by the + symbols.

Figure 8

Model of ESX-5 regulation by the Pst/SenX3-RegX3 system in response to phosphate availability. When the external P_i concentration is high, the Pst system inhibits the SenX3-RegX3 two-component system, esx-5 genes are transcribed at a basal level, there is no induction of secretion and ESX-5 is ‘off’. When external P_i is limiting, inhibition of SenX3-RegX3 by the Pst system is relieved, turning the ESX-5 system ‘on’. SenX3 activates RegX3 via phospho-transfer, allowing RegX3 to bind a promoter within the esx-5 locus to initiate transcription, leading to increased secretion of ESX-5 substrates.