Abstract
The repeating unit of the C. difficile Toxin A (rARU, also known as CROPS [combined repetitive oligopeptides]) C-terminal region, was shown to elicit protective immunity against C. difficile and is under consideration as a possible vaccine against this pathogen. However, expression of recombinant rARU in E. coli using the standard vaccine production process was very low. Transcriptome and proteome analyses showed that at restricted dissolved oxygen (DO) the numbers of differentially expressed genes (DEGs) was 2.5-times lower than those expressed at unrestricted oxygen. Additionally, a 7.4-times smaller number of ribosome formation genes (needed for translation) were down-regulated as compared with unrestricted DO. Higher rARU expression at restricted DO was associated with up-regulation of 24 heat shock chaperones involved in protein folding and with the up-regulation of the global regulator RNA chaperone hfq. Cellular stress response leading to down-regulation of transcription, translation, and energy generating pathways at unrestricted DO were associated with lower rARU expression. Investigation of the C. difficile DNA sequence revealed the presence of cell wall binding profiles, which based on structural similarity prediction by BLASTp, can possibly interact with cellular proteins of E. coli such as the transcriptional repressor ulaR, and the ankyrins repeat proteins. At restricted DO, rARU mRNA was 5-fold higher and the protein expression 27-fold higher compared with unrestricted DO. The report shows a strategy for improved production of C. difficile vaccine candidate in E. coli by using restricted DO growth. This strategy could improve the expression of recombinant proteins from anaerobic origin or those with cell wall binding profiles.
Highlights
Clostridium difficile (C. difficile) is an anaerobic bacterial pathogen responsible for diarrhea and pseudomembranous colitis[1,2]
We focused on improving the expression of the repeating unit region of the C. difficile toxin A
Maximum rARU expression of 4.36 μg/optical density (OD) was obtained when the cells grew in restricted dissolved oxygen (DO), and 0.16 μg/OD when the culture grew at unrestricted DO (Fig. 1B)
Summary
Clostridium difficile (C. difficile) is an anaerobic bacterial pathogen responsible for diarrhea and pseudomembranous colitis[1,2]. The bacteria produces two high molecular weight toxins, toxin A (308 kDa) and toxin B (269 kDa)[3,4]; both contain a repeating region called rARU (CROPs) (104 kDa) in subunit A and rBRU (70 kDa) in subunit B5,6. Both Toxin A and Toxin B are responsible for clinical symptoms and are candidates for vaccine development[7,8]. The importance of rARU was shown in animal models where serum neutralizing antibodies to toxin A conferred immunity to this pathogen and antiserum against nontoxic recombinant peptide, containing the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. This independent repeating unit region was found to be an efficient carrier for conjugated polysaccharide vaccines[15,16], and as a result of this observation, the efficient expression of the subunits of toxins A and B for clinical experiments is needed
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