Exogenous Glutathione Completes the Defense against Oxidative Stress in Haemophilus influenzae

Abstract

Since they are equipped with several strategies by which they evade the antimicrobial defense of host macrophages, it is surprising that members of the genus Haemophilus appear to be deficient in common antioxidant systems that are well established to protect prokaryotes against oxidative stress. Among others, no genetic evidence for glutathione (␥-Glu-Cys-Gly) (GSH) biosynthesis or for alkyl hydroperoxide reduction (e.g., the Ahp system characteristic or enteric bacteria) is apparent from the Haemophilus influenzae Rd genome sequence, suggesting that the organism relies on alternative systems to maintain redox homeostasis or to reduce small alkyl hydroperoxides. In this report we address this apparent paradox for the nontypeable H. influenzae type strain NCTC 8143. Instead of biosynthesis, we could show that this strain acquires GSH by importing the thiol tripeptide from the growth medium. Although such GSH accumulation had no effect on growth rates, the presence of cellular GSH protected against methylglyoxal, tert-butyl hydroperoxide (tBuOOH), and S-nitrosoglutathione toxicity and regulated the activity of certain antioxidant enzymes. H. influenzae NCTC 8143 extracts were shown to contain GSH-dependent peroxidase activity with t-BuOOH as the peroxide substrate. The GSH-mediated protection against t-BuOOH stress is most probably catalyzed by the product of open reading frame HI0572 (Prx/Grx), which we isolated from a genomic DNA fragment that confers wild-type resistance to t-BuOOH toxicity in the Ahp-negative Escherichia coli strain TA4315 and that introduces GSH-dependent alkyl hydroperoxide reductase activity into naturally GSH peroxidase-negative E. coli. Finally, we demonstrated that cysteine is an essential amino acid for growth and that cystine, GSH, glutathione amide, and cysteinylglycine can be catabolized in order to complement cysteine deficiency.

Characterization of the Bifunctional γ-Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida

“…In this respect, our previous results with H. influenzae were surprising, as this ␥-proteobacterium of the family Pasteurellaceae acquires glutathione solely via a highly specific import system (27). The motivation for the present research was to evaluate whether other Pasteurellaceae members were atypical also with respect to glutathione acquisition.…”

“…Fusion Protein GshF-In an earlier report, we showed that H. influenzae is not able to synthesize glutathione but instead acquires the thiol-tripeptide by import (27). To determine whether this type of glutathione acquisition is common among other Pasteurellaceae members, we analyzed the translated genome sequences (BLAST search at NCBI (37)) of Haemophilus somnus 129PT, H. somnus 2336, Haemophilus ducreyi 35000HP, Actinobacillus pleuropneumoniae serovar 1 strain 4074, Mannheimia succiniciproducens MBEL55E, and P. multocida subsp.…”

“…Indeed, P. multocida cells grown overnight on BHI agar plates were found to contain 72 Ϯ 12 nmol of glutathione/mg of protein, as determined by a highly specific glutathione reductase-based enzymatic recycling assay. To verify that P. multocida is able to synthesize rather than simply take up glutathione, as has been demonstrated for its close relative H. influenzae (27), cells were grown up to the early exponential phase of growth in a chemically defined liquid medium, either supplemented or not with 100 M GSSG, and analyzed for intracellular glutathione. The total glutathione content of GSSG-supplemented cultures (170 Ϯ 20 nmol of glutathione/mg of protein) was found to be 7.4-fold that of cultures grown in the absence of the oxidized tripeptide (23 Ϯ 5 nmol of glutathione/milligram of protein).…”

“…In an earlier report, we have shown that Haemophilus influenzae does not accumulate glutathione by biosynthesis but efficiently imports the tripeptide from the growth medium (27). In contrast to other Gram-negatives such as E. coli, the imported glutathione was found to be crucial for H. influenzae, as it scavenges its interior from a wide variety of peroxide chemicals (28,29).…”

Purification and Characterization of a Chimeric Enzyme fromHaemophilus influenzae Rd That Exhibits Glutathione-dependent Peroxidase Activity

“…When n equals 1, the v/[S] versus v is linear, as is the case for t-BOOH, but when n is greater than 1, the plot is curved, as shown for GSH (23). DISCUSSION We have recently shown that a subgenomic fragment from H. influenzae Rd, bearing the open reading frame HI0572, is capable of complementing t-BOOH and H 2 O 2 sensitivity of an Ahp-and catalase-negative E. coli strain, respectively, and of delivering GSH-dependent alkyl hydroperoxide reductase activity to a naturally GSH peroxidase-negative E. coli (24). It was envisioned that these properties could be attributed to the unusual primary structure of the gene product, consisting of a peroxidase region at the N terminus, showing homology with type C Prx, and a C-terminal region, showing homology with Grx.…”