Basal-Level Effects of (p)ppGpp in the Absence of Branched-Chain Amino Acids in Actinobacillus pleuropneumoniae

Abstract

The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory mechanism in bacterial pathogens, enabling adaptation to adverse environments, and is linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs of pigs, its only known natural host. Pig lungs are known to have a low concentration of free branched-chain amino acids (BCAAs) compared to the level in plasma. We had investigated the role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae Now, we sought to determine whether (p)ppGpp was a trigger signal for the SR in A. pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic analyses of the wild type (WT) and an relA spoT double mutant [which does not produce (p)ppGpp], we found that (p)ppGpp could repress de novo purine biosynthesis and activate antioxidant pathways. There was a positive correlation between GTP and endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology, and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the biosynthesis of BCAAs were constitutively upregulated, regardless of the existence of BCAAs, without accumulation of (p)ppGpp beyond a basal level. Collectively, our study shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. pleuropneumoniae (p)ppGpp-mediated regulation in A. pleuropneumoniae is different from that described for the model organism Escherichia coli Further work will establish whether the (p)ppGpp-dependent SR mechanism in A. pleuropneumoniae is conserved among other veterinary pathogens, especially those in the Pasteurellaceae family.IMPORTANCE (p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional challenges. Here, we present transcriptional and phenotypic differences of A. pleuropneumoniae grown in different chemically defined media in the absence of (p)ppGpp. We show that the deprivation of branched-chain amino acids (BCAAs) does not elicit a change in the basal-level (p)ppGpp, but this level is sufficient to regulate the expression of BCAA biosynthesis. The mechanism found in A. pleuropneumoniae is different from that of the model organism Escherichia coli but similar to that found in some Gram-positive bacteria. This study not only broadens the research scope of (p)ppGpp but also further validates the complexity and multiplicity of (p)ppGpp regulation in microorganisms that occupy different biological niches.

Keywords: (p)ppGpp; Actinobacillus pleuropneumoniae; BCAAs; GTP; stringent response.

FIG 1

Synthesis of (p)ppGpp and growth profiles of the WT and (p)ppGpp⁰ grown in rich medium. (A) Accumulation of (p)ppGpp in the WT and (p)ppGpp⁰ mutant. Cells were labeled with [³²P]H₃PO₄ in MOPS under starvation conditions; the formic acid extracts of the cells were subjected to TLC analysis as described in Materials and Methods. (B) Growth curves of the WT and (p)ppGpp⁰ mutant. Growth was monitored by OD₆₀₀ at various time points. (C) Cell viability of the WT and (p)ppGpp⁰ mutant. Overnight cultures were serially diluted with fresh medium and spotted onto the TSA plate. (D) (p)ppGpp⁰ failed to survive sudden starvation. The WT and (p)ppGpp⁰ mutant were treated with 0.5 mg/ml RHX for the indicated times and plated on TSA. Percent survival was calculated by counting the numbers of colonies and normalized to the number at time zero. All data are shown as arithmetic means and standard deviations from three replicates.

FIG 2

Growth and accumulation of the (p)ppGpp in WT and (p)ppGpp⁰ mutant under different growth conditions. (A and B) Growth curves of the WT and (p)ppGpp⁰ mutant in CDM and CDM-BCAA. The value of the OD₆₀₀ was monitored at various time points. (C and D) The viability of the WT and (p)ppGpp⁰ mutant in CDM and CDM-BCAA. Overnight cultures were serially diluted by fresh medium and spotted onto the TSA plate. (E) Determination of the production of (p)ppGpp by the WT in CDM and CDM-BCAA. Values shown in panels A and B are arithmetic means and standard deviations of results from at least three independent experiments.

FIG 3

Morphology of the A. pleuropneumoniae WT and (p)ppGpp⁰ mutant grown in different media as determined by scanning electron microscopy.

FIG 4

Transcriptome analysis of (p)ppGpp-dependent genes during growth in CDM or CDM-BCAA. (A) Schematic of differentially expressed genes affected by (p)ppGpp in different media. (B) Venn diagrams showing the number of genes that are upregulated and downregulated in a (p)ppGpp⁰-dependent manner under CDM and CDM-BCAA growth conditions. (C) Functional categories of genes differentially expressed in (p)ppGpp⁰ compared with the levels in the WT in the absence of BCAAs. The number of genes whose expression is differentially expressed in (p)ppGpp⁰ compared with the level in the WT is presented according to the functions assigned by the browser of CLRNA-Seq data analysis software.

FIG 5

Schematic of the pathways affected by (p)ppGpp. Starvation-induced changes in the purine biosynthesis pathway. The indicated genes are involved in the purine de novo and salvage biosynthesis pathways. Red, genes with more than 2-fold upregulation; gray, genes with no detectable change in transcript levels.

FIG 6

Sequence characteristics of promoters regulated by transcription factors. (A) Consensus promoter elements. Sequence conservation in the −35 element (B and C), extended −10 region (D and E), −10 element (F and G), discriminator element (H and I), transcription start site (TSS) region (J and K), and −10/−35 spacer length (L). Promoters of genes involved in purine biosynthesis pathway were aligned to create sequence logos (B, D, F, H, and J) and histograms of base distributions (C, E, G, J, I, K, and L) for each position.

FIG 7

Enhanced H₂O₂ production by the (p)ppGpp⁰ mutant. The cells of the WT and (p)ppGpp⁰ mutant grown in CDM and CDM-BCAA were harvested and washed in PBS. The washed cell suspensions and medium were mixed with an equal volume of buffer to determine cytoplasmic H₂O₂ production, according to the instructions of an H₂O₂-peroxidase assay kit.

FIG 8

Growth, viability, and H₂O₂ accumulation affected by addition of decoyinine and guanosine. Cells were grown to early log phase (2 h) in CDM and supplemented with 5 mM guanosine and 50 μg/ml decoyinine. (A and B) The growth curves of the WT and (p)ppGpp⁰ mutant were determined by monitoring the value of the OD₆₀₀ at various time points. (C and D) The viability of the WT and (p)ppGpp⁰ mutant was determined by counting the CFU at various time points. (E) The accumulation of H₂O₂ was determined by using a H₂O₂-peroxidase assay kit.