Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group AStreptococcus

Abstract

SummaryCatabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in non-pathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIPseq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIPseq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.Data sharing and data availabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.

Carbon catabolite repression on the Rgg2/3 quorum sensing system in Streptococcus pyogenes is mediated by PTSMan and Mga

“…Carbon catabolite repression can also be achieved in a CcpAindependent manner, one that involves the PTS components of EI and HPr phosphorylating a catabolic enzyme or a transcriptional regulator (Deutscher et al, 2014). Prior studies determined that an HPr deletion is not tolerated by GAS (DebRoy et al, 2021;Le Breton et al, 2015) and our attempts to generate an EI (ptsI) mutant were also unsuccessful. We therefore focused on the end point of this regulatory network, transcriptional regulators subject to PTS modulation that contains phosphorylatable histidine residues within PRD.…”

“…Besides participating in the uptake of carbohydrates, the phosphor‐carrier protein HPr is involved in regulation of genes that encode catabolic enzymes and carbohydrate transports, including PTS systems. This is achieved in conjunction with carbon catabolite protein A (CcpA), primarily a transcriptional repressor but can also function as a transcriptional activator of a small subset of genes within its regulon (DebRoy et al., 2016, 2021; Henkins, 1996). This regulatory network is unique to Gram‐positive Firmicutes, which requires phosphorylation of HPr on a serine residue, catalyzed by a kinase (HprK) with ATP as a phospho‐donor (reviewed in Deutscher et al., 2005).…”

“…This regulatory network is unique to Gram‐positive Firmicutes, which requires phosphorylation of HPr on a serine residue, catalyzed by a kinase (HprK) with ATP as a phospho‐donor (reviewed in Deutscher et al., 2005). The phosphorylation of the serine residue (HPr‐Ser46‐P) allows it to be a cofactor to CcpA, which increases its DNA‐binding activity for binding to cre (Görke & Stülke, 2008) or cre2 (Willenborg et al., 2014) elements to control gene transcription (Figure 1; DebRoy et al., 2016, 2021; Henkins, 1996). The regulon of CcpA is species specific, but typically includes genes‐encoding enzymes and transporters, such as the PTS system, for the import and catabolism of specific and less preferred carbohydrates.…”

“…Besides participating in the uptake of carbohydrates, the phosphor-carrier protein HPr is involved in regulation of genes that encode catabolic enzymes and carbohydrate transports, including PTS systems. This is achieved in conjunction with carbon catabolite protein A (CcpA), primarily a transcriptional repressor but can also function as a transcriptional activator of a small subset of genes within its regulon (DebRoy et al, 2016(DebRoy et al, , 2021Henkins, 1996).…”

Elucidation of Independently Modulated Genes in Streptococcus Pyogenes Reveals Carbon Sources that Control its Expression of Hemolytic Toxins

“…An underlying transcriptional regulatory network (TRN) alters the physiological state of a bacterium to adapt to unique challenges presented by each host environment [3][4][5][6][7] . S. pyogenes regulons have been determined based on direct molecular methods, including transcriptomics performed on single gene knockout mutants 3,5,6,[8][9][10][11][12][13][14][15][16][17][18][19][20][21] (PMIDs are listed in Supplementary_Data_3), and chromatin immunoprecipitation sequencing (ChIP-seq) 22,23 . At least 13 S. pyogenes twocomponent regulatory systems and 30 transcriptional regulators are known 24 .…”