Caridad Rodríguez

A heterodimer of EsxA and EsxB is involved in sporulation and is secreted by a type VII secretion system in Streptomyces coelicolor

An esx locus, related to the multiple esx loci of Mycobacterium tuberculosis, is conserved in all sequenced Streptomyces genomes, where it is associated with the developmental regulatory gene bldB. Here we demonstrate that the esxBA operon, comprising part of the locus, has a novel morphogenetic function in the model species Streptomyces coelicolor. This operon encodes two proteins belonging to the WXG-100 superfamily that can form a heterodimer and are secreted in the absence of signal sequences. A mutation in esxBA results in a delay in sporulation, with eventual development of aerial hyphae with chains of abnormally sized spore compartments possessing irregular DNA contents. During early sporulation, expression of the operon is elevated in a bldB mutant. Other genes in the locus, notably SCO5734 and SCO5721, encode components of a type VII secretion system. Disruption of either of these genes prevents secretion of EsxAB but has no effect on sporulation. To explain the morphogenetic function of EsxAB, we propose that the heterodimer sequesters a regulator of expression of genes involved in nucleoid organization during sporulation.

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.

Synthesis and Practical Applications of Brassinosteroid Analogs

Brassinosteroids, a new class of plant hormones of steroidal nature, show intriguing biological properties being now recognized as useful compounds in growth regulation, increasing crop yield and tolerance to abiotic stresses. Therefore, an extensive research work was planned on chemistry, biochemistry, physiology, molecular action and the synthesis of these compounds and their analogs, as the basis for further practical applications. Consequently, in Cuba, for fifteen years, the Laboratory of Natural Products from the University of Havana has been searching for new brassinosteroid analogs with biological activity that may be successfully applied to agriculture. This chapter summarizes the synthesis of several brassinosteroid analogs with structural variations in different parts of the molecules, starting from available steroids such as diosgenin, hecogenin, solasodine, solanidine and bile acids. Besides, the main results of practical applications of some formulations, which have spirostanic analogs as active ingredients, to agricultural fields and plant biotechnology are discussed.

Synthesis and biological activity of three new 5a-hydroxy spirostanic brassinosteroid analogues

Tres novos analogos espirostânicos de brassinosteroides foram sintetizados pela primeira vez a partir de diosgenina: (25R)-2a,3a-epoxi-5a-hidroxiespirostan-6-ona (3), (25R)-2b,3a,5a-triidroxiespirostan-6-ona (5) e (25R)-2b-metoxi-3a,5a-diidroxiespirostan-6-ona (6). A substância 3 mostrou acentuada atividade promotora de crescimento vegetal no bioensaio de elongacao do hipocotilo e expansao do cotiledone de rabanete, enquanto a substância 6 mostrou-se fitotoxica.

Lipopolysaccharide aggregates in native agarose gels detected by reversible negative staining with imidazole and zinc salts.

We investigated the use of imidazole and zinc salts for the detection of lipopolysaccharide (LPS) aggregates separated by native agarose gel electrophoresis (NAGE). As a result, a new staining procedure was established by which as little as 1.5 μg of Escherichia coli O55:B5 LPS aggregates was detected by means of inducing a clear, transparent pattern, contrasted against an opaque background. E. coli O55:B5 LPS preparations treated with nucleases and proteinase K proved that the reverse-stained LPS pattern is not related to any potential artifacts caused by unrelated biomolecules (e.g., nucleic acids, proteins). After this, we showed that the procedure is applicable to two-dimensional LPS separation using NAGE/SDS-PAGE, while at the same time confirming that real polydisperse LPS aggregates are represented by the stained profile. Also, we demonstrated the general applicability of this stain to the detection of different NAGE-separated LPS aggregates (e.g., from E. coli 026:B6, E. coli 0111:B4, Salmonella minnesota Re595). Finally, using lysozyme as a model protein, we found that imidazole-zinc may be combined with Coomassie brilliant blue R-250 into a double-staining process to enable the use of NAGE for investigating the interaction of cationic proteins and LPS aggregates and protein or LPS concentration effects on protein-LPS binding.

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.

A DNA fragment from Streptomyces peucetius activates biosynthesis of actinorhodin in Streptomyces lividans and daunomycin in the original strain

A chromosomal DNA fragment from Streptomyces peucetius SGF-107 (a producer of daunomycin) which activates the biosynthesis of two polyketide antibiotics, daunomycin and actinorhodin, has been cloned and sequenced. Partial DNA sequencing of the activating fragment revealed the presence of two open reading frames whose deduced products exhibit similarities with that of other known transcriptional regulators of the MarR and ArsR family, respectively.

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.

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

A prerequisite to the discovery and characterization of lipopolysaccharide (LPS) interaction with specific receptors and the resulting pathophysiological effects is the comprehensive structural analysis of LPS species. This brief review is aimed to summarize the use of gel electrophoresis linked to other biochemical technologies for detecting and characterizing LPSs. Lipopolysaccharide aggregates alone or mixtures containing LPSs and proteins/peptides can be separated by native agarose gel electrophoresis (NAGE), after which LPSs are detected with imidazole and zinc salts. A double-staining process with Coomassie brilliant blue R-250 enables the use of NAGE for detecting and studying protein-LPS interactions. For compositional analysis, the LPS aggregates are separated, with high resolution, by surfactant-polyacrylamide gel electrophoresis. After reverse staining with zinc-imidazole and elution from gel microparticles, glycoform-specific LPSs are ready for structural and biological analysis. For sequence analysis based on tandem electrospray ionization mass spectrometry (ESI-MS/MS) of oligosaccharides, LPSs are subjected to mild acid hydrolysis, dephosphorylation and permethylation. Also, O-deacylated LPS forms can be analyzed by matrix-assisted laser desorption/ionization-time of flight-MS. By comparison to spectra of unpurified LPSs, mass spectra of the micropurified LPSs show reduce heterogeneity and increased signal-to-noise ratios. Furthermore, the micropurification of LPSs prior to MS allows a higher sensitivity of detection for less abundant LPS glycoforms. The micropurified LPS fractions can be used to form self-assembled nanoaggregates which may be detected by dynamic light scattering. The effect of the O-side chain length on the Z-potential of LPS aggregates may be estimated by measurements based on laser Doppler electrophoresis. The thus obtained glycoform-specific LPSs are not only intact chemically but also biologically active as tested by e.g. Limulus amoebocyte lysate test, TNF-α assay and agonistic effect on human Toll-like receptor 4.