Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Identificación auxotípica de cepas de Pseudomonas aeruginosa aisladas de pacientes con fibrosis quística o VIH/SIDA / Auxotypic identification of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis or HIV/AIDS

Objetivo: Identificar la presencia del fenotipo auxotrofo en cepas de P. aeruginosa aisladas de pacientes con fibrosis quística o con VIH/SIDA. Métodos: Se realizó un estudio observacional descriptivo longitudinal utilizando la metodolog¡a de Barth y Pitt. En la determinación de la auxotrofía se realizó la siembra de la dilución 1:100 de cada cepa en un medio nutricionalmente completo agar Pseudomonas P (King A) y en un medio incompleto Agar Medio Mínimo y para la determinación del requerimiento específico en las auxotrofas se utilizaron 22 soluciones madre de aminoácidos en su forma L. Resultados: Se identificaron 20 cepas auxotrofas, once procedentes de pacientes VIH/SIDA y nueve de pacientes con fibrosis quística. Todos los aislamientos nutricionalmente dependientes proced¡an de muestras de esputo. El aminoácido simple de mayor requerimiento fue la metionina (7 de 20 aislamientos auxotrofos) seguido de arginina, lisina y glutamina. Conclusiones: Estos resultados sugieren que la P. aeruginosa auxotrofa coloniza a los pacientes VIH/SIDA durante el curso de la infección pulmonar al igual que en los pacientes con fibrosis quística. Confirmamos la auxotrofía como un fenotipo exclusivo de infecciones respiratorias.

Objective: To identify the presence of auxotrophic phenotype in strains of P. aeruginosa isolated from patients with cystic fibrosis or HIV/AIDS. Methods: We conducted a longitudinal descriptive study using the methodology of Barth and Pitt. For determination of auxotrophy, a seeding of a 1:100 dilution of each strain was made on nutritionally complete medium P Pseudomonas agar (King A) and in the incomplete medium minimal medium agar. For determining the specific requirement of auxotrophs, 22 amino acids solutions in their L-form were used. Results: We identified 20 auxotrophic strains, eleven from HIV/AIDS patients and nine frompatients with cystic fibrosis. All isolates that were nutritionally dependent came from sputum samples. The amino acid with the highest requirement was methionine (7 of 20 auxotrophs isolates) followed by arginine, lysine and glutamine. Conclusions: These results suggest that auxotrophic P. aeruginosa colonizes HIV/AIDS patients during the course of pulmonary infection, as in patients with cystic fibrosis. Auxotrophy was confirmed as a unique phenotype of respiratory infections.

Construction and identification of the double auxotrophic Candida parapolymorpha / 中华微生物学和免疫学杂志

Objective To construct a genetically-stable double auxotrophic,in which the uracil and leucine were mutated,using the Candida parapolymorpha ATCC26012 as materials.Methods Based on the physical and genetic engineering methods,the chromosome of the C.parapolymorpha strain was modified,where the ura3 and leu2 genes were directly mutated,to obtain the uracil and leucine double auxotrophic strain.Then the constructed strain was identified by the analysis of its biological properties,such as genetic stability,the change of the genes,and the physiologic and biochemical characteristics.Results The uracil and leucine double auxotrophic strain is obtained by screening.The biological identification results show that the obtained strain is genetically stable and the targeted genes are directly altered.In addition,the physiologic and biochemical analyses indicate that the auxotrophic can utilize various kinds of carbon and nitrogen nutrient sources,and its growth is good.Conclusion The successful construction of double auxotrophic mutant strains facilitated the genetic studies on C.parapolymorpha to meet various investigational purposes.Moreover,the constructed auxotrophic strains can be applied as advantageous host cells to express multiple proteins/antigens simultaneously,which is of great significance in the development of vaccines.

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

The Efficient Transformation of Pleurotus ostreatus using REMI Method

Restriction enzyme-mediated integration (REMI) was used to transform uracil auxotrophs of Pleurotus ostreatus to prototrophy. When protoplasts of Pleurotus ostreatus were treated by the reaction mixture containing 10 units of BamHI, the frequency of REMI was about 64 transformants per 1 microg of DNA. This efficiency was increased by 14.2 times compared with that of the conventional PEG transformation. The optimal condition for REMI of P. ostreatus was achieved when 1 microg of linearized pTRura3-2 DNA was added into 1x10(7) protoplasts along with 10 units BamHI. Southern blot analysis revealed that about 50% of transformants examined were caused by REMI event and 30% carried single copy insertion at the genome. This suggested that the REMI method might be a useful tool for efficient transformation and tagging mutagenesis of P. ostreatus.