Characterization of heat, oxidative, and acid stress responses in Brucellamelitensis

Brucella melitensis is a facultative intracellular pathogen which is able t o survive and replicate within phagocytic cells. Therefore, it has to adapt to a range of different hostile environments. In order to understand the m echanisms of intracellular survival employed by virulent B. melitensis 16M, an initial approach consisting of analysis of the differences in patterns of protein synthesis in response to heat, oxidative, and acid pH stresses b y two-dimensional (2-D) polyacrylamide gel electrophoresis was used. Depend ing on the stress, this involved about 6.4 to 12% of the 676 protein spots detected in 2-D gel electrophoresis. On the basis of N-terminal sequence an alysis and database searching, 19 proteins whose level of synthesis was up- or down-regulated by stress conditions were identified. Some of them were previously reported for Brucella, such as BvrR, DnaK, GroEL, and Cu-Zn supe roxide dismutase (SOD). Eight other proteins closely matched proteins found in other bacteria: AapJ, alpha-ETF, ClpP, Fe and/or Mn SOD, malate dehydro genase, IalB, 30S ribosomal protein S1, and pyruvate dehydrogenase E1 compo nent beta subunit, Results indicated that B. melitensis could bring specifi c regulatory mechanisms into play in response to stress conditions. For exa mple, the ribosome releasing factor in B. melitensis appeared to be a heat shock protein, whereas the ClpP protein, described as a heat shock protein for Escherichia coli, was strongly down-regulated in B. melitensis in respo nse to heat stress. Some of the identified proteins and their potential spe cific regulation could be required for the adaptation of B. melitensis to e nvironmental stresses encountered in phagocytic cells and possibly for bact erial virulence.