A Multi-Serotype Approach Clarifies the Catabolite Control Protein A Regulon in the Major Human Pathogen Group A Streptococcus

Abstract

Catabolite control protein A (CcpA) is a highly conserved, master regulator of carbon source utilization in gram-positive bacteria, but the CcpA regulon remains ill-defined. In this study we aimed to clarify the CcpA regulon by determining the impact of CcpA-inactivation on the virulence and transcriptome of three distinct serotypes of the major human pathogen Group A Streptococcus (GAS). CcpA-inactivation significantly decreased GAS virulence in a broad array of animal challenge models consistent with the idea that CcpA is critical to gram-positive bacterial pathogenesis. Via comparative transcriptomics, we established that the GAS CcpA core regulon is enriched for highly conserved CcpA binding motifs (i.e. cre sites). Conversely, strain-specific differences in the CcpA transcriptome seems to consist primarily of affected secondary networks. Refinement of cre site composition via analysis of the core regulon facilitated development of a modified cre consensus that shows promise for improved prediction of CcpA targets in other medically relevant gram-positive pathogens.

Figure 1. Characterization of the GAS M serotype strains and their ccpA derivatives.

(a) Whole genome phylogeny of sequenced GAS strains. Complete GAS genomes of indicated M serotypes (blue) were obtained from NCBI. Relationships were inferred from 68,084 core single nucleotide polymorphism (SNP) loci using SplitsTree. Location of parental strains used in this work are shown in red. (b) Growth curves for wild type, ccpA-inactivated and complemented strains in THY. Data points are mean and standard deviations from duplicate samples of each strain measured on two independent occasions.

Figure 2. Effect of ccpA deletion on virulence of GAS serotype strains in mouse models of infection.

CD-1 swiss mice were inoculated with GAS strains by intraperitoneal (a) or intranasal (b) route. Mice were monitored to near-mortality for the bacteremia model (a) and survival was graphed. For the oropharyngeal model (b), throat swabs were obtained daily and the bacterial density was graphed. P values were derived from repeated measures analysis (see Methods).

Figure 3. The effect of ccpA deletion on the GAS transcriptome.

(a) Linear representation of the genome of GAS serotype strains showing the effect of ccpA-inactivation on gene expression depicted as log fold change. (b) A weighted Venn diagram displaying the distribution and overlap of the CcpA-affected genes in all three serotypes was generated using the BioVenn application. Arrows indicate the number of genes repressed (down) and activated (up) by CcpA in individual serotypes and in the core regulon. (c) Genes affected by CcpA in each serotype are shown by order of COG categories. Each colored circle denotes the enrichment of a specific COG in the serotype strain. [C] Energy production and conversion; [E] Amino acid transport and metabolism; [G] Carbohydrate transport and metabolism; [I] Lipid transport and metabolism; [J] Translation, ribosomal structure and biogenesis; [L] Replication, recombination and repair; [M] Cell wall/membrane/envelope biogenesis; [N] Cell motility; [O] Posttranslational modification, protein turnover, chaperones; [R] General function prediction only; [S] Function unknown; [U] Intracellular trafficking, secretion, and vesicular transport.

Figure 4. Distribution of cre and cre2 motifs.

(a) Percentage of cre and cre2-bearing operons that exhibit CcpA-dependent differential expression (DE) or do not (not), in the three GAS serotype strains. (b) Distribution of the core regulon genes based on the presence (cre) or absence (non-cre) of a cre site in the promoter. (c) Differentially regulated genes containing cre or cre2 operators displayed by relative percentages that are repressed (up) or activated (down) in ΔccpA. (d) Number of cre and cre2 sites in the GAS core CcpA regulon depicted as a Venn diagram.

Figure 5. The improved GAS cre motif.

Weblogo representation of the GAS cre consensus motif. The frequency of occurrence of nucleotides for all core GAS cre sites is compared to that reported for B. subtilis. Positions displaying notable differences between S. pyogenes and B. subtilis are marked with red boxes.

Figure 6. Prediction of cre operators in other gram-positive pathogens.

The suite of cre sites predicted for (a) S. pneumonia and (b) S. aureus using the cre_GAS and by RegPrecise (RP) are compared and displayed as Venn diagrams (calls). The RegPrecise database was accessed in November, 2015. Cre sites that have reported experimental evidence of CcpA-dependent expression are displayed in the lower panel (regulated genes).