Identification of a Thiol-Disulfide Oxidoreductase (SdbA) Catalyzing Disulfide Bond Formation in the Superantigen SpeA in Streptococcus pyogenes

Abstract

Mechanisms of disulfide bond formation in the human pathogen Streptococcus pyogenes is currently unknown. To date, no disulfide bond forming thiol-disulfide oxidoreductase (TDOR) has been described and at least one disulfide bonded protein is known in S. pyogenes . This protein is the superantigen SpeA, which contains 3 cysteine residues (Cys 87, Cys90, and Cys98), and has a disulfide bond is formed between Cys87 and Cys98. In this study, candidate TDORs were identified from the genome seuence of S. pyogenes MGAS8232. Using mutational and biochemical approaches, one of the candidate proteins, SpyM18_2037 (named here SdbA), was shown to be the catalyst that introduces the disulfide bond in SpeA. SpeA in the culture supernatant remained reduced when sdbA was inactivated and restored to the oxidized state when a functional copy of sdbA was returned to the sdbA -knockout mutant. SdbA has a typical C 46 XXC 49 active site motif commonly found in TDORs. Site-directed mutagenesis experiments showed that the cysteines in the CXXC motif were required the disulfide bond in SpeA to form. Interactions between SdbA and SpeA were examined using cysteine variant proteins. The results showed that SdbA C49A formed a mixed disulfide with SpeA C87A , suggesting that the N-terminal Cys46 of SdbA and the C-terminal Cys98 of SpeA participated in the initial reaction. SpeA oxidized by SdbA displayed biological activities suggesting that SpeA was properly folded following oxidation by SdbA. In conclusion, formation of the disulfide bond in SpeA is catalyzed by SdbA and the findings represent the first report of disulfide bond formation in S. pyogenes . IMPORTANCE Here, we reported the first example of disulfide bond formation in Streptococcus pyogenes . The results showed that a thiol-disulfide oxidoreductase, named SdbA, is responsible for introducing the disulfide bond in the superantigen SpeA. The cysteine residues in the CXXC motif of SdbA are needed for catalyzing the disulfide bond in SpeA. The disulfide bond in SpeA and neighboring amino acids form a disulfide loop that is conserved among many superantigens, including those from Staphylococcus aureus . SpeA and staphylococcal enterotoxins lacking the disulfide bond are biologically inactive. Thus, the discovery of the enzyme that catalyzes the disulfide bond in SpeA is important to understanding the biochemistry of SpeA production and presents a target for mitigating the virulence of S. pyogenes .

The Role of Pseudomonas aeruginosa DsbA-1 in Bacterial Pathogenesis: Current Research and Future Prospects

“…This is likely due to the lack of known and tractable model organisms and/or the lack of genetic tools for analysis [4]. Peculiar characteristics of gram-positives will be discussed later in the text [5]. Scientists study DsbA function by disrupting the coding sequence of the dsbA gene.…”

Interactomics of CXXC proteins involved in epigenetic regulation of gene expression

“…Таблица 6. Список # потенциальных белковых партнеров CFP1, кодируемого геном CXXC1, выделенных из лизата клеточной линии HepG2 Примечание: * -коэкспрессия генов (коэффициент корреляции Пирсона >0,8), кодирующих CFP1 и его потенциальные партнеры по взаимодействию, согласно порталу GeneFriends[68]; ** -белки, которые кофракционируются с CFP1, согласно литературным данным, гель-хроматографического профилирования клеточных лизатов HEK293[64], HeLa-CCL2[65], U2OS[66], HeLa-S3 и HEK293[63]; # -32 белка осталось после вычитания известных контаминирующих белков по данным CRAPome[67] и белков с несоответствующим CFP1 паттерном субклеточной локализации; всего было выделено из лизата клеток HepG2 144 белка (p<0,01 и Mascot score >30).что взаимодействие DNAJC10 с CFP1 способствует правильному образованию дисульфидных связей в последнем[85]. Упоминания о взаимодействиях между катепсином В (CTSB) и CFP1 не встречаются в литературе.…”