The effect of heat shock, acid shock and macrophage phagocytosis on gene expression in Brucella abortus

Abstract

Brucella abortus is an intracellular pathogenic bacterium that replicates within macrophages and resists macrophage microbicidal mechanisms. To study the defense mechanisms of B. abortus and mimic the conditions that B. abortus may encounter during infection, protein synthesis by B. abortus was examined using pulse-labeling techniques when bacteria were stressed by heat, acid pH, or infection of macrophages. The most prominent proteins induced following heat shock have estimated molecular weights of 70. 62. and 10 kDa. BaHSP62 and BaHSP10 have been shown to be related to the GroEL and GroES of several other bacteria. The overall amino acid similarities of BaHSP10 with GroES and BaHSP62 with GroEL range from 83% (Rhizobium meliloti) to 40% (Chlamydia psittaci) and 87.2% (Rhizobium meliloti) to 53.5% (mouse HSP60), respectively. The organization of the groE operon in B. abortus most resembles E. coli, in which the two genes represent a single transcription unit and are separated by 44 base pairs. Western blot analysis of BaHSP62 with bovine serum indicates that BaHSP62 is a major target of the humoral response with the immunodominant site confined to amino acids 317 to 433 region. Mice immunized with BaHSP62 induce an humoral response to BaHSP62, but fail to elicit protective immunity to challenge with B. abortus S2308. The most prominent proteins induced following acid shock have estimated molecular weights of 60 and 24 kDa. Optimum expression of these two proteins occurs during the first three hours following a pH shift from pH 7.3 to 3.8, which correlates with the period of maximum survival. The 60 kDa protein appears to be related to BaHSP62 as shown by immunoprecipitation using anti-BaHSP62 serum. The exact relationship between the two proteins remains uncertain. The most prominent proteins induced following macrophage engulfment have estimated molecular weights of 62, 28 and 17 kDa. The 62-kDa protein was shown to be BaHSP62. The nature of the 28 and 17 kDa proteins is under investigation. Taken together, the data reveal that environmental stresses induce multiple virulence factors in B. abortus, the nature of these gene products and the mechanisms controlling their expression is the key to understanding the pathogenesis of this organism.