A PTS EII mutant library in Group A Streptococcus identifies a promiscuous man‐family PTS transporter influencing SLS‐mediated hemolysis

Abstract

Summary The group A streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)-mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared to parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC-encoding genes in the GAS MGAS5005 genome and subjected this library to metabolic and hemolysis assays to functionally characterize each EIIC. We found that a few EIIs had a very limited influence on PTS sugar metabolism, whereas others were fairly promiscuous. The mannose-specific EII locus, encoded by manLMN, was expressed as a mannose-inducible operon that exhibited the most influence on PTS sugar metabolism, including mannose. Importantly, components of the mannose-specific EII also acted to prevent the early onset of SLS-mediated hemolysis. Interestingly, these roles were not identical in two different M1T1 GAS strains, highlighting the possible versatility of the PTS to adapt to strain-specific needs.

Route of Glucose Uptake in the Group a Streptococcus Impacts SLS-Mediated Hemolysis and Survival in Human Blood

“…Streptolysin S (SLS), a GAS toxin that lyses host tissues and blood cells, was shown to be under the influence of the PTS, as a PTS-null (Δ ptsI ) mutation led to earlier SLS activity as compared to the parental strain MGAS5005 (Gera et al, 2014 ). We screened an annotated EIIC mutant library in a previous study, and discovered 7 out of 14 EIIC mutants that had earlier hemolysis as compared to MGAS5005 (Sundar et al, 2017 ). We carried out this screen in an effort to determine if the lack of utilization of a particular PTS carbon source was responsible for the dysregulation of timely SLS-mediated hemolysis.…”

“…We carried out this screen in an effort to determine if the lack of utilization of a particular PTS carbon source was responsible for the dysregulation of timely SLS-mediated hemolysis. There was no carbohydrate whose metabolism was commonly effected in all 7 EIIC mutants which displayed early hemolysis (Sundar et al, 2017 ). However, all of these EIIs do appear in the CcpA regulon (DebRoy et al, 2016 ).…”

“…In GAS, glucose is hypothesized to be transported by the PTS. Several EIIs in other bacteria have been shown to transport glucose, and deletion of certain PTS transporters in GAS leads to altered metabolism of glucose (Castro et al, 2009 ; Vitko et al, 2016 ; Sundar et al, 2017 ). However, the specific EIIs responsible for glucose transport in GAS are unknown, as the only annotated glucose-specific EII gene malT (formerly ptsG ) has been shown to transport maltose instead (Shelburne et al, 2008a ).…”

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

“…Although both MGAS2221 and HSC5, the strain used in the aforementioned study (Kietzman & Caparon, 2009), both contain the sagA cre site we identified, the area upstream of the cre site is quite divergent for the two strains which could explain the differential in vivo binding results. It has been shown that the manLMN operon and the β-glucoside PTS (Table 3), which we identified herein as being directly and indirectly regulated by CcpA respectively, impacts streptolysin production (Braza et al, 2020, Sundar et al, 2017 suggesting that CcpA may regulate streptolysin S production both directly and indirectly. In concert with our data, CcpA was identified as binding in vivo to the gene encoding suilysin, the pore-forming toxin of S. suis, (Willenborg et al, 2014) and to bind in vitro to the sagA promoter of S. anginosus (Bauer et al, 2018).…”

Crystal Structure of Mannose Specific IIA Subunit of Phosphotransferase System from Streptococcus pneumoniae

“…Because PTSs are central to energy metabolism and unique to bacteria, the system components have been suggested as suitable drug targets [ 20 ]. Interestingly, nature has already provided a validation of this concept—recent studies have demonstrated Man-PTS as a major receptor of class II bacteriocins (natural bactericidal peptides) [ 21 , 22 ]. So far, a number of three-dimensional structures of cytoplasmic components of the mannose-PTS have been determined both by X-ray and NMR methods.…”