Streptococcus pneumoniae uses glutathione to defend against oxidative stress and metal ion toxicity

Abstract

The thiol-containing tripeptide glutathione is an important cellular constituent of many eukaryotic and prokaryotic cells. In addition to its disulfide reductase activity, glutathione is known to protect cells from many forms of physiological stress. This report represents the first investigation into the role of glutathione in the Gram-positive pathogen Streptococcus pneumoniae. We demonstrate that pneumococci import extracellular glutathione using the ABC transporter substrate binding protein GshT. Mutation of gshT and the gene encoding glutathione reductase (gor) increases pneumococcal sensitivity to the superoxide generating compound paraquat, illustrating the importance of glutathione utilization in pneumococcal oxidative stress resistance. In addition, the gshT and gor mutant strains are hypersensitive to challenge with the divalent metal ions copper, cadmium, and zinc. The importance of glutathione utilization in pneumococcal colonization and invasion of the host is demonstrated by the attenuated phenotype of the gshT mutant strain in a mouse model of infection.

Fig 1

Characterization of S. pneumoniae D39gshT and D39gor mutant strains. (A) Total intracellular glutathione levels of D39 and D39gshT strains; (B) growth of D39 and D39gshT strains after 12 h in a chemically defined medium (CDM) (white bars), in CDM with cysteine omitted (gray bars), and in CDM lacking cysteine supplemented with 3.2 mM glutathione (black bars); (C) glutathione reductase activity of cell extracts of D39 and D39gor strains. Bars represent the mean values from triplicate cultures, and error bars represent the standard deviations from the means.

Fig 2

Growth of S. pneumoniae D39, and either D39gshT (A) or D39gor (B), with or without the addition of 0.5 mM paraquat (PQ). Curves represent the means from triplicate cultures, and error bars represent the standard deviations from the means.

Fig 3

Growth of S. pneumoniae D39 and D39gshT with or without the addition of 250 μM copper sulfate (Cu) (A), 10 μM cadmium chloride (Cd) (B), and 100 μM zinc sulfate (Zn) (C). Curves represent the means from triplicate cultures, and error bars represent the standard deviations from the means.

Fig 4

Growth of S. pneumoniae D39 and D39gor with or without the addition of 250 μM copper sulfate (Cu) (A), 10 μM cadmium chloride (Cd) (B), and 100 μM zinc sulfate (Zn) (C). Curves represent the means from triplicate cultures, and error bars represent the standard deviations from the means.

Fig 5

The number of S. pneumoniae D39 and D39gshT cells recovered from blood (A), lungs (B), nasal wash (C), and nasal tissue of mice (D) 24, 48, and 72 h following intranasal challenge. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to WT. The horizontal dotted line denotes the limit of detection. NB, two mice challenged with WT succumbed to infection prior to 72 h, and so only three mice from this group could be processed at this time point.