Overexpression of the Rv0805 phosphodiesterase elicits a cAMP-independent transcriptional response

Abstract

The Rv0805 gene in Mycobacterium tuberculosis encodes a metallophosphoesterase which shows cAMP-hydrolytic activity. Overexpression of Rv0805 has been used as a tool to lower intracellular cAMP levels and thereby elucidate the roles of cAMP in mycobacteria. Here we show that levels of cAMP in M. tuberculosis were lowered by only ∼30% following overexpression of Rv0805, and transcript levels of a number of genes, which include those associated with virulence and the methyl citrate cycle, were altered. The genes that showed altered expression were distinct from those differentially regulated in a strain deleted for the cAMP-receptor protein (CRP(Mt)), consistent with the relatively low dependence on cAMP of CRP(Mt) binding to DNA. Using mutants of Rv0805 we show that the transcriptional signature of Rv0805 overexpression is a combination of catalysis-dependent and independent effects, and that the structurally flexible C-terminus of Rv0805 is crucial for the catalysis-independent effects of the protein. Our study demonstrates the dissociation of Rv0805 and cAMP-regulated gene expression, and reveals alternate functions for this phosphodiesterase from M. tuberculosis.

Keywords: Cyclic AMP; Microarray; Mycobacterium tuberculosis; Phosphodiesterase; Rv0805.

Figure 1

Overexpression of Rv0805 elicits changes in gene expression. A. Confirmation of Rv0805 overexpression in M. tuberculosis by immunoblotting using Rv0805-specific antibodies. Equal amounts of protein from cell lysates of strains containing empty vector (Control) or Rv0805 overexpression constructs (Rv0805) were used for western blotting. Robust overexpression of wild type Rv0805 was observed in three independent transformants (colonies A, B, C). B. Measurement of intracellular cAMP levels in M. tuberculosis overexpressing Rv0805 (Rv0805) compared to the empty vector (control). Bacterial cAMP was measured in the exponential phase of growth (Light transmittance at 600 nm ∼ 0.6). Expression of wild type Rv0805 led to decreased intracellular cAMP levels. Mean ± SEM of measurements done in triplicates from three independent biological replicates is plotted. C. Validation of microarray results by qRT-PCR was performed for three up-regulated (Rv1623c, Rv0287 and Rv2057c) and three down-regulated genes (Rv3197A, Rv1318c and Rv0467). rrs, coding for 16S rRNA, was used as the normalizing gene. Bars represent mean ± SEM of expression level of genes in M. tuberculosis-Rv0805 relative to M. tuberculosis-VC as measured from triplicate cultures. Numbers in parentheses represent fold regulation from microarray results. D. Analysis of cellular pathways affected by Rv0805 overexpression. Genes with altered expression levels (>±2 fold) on expression of Rv0805 were assigned functional categories according to Tuberculist (www.tuberculist.epfl.ch) annotation. Numbers represent percentage of total altered genes.

Figure 2

Comparison of changes in the transcriptome following Rv0805 overexpression and in the Δcrp mutant A. Comparison of Rv0805 overexpression gene set and the CRP^Mt regulon. Fold regulation of 160 genes from the two data sets were compared on a scatter plot. Pearson's correlation analysis was performed using Graphpad Prism and was found not to be statistically significant (p-value 0.49). B. Comparison of cellular pathways affected by Rv0805 overexpression (outer donut) and CRP^Mt deletion (inner donut). Genes with altered expression levels (>±2 fold) were assigned functional categories according to Tuberculist (www.tuberculist.epfl.ch) annotation. Numbers represent percentage of total genes altered. C. Comparison of genes altered by Rv0805 overexpression and CRP^Mt deletion. Genes dysregulated upon Rv0805 overexpression (upper panel) do not show significant alteration in expression levels in the CRP^Mt knockout and vice-versa (lower panel). Red boxes indicate up-regulation and blue boxes indicate down-regulation compared to appropriate reference strains. Numbers indicate fold change in expression level as obtained from microarray analyses. ‘-’ indicates no change in expression levels (<2.0 fold).

Figure 3

A. Role of the C-terminus of Rv0805 in gene expression changes. Overexpression of Rv0805N97A and Rv0805(1-278)N97A. Immunoblot for lysates of M. tuberculosis harboring empty vector (control) or plasmid for overexpression of Rv0805, Rv0805N97A or Rv0805(1-278)N97A. Polyclonal antibodies against Rv0805 or a C-terminus specific monoclonal antibody (1G5B5) were used to distinguish between the full length and C-terminally truncated proteins based on differential mobility and loss of immunoreactivity respectively. B. Intracellular cAMP levels in M. tuberculosis overexpressing Rv0805, Rv0805N97A or Rv0805(1-278)N97A. Mean ± SEM of at least two biological replicates are plotted. (n.s. indicates that the two groups compared are statistically not significant (p-value > 0.05)) C. Heat map showing expression levels of genes in M. tuberculosis-Rv0805 compared with a strain harboring empty vector (control) or overexpressing Rv0805N97A. Two distinct gene sets (A and B) are clearly discernible. D. Genes showing altered regulation in the Rv0805 overexpression strain compared to M. tuberculosis harboring empty vector (control) or overexpressing Rv0805N97A or Rv0805(1-278)N97A. Presence of the C-terminus of Rv0805 is essential for manifestation of catalysis-independent gene expression changes. Red boxes indicate up-regulation and blue boxes indicate down-regulation in M. tuberculosis-Rv0805 compared to the indicated strains. Numbers indicate fold change in expression level as obtained from microarray analyses. ‘-’ indicates no change in expression levels (<2.0 fold).