Molecular And Biochemical Study On A Novel Thiol-Disulphide System In Bacteria And Its Possible Application In Development Of Antimicrobial Assay

Abstract

Thiol disulfide exchange reactions have been reported to play a key role in many metabolic processes. Cellular sulfhydryl content consists of two major pools, the protein sulfhydryl and low molecular weight sulfhydryl. Protein sulfhydryl (thioredoxin and glutaredoxin systems) are essential for enzyme activity, while low molecular weight thiols (glutathione, cysteine) provide reducing environment in the cytoplasm. Components of Dsb system (protein sulfhydryl) are DsbA, DsbB, DsbC and DsbD are responsible for disulfide bond formation in newly synthesized and translocated proteins. In this study, it was reported that different bacterial strains do not build up any significant amounts of extracellular sulfhydryl compounds under normal physiological conditions; they however responde vigorously to disulfide compounds producing measurable amounts of extracellular reduced thiol compounds. Incubation of growing bacterial cultures with disulfide compounds: DTNB, cystine and oxidized glutathione resulted in increased extracellular total thiol contents. Cystine produced the highest extracellular response among Gram negative strains followed by DTNB then glutathione, while in Gram positive strains DTNB produced the highest extracellular response followed by cystine. Glutathione did not produce an extracellular response in Gram positive strains. Intracellular thiol contents increased when cells were incubated with cystine, followed by oxidized glutathione. On the other hand, DTNB did not result in any increase in the intracellular thiol contents, confirming that it is a membrane impermeant reagent. Incubation of growing bacterial cultures in broth media containing DTNB (a disulfide compound) led to chromogen development indicating reduction of DTNB into the colored anion (TNB). The intensity of color was time and inoculum size dependant. Reduction of DTNB by different bacterial strains was studied and was found to be a common phenomenon among Gram negative and Gram positive tested strains. DTNB was more efficiently reduced by Gram negative than Gram positive strains. Similarity between turbidimetric and colorimetric DTNB -growth curves, supported evidence for the correlation between active growth and DTNB reduction and suggested its use as a metabolic or growth indicator. DTNB was proven to be sensitive to monitor the growth of bacterial cultures even at small inoculum densities and was used to develop a surrogate assay for growth monitoring. DTNB reduction was very high in metabolizing cells for all the tested strains, than in starved cells. This indicated that DTNB reduction requires high level of metabolic energy and that decrease in metabolic energy induced by starvation conditions markedly affect DTNB reduction. E. coli Mutants missing some reducing properties, namely gshA, TrxA, DsbC, DsbD and the transcription activator of the cysteine pathway CysB, were tested for DTNB reduction. All mutant strains, were able to reduce DTNB equally efficiently as their parent strains under metabolizing conditions. We excluded periplasmic proteins: DsbC, DsbD and cytoplasmic proteins TrxA, CysB and gshA from having any contribution in DTNB reduction by intact cells. E. coli cells subjected to osmotic shock, were able to reduce DTNB, confirming that periplasmic proteins do not have any role in DTNB reduction. HPLC analysis of the derivatized compound obtained by reaction of growing cells with DTNB revealed no peaks of reduced thiols, confirming that DTNB reduction was mediated exclusively by a membrane bound –disulfide reductase protein. Cell extract of different bacterial strains reduced DTNB. DTNB reduction was found to be NADH-linked. DTNB was reduced by higher rates by cell extracts of E. coli wild and mutant type strains than by that of S. aureus. Incubation of growing bacterial cultures to other disulfide compounds such as cystine, resulted in the increase in the extracellular reduced thiol content in both metabolizing and starvation conditions. Gram negative strains showed higher response to exogenous cystine than tested Gram positive strains, although a comparable rate of cystine reduction was obtained by cell extracts of E. coli and S. aureus indicating a defect in cystine transport system in Gram positive bacteria. Two mutants strains showed altered response towards cystine: gshA and CysB. However cell extracts of these two mutants showed cystine reduction rates comparable to their parent strains, indicating that defect of intact cells in their cystine response was due to impairment in transport activity but not to the reductase activity. Removal of Periplasmic proteins by osmotic shock significantly affected response of E. coli to cystine which confirmed the role of periplasmic cystine-binding proteins (components of ATP-type transport system found in Gram negative bacteria) in uptake of cystine which was further reduced and exported back extracellularly. Spectroscopic identification and HPLC analysis of the reduced thiol obtained by incubation of 2 with cystine, revealed a typical characteristic cysteine peak. Cystine transport was inhibited by azide (66.6%) and by 2,4 dinitrophenol (91.7%) indicating that the transport is an energy dependent process. DTNB reduction was found to be inhibited by different antibiotics. We developed a new antimicrobial susceptibility assay based on colorimetric –DTNB reduction. Ten antibiotics representing different classes were tested against 9 ATCC bacterial strains using inoculum size of 7.5 x 10 149 6 cfu/ml. MIC values were determined by colorimetric 6 hours DTNB assay and traditional overnight assay. There was excellent agreement between MIC values determined by both methods. Inclusion of cystine along with DTNB markedly enhanced DTNB reduction (with no inhibitory effect on growth) in Gram negative strains and moderately in Gram positive strains. This prompted the incorporation of cystine in the developed assay for growth determination to enhance the sensitivity and reduce the time of the assay for Gram negative strains. Readability of MIC for Gram positive strains however could not be achieved by the 3 hour assay method and the incubation of culture for 6 hour remained to be required.