Molecular and structural basis of glutathione import in Gram‐positive bacteria via GshT and the cystine ABC importer TcyBC of Streptococcus mutans

Abstract

SummaryGlutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram-negative bacteria, the molecular and structural basis of GSH import into Gram-positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS-Ileg of GSSG is accommodated tightly via extensive interactions contributed by the N-and C-terminal lobes of GshT, while the GS-II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine-sulfur or the glycine-carboxylate. Finally, we show that GshT primes glutathione import via the L-cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of L-cystine.

Utilization of host‐derived cysteine‐containing peptides overcomes the restricted sulphur metabolism of Campylobacter jejuni

“…However, this uptake is only detectable in C. jejuni without active GGT. The intracellular GSH level in the C. jejuni 81‐176 ggt mutant is significantly lower (about 20 nmol GSH per mg protein) than the GSH concentrations measured, for example, in Streptococcus mutans (about 120 nmol GSH per mg protein) (Vergauwen et al ., ). This difference may be due to cleavage of GSH by intracellular peptidases in the C. jejuni ggt mutant or other ggt ‐negative C. jejuni strains.…”

“…This difference may be due to cleavage of GSH by intracellular peptidases in the C. jejuni ggt mutant or other ggt ‐negative C. jejuni strains. In S. mutans , the periplasmic protein GshT of the type 3 solute‐binding protein (SBP) family mediates the import of GSH (Vergauwen et al ., ). The periplasmic binding proteins (PBPs) CjaC, CjaA, GlnH and Peb1A of C. jejuni belong to the same SBP family as GshT, but our study clearly revealed that inactivation of any of these four PBPs did not abolish the growth of C. jejuni 81‐176 with GSH.…”

Redox regulation by reversible protein S-thiolation in bacteria

“…Interestingly, Lactococcus lactis, Streptococcus pneumoniae and Haemophilus influenzae do not synthesize GSH, but encode GSH-uptake mechanisms. In S. pneumoniae , the GSH-uptake from the host is mediated by the ABC transporter binding protein GshT (Potter et al, 2012 ; Vergauwen et al, 2013 ). In addition, the cystine importer TcyBC was shown to be primed for GSH import by GshT.…”

Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF)

“…The GSH import protein is also active in Gram‐positive prokaryotes (Vergauwen et al . ) and thus the import ability was also tested by growing cultures in GSH‐supplemented CDM followed by the assessment of the cytoplasmic content of GSH.…”

“…The use of CDM devoid of GSH is essential for the establishment of de novo GSH biosynthesis ability of cultures as rich media, such as MRS and M-17, have considerable GSH content (Pophaly et al 2012) and its import interferes in independent assessment of biosynthesis ability. The GSH import protein is also active in Gram-positive prokaryotes (Vergauwen et al 2013) and thus the import ability was also tested by growing cultures in GSH-supplemented CDM followed by the assessment of the cytoplasmic content of GSH.…”