Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator

Abstract

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

Fig 1. Global features of the transcriptomes and proteomes of the M. marinum and M. tuberculosis esx-1 mutant strains.

Volcano plots obtained from RNA-seq analysis of the wild-type M. marinum E11 strain vs. the eccCb₁ transposon mutant (A) and of M. tuberculosis mc²6020 vs. the esx-1 mutant strain (B). Each dot indicates the expression value of a gene. Red dots indicate statistical significance (q < 0.05), and black dots indicate a lack of statistical significance. Selected genes that are most down- or up-regulated in the esx-1 mutant strains are highlighted. (C) Venn diagram of the number of differentially expressed transcripts and proteins quantified of M. marinum eccCb₁ mutant using RNA-seq and quantitative proteomics, respectively. Scatterplots of the relationship between differentially expressed genes of M. marinum eccCb₁ transposon mutant and those of the isogenic wild-type strain E11, quantified in both data sets and classified into the following categories: (D) lipid metabolism, (E) regulatory proteins and (F) cell wall and cell process. Scatterplots and bar chart show the rectilinear equation and the Pearson correlation coefficient (R²).

Fig 2. Top differentially expressed genes of M. marinum and M. tuberculosis, when grown in culture medium, grouped into broad functional categories.

Within each group, genes are ranked in ascending order by p-value. (Red) Top 100 annotated M. marinum E11 genes that exhibit greatest differential expression in the M. marinum eccCb₁ transposon mutant compared to the isogenic wild-type strain E11 during growth in 7H9 culture medium. Bar chart of log2-fold change for individual genes (RNA, blue; protein, red; locus tags, outer). (Green) Top 100 annotated M. tuberculosis genes that exhibit greatest differential expression in the auxotrophic M. tuberculosis RD1 deletion mutant strain mc²6030 compared to the isogenic control strain mc²6020 during growth in 7H9 culture medium. Bar chart of log2-fold change for individual genes. The genes rv3872-rv3878 are not included as these genes are deleted in the RD1 mutant strain.

Fig 3. Effect of ESX-1 disruption (M. marinum eccCb₁ Transposon Mutant) on gene transcription during infection (Indicated as ínt’) and growth in culture medium in M. marinum compared to that in the wild-yype strain E11 during growth in 7H9 culture medium.

(A) Relative transcript expression levels of the ESX-1 secretion system-associated genes, including the main esx-1 locus as well as the EspR regulator and accessory factors in the espA operon, which is encoded outside the RD-1 region. (B) Gene expression levels, as measured by qRT-PCR, were compared to those of the wild-type strain E11 grown in similar conditions. Values represent mean ± standard error of the mean of two biological replicates. (C, D) Regulation of genes associated with cell wall synthesis, including genes involved in mycolic acid synthesis (C) and PDIMs (D).

Fig 4. Esx-1 transposon mutants have similar gene transcription profiles.

Gene expression levels for M. marinum eccB₁, eccCa₁, eccCb₁, eccD₁ and eccE₁ transposon mutants as measured by qRT-PCR. All strains were grown in 7H9 culture medium, and gene expression levels were compared to those of the wild-type strain E11. Values represent mean ± standard error of the mean of at least three biological replicates.

Fig 5. Regulation of the ESX-1 secretion system.

(A) Down-regulation of espL is the result of a regulatory process. A functional copy of espL was introduced into wild-type and eccCb1 mutant strains of M. marinum, and the espK and espL gene expression levels were measured by qRT-PCR. Gene expression levels were compared to those of the wild-type strain E11. Values represent mean ± standard error of the mean of two biological replicates. (B) Introduction of PE35/PPE68_1 result in increased EsxA secretion but not in gene regulation. Pellet (p), cell wall extract (cw), and supernatant (s) fractions of the wild-type and eccCb1 mutant strains of M. marinum expressing PE35/PPE68_1; PE35/PPE68, containing a C-terminal deletion of PPE68_1; or PE35/PPE68_1, containing a 15-amino-acid C-terminal deletion of PE35, were analysed for the presence of EspE, EsxA and the introduced PE35 by immunoblotting. Fractions represent 0.5, 1 or 2 OD units of original culture. In all cases, PE35 contained a C-terminal HA tag. (C) EspG1, EspI and PE35/PPE68_1 do not regulate the transcription of selected esx-1-associated genes. EspG1, EspI or PE35/PPE68_1 were overexpressed in the M. marinum eccCb1 mutant strain, and the expression levels of espK, espL, esxA, pe_pgrs1 and eccD1 were measured by qRT-PCR. Gene expression levels were compared to those of the wild-type strain E11. Values represent mean ± standard error of the mean of at least two biological replicates. (D) WhiB6 is involved in transcriptional regulation of ESX-1 substrates and associated genes. The whib6 gene was overexpressed in the M. marinum eccCb1 mutant strain, and transcript levels of espK, espL, esxA, pe_pgrs1 and eccD1 were measured by qRT-PCR. Gene expression levels were compared to those of the eccCb1 mutant strain. Values represent mean ± standard error of the mean of two biological replicates.

Fig 6. Gene expression profiles (Log2 Fold Change) of the complementary strains (M. marinum M^USA-complementary and M. marinum M^VU-complementary) and knock-out strains (M. marinum M^USA -⊿whiB6 and M. marinum M^VU-⊿whiB6) compared with those of the corresponding control strains (M. marinum M^USA-empty vector strain and M. marinum M^VU-empty vector).

The red colour represents up-regulate genes and green colour represents down regulate genes compared with the control strains. The heat map of expression of the whiB6-activated gene set is shown in (A); Expression of the esx-1 locus is shown in (B); and (C) shows the WhiB6-repressed gene set.