Cloning and functional characterization of thiol disulfide interchange system proteins from Staphylococcus aureus

Klopper, Sunette Laurika (2013-12)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: The 21st century has seen the re-emergence of infectious diseases thought previously conquered by antibiotics. The human pathogen, Staphylococcus aureus, is one such example, with global reports listing resistance to antibiotics from methicillin to vancomycin, the so-called “drug of last resort”. The speed at which such pathogens acquire antibiotic resistance has led to the questioning of the current strategy of developing drugs that kill infectious agents directly. Instead, new tactics must be employed that rely more heavily on the host’s own immune defences. S. aureus circumvents the human immune system’s oxidative killing mechanisms through the actions of a thiol-disulfide interchange system which maintains intracellular redox balance within the pathogen. Disruption of the system and its players thus represents a potential target for the development of novel antistaphylococcal agents. This study set out to characterise three proteins suggested to be involved in the thiol-disulfide interchange system so as to assess their roles and their viability as effective drug targets. This study had two major aims: the functional characterisation of the MerA and YpdA flavin disulfide reductase homologs, and the assessment of the substrate promiscuity of the TrxB and TrxA proteins. The characterisation of MerA and YpdA involved activity assays with the LMW thiol disulfide substrates involved in S. aureus redox balance. While failing to show demonstrable disulfide reductase activity, MerA instead was found to have the ability to reduce two biologically-relevant transition metal ions without the aid of an interacting protein partner: Hg2+ and Fe3+. Fe3+ was further shown to be favoured over Hg2+ as a substrate of MerA, which has implications for the protein’s role in oxidative stress resistance. YpdA was shown to be incapable of reducing any of the LMW thiol disulfides found in S. aureus, even in the presence of a generic TFP protein, TrxA. This suggests that YpdA may be unable to perform catalytic functions in the absence of its correct interacting partner. Alternatively, the protein may perform a different cellular function unrelated to LMW thiol disulfide reduction or oxidative stress resistance. The substrate promiscuity of the thioredoxin system proteins of S. aureus, TrxB and TrxA, was demonstrated through their measurable reduction of three LMW thiol disulfides. However, the poor kinetic parameters determined for the reactions indicate that these proteins may be more likely to reduce the LMW thiol disulfides under critical conditions, as opposed to acting as their primary reducing system. Importantly, this study has demonstrated a role in the maintenance of S. aureus redox balance beyond the usual cellular functions of TrxB and TrxA. These findings provide additional support for TrxB as a viable target for the development of novel antibiotics.

AFRIKAANSE OPSOMMING: Die 21ste eeu het die terugkeer gesien van aansteeklike siektes wat voorheen deur antibiotika beheer kon word. Die menslike patogeen, Staphylococcus aureus, is een so 'n voorbeeld, met internasionale verslae wat weerstandigheid lys teen geneesmiddels van methicillin tot vancomycin, die sogenaamde "geneesmiddel van laaste uitweg". Die spoed waarteen sulke patogene weerstand ontwikkel teen antibiotika lei tot die bevraagtekening van die huidige strategie van geneesmiddels ontwikkel om bakterieë te dood. In teenstelling word nuwe taktiek benodig wat eerder staatmaak op die mens se eie immuunstelsel. S. aureus vermy die menslike immuunstelsel se oksidatiewe verdedigingsmeganismes deur die gebruik van 'n tiol-disulfied uitruilingstelsel wat intrasellulêre redoksbalans beheer binne die patogeen. Ontwrigting van die stelsel verteenwoordig dus 'n potensiële teiken vir die ontwikkeling van nuwe antistafilokokkale agente. Die doel van hierdie studie was om drie proteïene te karakteriseer wat betrokke is in die tiol-disulfied uitruilingstelsel, om sodoende hulle rolle te assesseer en hulle lewensvatbaarheid as teikens vir antibiotika te evalueer. Hierdie studie het twee hoofdoelwitte gehad: die funksionele karakterisering van die MerA en YpdA flavoproteïene, en die assessering van substraat-promiskuïteit van die TrxB en TrxA proteïene. Die karakterisering van MerA en YpdA het bestaan uit die uitvoering van aktiwiteitstoetse met die tiol disulfide substrate wat betrokke is in die handhawing van S. aureus se redoksbalans. Hoewel MerA geen bewese disulfide reduktase aktiwiteit gedemonstreer het nie, het die proteïen eerder die vermoë gedemonstreer om twee biologies-relevante metaal ione te reduseer sonder die hulp van 'n interaktiewe proteïenvennoot: Hg2+ en Fe3+. Fe3+ is verkies bo Hg2+ as 'n substraat van MerA, wat implikasies het vir die proteïen se rol in oksidatiewe stress-weerstand. Daar is bewys dat die YpdA proteïen geen van die tiol disulfiedes kon reduseer nie, selfs in die teenwoordigheid van 'n generiese TFP proteïen, TrxA. Dit dui daarop dat YpdA nie in staat mag wees om katalitiese funksies uit te voer nie, in die afwesigheid van die korrekte interaksie vennoot. Die substraat-promiskuïteit van die thioredoxin-sisteem proteïene van S. aureus, TrxB en TrxA, is gedemonstreer deur die meetbare redusering van drie tiol disulfied substrate. Tog dui die swak kinetiese parameters vir hierdie spesifieke reaksies daarop dat hierdie proteïene nie as primêre reduseeringsstelsel vir hierdie verbindings op te tree nie. Hierdie studie het 'n rol vir TrxB en TrxA in die handhawing van S. aureus redoksbalans gedemonstreer wat buite die proteïene se gewone sellulêre funksies is. Hierdie bevinding gee addisionele ondersteuning aan TrxB as 'n doeltreffende teiken vir die ontwikkeling van nuwe antibiotika.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/95479
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