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

Abstract

Catabolite 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 nonpathogenic 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/ChIP‐seq 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 ChIP‐seq 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.

Characterization of the Type I Restriction Modification System Broadly Conserved among Group A Streptococci

“…Cells were harvested at mid-exponential phase. RNA was prepared using the Qiagen RNeasy kit and processed as described earlier ( 68 ). Primers and probes used are listed in Table S3 .…”

Expanding the genetic toolbox for the obligate human pathogenStreptococcus pyogenes

“…We hypothesize that glucose utilization by GAS led to downregulation of this promoter during exponential growth. Previous research suggests that the trehalose operon is repressed by the catabolite control protein A (CcpA), despite a direct binding by CcpA could not be proven by ChIP-seq analysis (84). Potentially, induction could be observed in a growth medium devoid of glucose or fructose, which were shown to be the preferred carbon source for GAS (85).…”

LiaR-dependent gene expression contributes to antimicrobial responses in group AStreptococcus

“…High-throughput sequencing on prepared sample libraries was performed by Genewiz (Azenta Life Sciences) to generate a minimum of 50M 150-bp paired reads per replicate/input sample. Analysis was performed as previously described (Horstmann et al 2023; DebRoy et al 2021) using CLC Genomics Workbench (v 23) Transcription Factor ChIP-seq module.…”

“…Briefly, strains were grown to mid-logarithmic phase (OD600 ~0.4) in 40 mL THY, proteins crosslinked to DNA with 1% formaldehyde, cells harvested by centrifugation, flash-frozen, and stored at -80 o C until processed. Processing of replicate samples for ChIP-seq was performed as previously described (Horstmann et al 2023;DebRoy et al 2021). High-throughput sequencing on prepared sample libraries was performed by Genewiz (Azenta Life Sciences) to generate a minimum of 50M 150-bp paired reads per replicate/input sample.…”