An unusual thioredoxin system in the facultative parasite Acanthamoeba castellanii

Abstract

The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii’s response to oxidative stress. The role of the large TrxR, however, remains elusive.

Transcriptional changes of proteins of the thioredoxin and glutathione systems in Acanthamoeba spp. under oxidative stress – an RNA approach

“…2b ). As already reported by Leitsch et al [ 22 ], in strain Neff TrxR-S expression increases approximately by a factor 50, when exposed to H 2 O 2 . Obviously even a short time under different culture conditions, in this case on agar plates coated with E. coli , has a strong impact on the TrxR-S response to H 2 O 2 , since NeffPl exhibited an increase in TrxR-S expression almost twice as high (fold change 97.5).…”

“…Fold changes for strains Neff TrxR-L, TrxR-S and GR were already published in Leitsch et al [ 22 ], but were included in this figure for comparison with investigated clinical isolates in this study.…”

“…The fact that Acanthamoeba possesses two different types of TrxR is quite intriguing, since only very few organisms were reported so far with this feature, for instance Naegleria fowleri [ 6 ]. While our primary focus lay on the TrxRs and GR in Acanthamoeba castellanii strain Neff [ 22 ], in this study we attempted a more detailed evaluation of other enzymes involved in the redox systems of acanthamoebae. Moreover, the expression patterns of clinical strains were investigated in order to evaluate whether pathogenic strains exhibit a distinctive response to oxidative stress.…”

“…RT-qPCRs targeting TrxR of the low-molecular-weight type (TrxR-S), TrxR of the high-molecular-weight type (TrxR-L), thioredoxin-1 (Trx-1), two peroxiredoxins (Prx-2, Prx-3), two glutaredoxins (Glx-1, Glx-2), glutathione-disulfide reductase (GR) and glutathione peroxidase (Gpx) were performed as described previously [ 22 ]. Information on primers used is provided in Table 1 .…”

“…In a previous study, we already established that both TrxRs in A. castellanii strain Neff are expressed on the protein level and localized in the cytosol. Interestingly, however, only TrxR-S was shown to be upregulated upon oxidative stress on the mRNA and protein level [ 22 ].…”