Expression and purification of 30 kilodalton protein antigen of Ara- Burkholderia pseudomallei.

The 30 kDa protein of B. pseudomallei is found in virulent Ara- but not avirulent Ara+ strain. The gene was cloned in Escherichia coli JM105 employing pInIII-C2 vector. The open reading frame was 870 nucleotides with a guanine plus cytosine content of 69.9%. Arginine was the most abundant amino acid in the protein, having a PI of 12.65. Nucleotide sequence of the gene was 96% identical to B. pseudomallei 1710b chromosome II sequence CP000125.1, encoding an oxidoreductase of the short chain dehydrogenase/reductase family. The 30 kDa antigen was expressed as a maltose-fusion protein with a yield of 5.25 mg/l of bacterial culture.

Distinct immunologic properties of Penicillium marneffei yeasts obtained from different in vitro growth conditions.

A dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis, a life-threatening disseminated disease in immunocompromised hosts predominantly found in southeast Asia and southern China. P. marneffei is the only known Penicillium that possesses a dimorphic characteristic. Since it is difficult to produce large amount of P. marneffei yeasts in vivo for experimentation purpose, yeast cells were produced in different in vitro conditions as alternatives. We interested in investigating the immunologic properties of yeast cells from different culture preparations. It was found that yeast cells obtained from brain heart infusion broth and Sabouraud dextrose broth did not resemble those resided in clinical specimens. A solution of 1% peptone, on the other hand, could induce a direct conidial transition into fission yeasts. Ability of yeast cells in each preparation to activate macrophages was determined by analyzing surface expression of CD40 and CD86 co-stimulatory molecules after two days of co-cultivation. Every P. marneffei yeast cell preparation demonstrated such ability. However, the ones from Sabouraud dextrose broth seemed to induce less phagocytosis. Additionally, although distinct antigenic profiles and lack of conformity in antigenic expression were observed among yeast cells from different culture conditions, most major immunogenic bands were present when Western analysis was performed using polyclonal antisera from penicilliosis patients. The results of the study raise attention on immunological and biochemical characteristics of P. marneffei yeasts if such preparations are to be used in future laboratory investigations.