Lable-free based comparative proteomic analysis of secretory proteins of rough Brucella mutants

Brucella rough mutants are reported to induce infected macrophage death, which is type IV secretion system (T4SS) dependent. T4SS and its secretory proteins play a major role in host-bacteria interactions, but the crucial secretory proteins to promote macrophage death during Brucella rough mutant infection have not been characterized. In this study, we found that T4SS components played no role for macrophage death induced by Brucella rough mutant infection, but some T4SS effectors did. Proteomics of secretory proteins from Brucella rough mutants ΔrfbE and ΔrfbEΔvirB123 was analyzed by liquid chromatography/tandem mass spectrometry and 861 unique proteins were identified, among which 37 were differential secretory proteins. Gene ontology and pathway analysis showed that differential secretory proteins involved in cellular process and metabolic process, distributed in the cell and membrane, possessed molecular function of catalytic activity and binding, and were associated with ribosome, NOD-like receptor signaling pathway, two-component system and bacterial secretion system. Cell death analysis showed that T4SS effector VceC, and two differential secretory proteins OmpW family protein (BAB1_1579) and protein BAB1_1185 were associated with Brucella cytotoxicity. This study provides new insights into the molecular mechanisms associated with Brucella cytotoxicity and valuable information for screening vaccine candidates for Brucella. SIGNIFICANCE: Brucella rough mutants induce infected macrophage death, which is T4SS dependent. In the present report, a comparative proteomics analysis revealed 37 differential secretory proteins between Brucella rough mutants ΔrfbE and ΔrfbEΔvirB123. Further study demonstrated OmpW family protein (BAB1_1579) and uncharacterized protein BAB1_1185, two differential secretory proteins, were associated with Brucella cytotoxicity. This study provides novel information of the secretory proteins from the Brucella rough mutants and their effects on the Brucella cytotoxicity.