Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker.

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.

[Ultrastructural immunochemical study of pathogenic Burkholderia capsule].

The capsular structures of Burkholderia pseudomallei, B. mallei, B. cepacia and their avirulent noncapsular mutants were studied with the use of electron ahd immunocytochemical techniques. For this purpose, antimelio-idosis monoclonal antibodies (McAb) G11 and 1 G2, epitope-aimed at capsular glycopyotein of 200 kD and outer-membrane proteins of 42 and 39 kD, were used. As revealed in this study, the typical causative agents of melioidosis and glanders formed the capsule and exhibited high virulence due to the antiphagocytic activity of 200 kD glycoprotein, whose epitopes were found to be incorporated into the capsule, in contrast to avirulent variants and B. cepacia, found to have no such structure. The recognition of the membrane determinants of McAb 1 G2 on the outer-membrane surface of the non-capsular variants of microbes known to be the causative agents of melioidosis and glanders was indicative of absence of the capsule in these microbial cells. These data concerning the role of 200 kD antigen in virulence, its structural and functional characteristics may be efffectively used in the study of the pathogenetic mechanisms of melioidosis and glanders, as well as in the construction of preparations for their immunodiagnostics and prophylaxis.