Identification of cyclic AMP-regulated genes in Mycobacterium tuberculosis complex bacteria under low-oxygen conditions

Abstract

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which kills approximately 2 million people a year despite current treatment options. A greater understanding of the biology of this bacterium is needed to better combat TB disease. The M. tuberculosis genome encodes as many as 15 adenylate cyclases, suggesting that cyclic AMP (cAMP) has an important, yet overlooked, role in mycobacteria. This study examined the effect of exogenous cAMP on protein expression in Mycobacterium bovis BCG grown under hypoxic versus ambient conditions. Both shaking and shallow standing cultures were examined for each atmospheric condition. Different cAMP-dependent changes in protein expression were observed in each condition by two-dimensional gel electrophoresis. Shaking low-oxygen cultures produced the most changes (12), while standing ambient conditions showed the fewest (2). Five upregulated proteins, Rv1265, Rv2971, GroEL2, PE_PGRS6a, and malate dehydrogenase, were identified from BCG by mass spectrometry and were shown to also be regulated by cAMP at the mRNA level in both M. tuberculosis H37Rv and BCG. To our knowledge, these data provide the first direct evidence for cAMP-mediated gene regulation in TB complex mycobacteria.

FIG. 1.

1-D SDS-PAGE gel showing protein changes after addition of 10 mM dbcAMP to BCG cultures grown shaking or standing in 1.3% O₂, 5% CO₂ (low O₂) or 20% O₂, 0% CO₂ (high O₂). Banding pattern changes between cultures grown in the presence (+) or absence (−) of exogenous cAMP (10 mM) are marked by dots.

FIG. 2.

Representative 2-D gel profile of BCG grown shaking in low oxygen (1.3% O₂, 5% CO₂) in the presence or absence of 10 mM dbcAMP. Circled protein spots represent protein changes consistently seen upon addition of dbcAMP (10 mM) in three biologically independent experiments. Numbers correspond to protein spot numbers in Tables 2 to 5.

FIG. 3.

RT-PCR quantitation data indicating cAMP-mediated transcriptional regulation of differentially expressed proteins identified from 2-D GE studies. BCG cultures were grown with (solid bars) or without (hatched bars) exogenous cAMP (10 mM) shaking in 1.3% O₂, 5% CO₂ (1.3/5) or 20% O₂, 5% CO₂ (20/5). Genes examined included Rv1265, Rv2971, fixB, GroEL3, mdh, and PE_PGRS6a. Controls included tuf and normalized 16S RNA.

FIG. 4.

Testing GroEL2 alternate transcriptional start site versus protein cleavage models. (A) Schematic of the GroEL2 mRNA transcript indicating the RT-PCR products obtained from the 5′ end (primers 1 and 2) or 3′ end (primers 3 and 4). (B) RT-PCR quantitation data indicating cAMP-mediated transcriptional upregulation from both the 5′ and 3′ ends of the GroEL2 transcript. BCG cultures were grown with (solid bars) or without (hatched bars) exogenous cAMP (10 mM) under shaking low-oxygen (1.3% O₂, 5% CO₂) conditions. (C) Schematic of the GroEL2 amino acid sequence, indicating the region of protein recognized by antibodies MC 4220 and MC 5205. (D) Western blot of shaking low-oxygen 2-D gel profiles (left panel) with (+) or without (−) exogenous cAMP (10 mM) and probed with either MC 4220 (middle panel) or MC 5205 (right panel) to detect possible cleavage products of GroEL2.

FIG. 5.

Representative 2-D gel of BCG cultures grown shaking in low oxygen (1.3% O₂, 5% CO₂) after a 4-day incubation with either 10 mM dbcAMP (A) or 5 mM butyrate (B). Circled protein spots indicate proteins upregulated upon addition of exogenous dbcAMP. Boxed spots indicate proteins where regulation is partially affected by the addition of exogenous butyrate, as well as by the addition of exogenous dbcAMP.