Transcriptome, Phenotypic, and Virulence Analysis of Streptococcus sanguinis SK36 Wild Type and Its CcpA-Null Derivative (ΔCcpA)

Abstract

Catabolic control protein (CcpA) is linked to complex carbohydrate utilization and virulence factor in many bacteria species, influences the transcription of target genes by many mechanisms. To characterize the activity and regulatory mechanisms of CcpA in Streptococcus sanguinis, here, we analyzed the transcriptome of Streptococcus sanguinis SK36 and its CcpA-null derivative (ΔCcpA) using RNA-seq. Compared to the regulon of CcpA in SK36 in the RegPrecise database, we found that only minority of differentially expressed genes (DEGs) contained putative catabolite response element (cre) in their regulatory regions, indicating that many genes could have been affected indirectly by the loss of CcpA and analyzing the sequence of the promoter region using prediction tools is not a desirable method to recognize potential target genes of global regulator CcpA. Gene ontology and pathway analysis of DEGs revealed that CcpA exerts an influence predominantly involved in carbon catabolite metabolism and some amino acid catabolite pathways, which has been linked to expression of virulence genes in many pathogens and coordinately regulate the disease progression in vivo studies. However, in some scenarios, differences observed at the transcript level could not reflect the real differences at the protein level. Therefore, to confirm the differences in phenotype and virulence of SK36 and ΔCcpA, we characterized the role of CcpA in the regulation of biofilm development, EPS production and the virulence of Streptococcus sanguinis. Results showed CcpA inactivation impaired biofilm and EPS formation, and CcpA also involved in virulence in rabbit infective endocarditis model. These findings will undoubtedly contribute to investigate the mechanistic links between the global regulator CcpA and the virulence of Streptococcus sanguinis, further broaden our understanding of the relationship between basic metabolic processes and virulence.

Manganese Depletion Leads to Multisystem Changes in the Transcriptome of the Opportunistic Pathogen Streptococcus sanguinis

“…However, 15 significant DEGs were in the same direction, and the remainder were unchanged in our study. Additionally, most of the DEGs we observed in our study did not overlap with those of Bai et al (2019). This comparison indicates that CcpA-dependent repression could be responsible for some of the changes in expression after Mn depletion, but it does not explain all of the observed results.…”

“…In our concurrent metabolomics study, we found that ssaACB cells accumulated FBP at T 50 after Mn depletion , which may explain the strong evidence for CcpA repression. As expected if CcpA were responsible for the changes in expression, we found that 48 out of the 169 DEGs found by Bai et al (2019) when comparing a ccpA mutant to the SK36 WT were changed in the opposite Gorke and Stulke (2008). In the top panel, normal Mn levels in BHI result in low FBP, which leads to less phosphorylation of HPr to HPr(Ser-P) by HPr kinase/phosphorylase (HPrK).…”

“…Many of these genes were differentially expressed ( Supplementary Figure S6), which may be due to the intertwined nature of amino acid and carbohydrate metabolism (Radin et al, 2016). We also observed decreased expression of large, contiguous loci encoding ethanolamine utilization (Fox et al, 2009;Kaval and Garsin, 2018;Kaval et al, 2019), a type IV pilus system (Chen et al, 2019), and CRISPR-associated proteins (Makarova et al, 2015;Gong et al, 2020; Supplementary Figure S7), which are all also tied to CcpA-regulation (Bai et al, 2019).…”

“…Surprisingly, expression of nearly all sugar transport systems decreased after Mn depletion ( Supplementary Table S2 ), despite constant levels of glucose in the cells ( Puccio et al, 2020 ). CcpA is known to repress its own expression in a glucose-dependent manner ( Bai et al, 2019 ), and yet much like the glucose transporters, ccpA expression was high at T – 20 and significantly decreased by T 50 ( Supplementary Table S2 ). Potential explanations could include: (i) 2 g/L glucose in BHI is not sufficient to induce CcpA repression; (ii) other regulatory mechanisms are preventing proper CCR under these conditions; or (iii) much like spxB , many other systems in S. sanguinis are subject to glucose-independent CcpA repression.…”

Time-course analysis ofStreptococcus sanguinisafter manganese depletion reveals changes in glycolytic, nucleotide, and redox metabolites

“…In our recent transcriptomics study, expression of punA and deoD were significantly decreased after Mn depletion while SSA_2046 expression was unchanged . The operon encoding deoD and punA has a carbon responsive element (cre) upstream of the first gene, rpiA (Bai et al, 2019), which is the recognition sequence for the carbon catabolite repression (CCR) regulator CcpA (Warner and Lolkema, 2003). As observed in , Mn depletion results in many changes in the CcpA regulon, which may explain the repression of this operon at T 50 .…”