Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses

Abstract

Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS) via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs) that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular homeostasis and metabolic balance under stress. In conclusion, our results provide a better understanding of the global metabolic adaptations of B. abortus associated with distinct environmental stresses. The identification of proteins necessary for stress resistance is crucial toward elucidating the infectious process in order to control brucellosis, and may facilitate the discovery of novel therapeutic targets and effective vaccines.

Proteomics of Brucella

“…The multiplexing of samples and the MS/MS-based quantification of iTRAQ reporter ions [35,36], or alternatively with Isotope Coded Protein Labeling (ICPL) [32] has been pursued. In-solution digestion [37][38][39][40][41], or in-gel digestion [42,43], and LC-MS/MS were sometimes employed.…”

“…As a result, 55 proteins were identified as differentially abundant, demonstrating the role of Hfq in coordination of the adaptive response to stress conditions. Recently, a label-free quantitative proteomic analysis to identify B. abortus proteins involved in bacterial response to distinct stresses encountered during its intracellular phase detected more than 1000 differentially expressed proteins belonging to diverse functional groups [38].…”

Proteomics of Brucella: Technologies and Their Applications for Basic Research and Medical Microbiology

“…Among them, 63 and 103 proteins were detected exclusively in the whole-cell extracts of B. abortus and B. melitensis, respectively [15]. The mass spectrometry-based label-free relative quantitative proteomics analysis of B. abortus was reported to have identified a total of 1221 differentially expressed proteins following multiple environmental stresses and adaptations [72]. Furthermore, the proteogenomic mapping of B. abortus 104 M human vaccine strains using high-resolution mass spectrometry revealed 1729 proteins and 218 hypothetical proteins [73].The liquid chromatography/tandem mass spectrometry was used to analyze secretory proteins from the Brucella rough mutants ΔrfbE and ΔrfbEΔvirB123.…”

Pan-Proteomic Analysis and Elucidation of Protein Abundance among the Closely Related Brucella Species, Brucella abortus and Brucella melitensis

“…Although it is broadly accepted in the field that the Type IV secretion system (T4SS) is in some way tied to virulence in Brucella species, Brucella generally lacks classical virulence factors and in order to explain its virulence and host specificity, a better understanding at the proteome and metabolome level is needed. Proteomic analyses of various strains of B. abortus and B. melitensis have been reported [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ], suffering from limitations in terms of technology and database coverage. In the present study, the label-free quantitative proteomic analysis includes the reference strains as well as strains isolated from infected animals analyzed in an earlier study [ 23 ].…”