Elsa Pimienta

Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans.

BackgroundStreptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.ResultsSK yield in the spent culture medium of S. lividans was higher when the Sec-dependent signal peptide mediates the SK translocation. Using a 1.5 L fermentor, the secretory production of the Vsi-SK fusion protein reached up to 15 mg SK/l. SK was partially purified from the culture supernatant by DEAE-Sephacel chromatography. A 44-kDa degradation product co-eluted with the 47-kDa mature SK. The first amino acid residues of the S. lividans-produced SK were identical with those of the expected N-terminal sequence. The Vsi signal peptide was thus correctly cleaved off and the N-terminus of mature Vsi-SK fusion protein released by S. lividans remained intact. This result also implicates that the processing of the recombinant SK secreted by Streptomyces probably occurred at its C-terminal end, as in its native host Streptococcus equisimilis. The specific activity of the partially purified Streptomyces-derived SK was determined at 2661 IU/mg protein.ConclusionHeterologous expression of Streptococcus equisimilis ATCC9542 skc-2 in Streptomyces lividans was successfully achieved. SK can be translocated via both the Sec and the Tat pathway in S. lividans, but yield was about 30 times higher when the SK was fused to the Sec-dependent Vsi signal peptide compared to the fusion with the Tat-dependent signal peptide of S. lividans xylanase C. Small-scale fermentation led to a fourfold improvement of secretory SK yield in S. lividans compared to lab-scale conditions. The partially purified SK showed biological activity. Streptomyces lividans was shown to be a valuable host for the production of a world-wide important, biopharmaceutical product in a bio-active form.

Immune Response to Streptomyces lividans in Mice: A Potential Vaccine Vehicle Against TB

The potentialities of Streptomyces lividans 1326 as new live vaccine vehicle strategy have been evaluated. Immunization of mice with the Streptomyces mycelium induced high levels of specific antibodies against proteins released in the culture supernatant of the analyzed strain. Splenocytes from the Streptomyces-immunized animals were able to secrete high levels of IFN-� (2103.9 pg/mL) and to proliferate in vitro on stimulation with proteins released by Streptomyces. The analysis of cross-reactivity against M. tuberculosis secreted proteins showed that the immunization of mice with Streptomyces led to a comparable level of cross-reacting antibodies as in the BCG immunized mice. Similarly sera antibodies from Streptomyces immunized group recognized whole BCG cells to the same degree as antibodies raised against BCG reacted with Streptomyces mycelium, indicating that these two actinobacteria cross-react immunologically. The Streptomyces lividans strain was highly immunogenic in mice showing an enduring Th1-dependent immune response. This is the first report demonstrating the potential of Streptomyces as an attractive vehicle for developing a live TB vaccine.

Assessment of an ELISA for serodiagnosis of active pulmonary tuberculosis in a Cuban population

Abstract Objective To explore the serodiagnostic potential of the five recombinant Mycobacterium tuberculosis antigens CFP-10 (Rv3874), ESAT-6 (Rv3875), APA (Rv1860), PstS-1 (Rv0934), Ag85A (Rv3804c) and their combination in a Cuban population with active pulmonary tuberculosis. Methods The serodiagnostic potential of the recombinant antigens rESAT-6, rCFP-10, rAPA, rPstS-1 produced in Escherichia coli , rAg85A produced in Streptomyces lividans and the combination of the five proteins was evaluated by an indirect ELISA. Humoral immune response was analysed in a group of 140 patients with active pulmonary tuberculosis (smear-, Mantoux- and culture-positive) and in a control group consisting of 34 bacillus Calmette-Guerin vaccinated, Mantoux-negative, healthy subjects. Results With the exception of CFP-10, the use of the separate recombinant antigens or the antigenic cocktail in ELISA-based serodiagnosis resulted in a significant difference in the mean optical densitiy values between sera of patients and healthy subjects. The highest sensitivity of the assay using single antigens, being 58.57%, was achieved with rPstS-1 compared to 27.14% with rCFP-10, 31.65% with Ag85A, 42.86% with rAPA and 44.29% with rESAT-6. Single antigen ELISAs provided high specificity values ranging from 94.12% to 97.06%. A cocktail of the aforementioned antigens increased the sensitivity to 87.14% and the specificity to 97.06%. Conclusions An ELISA using a multi-antigen mix containing recombinant immuno-dominant antigens of Mycobacterium tuberculosis , namely, rCFP-10, rESAT-6, rAPA, rPstS-1 and rAg85, increases the sensitivity and specificity compared with that using the single antigens and shows potential as a complementary tool for the diagnosis of active pulmonary tuberculosis in Cuba.