Zinc Tolerance Through Glutathione Import Strikes a Fine Balance Between Protection and Damage in Streptococcus...

Recently, our group showed that the dental pathogen Streptococcus mutans is inherently more tolerant to high zinc stress than other streptococci, a phenotype associated with the presence of a P-type ATPase exporter named ZccE, virtually unique to S. mutans. In addition to zccE, a previous transcriptome analysis revealed that S. mutans upregulates genes involved in glutathione uptake during initial exposure to zinc stress. Glutathione, a major supplier of organic sulfur that also plays key roles in antioxidant defense and xenobiotic detoxification, forms coordination complexes with a variety of metals, including zinc, thereby functioning as a buffer that protects cells from metal intoxication. To investigate the contribution of glutathione zinc tolerance in S. mutans, the gshT gene, which encodes the substrate-binding subunit of a glutathione transporter, was deleted in both the parent and ΔzccE strains and the ability of these mutants to overcome zinc stress through intracellular glutathione accumulation determined. Targeted metabolomics revealed that S. mutans accumulates glutathione in a GshT-dependent manner following zinc stress, a response that was strikingly amplified in the ΔzccE strain. Although glutathione supplementation had a minimal and non-significant impact on growth of either parent or mutant strains in sub-inhibitory zinc concentrations, the ΔgshT strain exhibited increased zinc sensitivity in a plate-based assay. However, the ΔzccEΔgshT mutant displayed enhanced zinc tolerance compared to the ΔzccE single mutant. While glutathione alone did not alter zinc levels in the UA159 or ΔzccE strains, the combination of zinc and glutathione nearly doubled intracellular zinc levels in ΔzccE compared to cells grown in zinc only. We conclude that while glutathione may play a minor role in S. mutans zinc tolerance, uncontrolled glutathione uptake observed in ΔzccE facilitates zinc entry, as glutathione:Zn²⁺ complexes inadvertently promote zinc intoxication via a Trojan horse mechanism.