Iron-dependent reconfiguration of the proteome underlies the intracellular lifestyle of Brucella abortus

Abstract

Brucella ssp. is a facultative intracellular pathogen that causes brucellosis, a worldwide zoonosis that affects a wide range of mammals including humans. A critical step for the establishment of a successful Brucella infection is its ability to survive within macrophages. To further understand the mechanisms that Brucella utilizes to adapt to an intracellular lifestyle, a differential proteomic study was performed for the identification of intracellular modulated proteins. Our results demonstrated that at 48 hours post-infection Brucella adjusts its metabolism in order to survive intracellularly by modulating central carbon metabolism. Remarkably, low iron concentration is likely the dominant trigger for reprogramming the protein expression profile. Up-regulation of proteins dedicated to reduce the concentration of reactive oxygen species, protein chaperones that prevent misfolding of proteins, and proteases that degrade toxic protein aggregates, suggest that Brucella protects itself from damage likely due to oxidative burst. This proteomic analysis of B. abortus provides novel insights into the mechanisms utilized by Brucella to establish an intracellular persistent infection and will aid in the development of new control strategies and novel targets for antimicrobial therapy.

Figure 1

Schematic representation of the workflow of the iTRAQ experiment.

Figure 2

Western blot validation of up and down-regulated protein identified by iTRAQ proteomic. (A) Quantification of the intensity of bands (below) of the Western blots analysis (top) of the whole cell lysates of intracellular Brucella (I) or Brucella growth in culture media (control) (C). Blots were probed with mouse sera specific against; (a) universal stress protein (UspA), (b) ferric uptake regulator (Irr), (c) choloylglycine hydrolase (Cgh) (d) proline racemase (PrpA) and (e) ribosome recycling factor (Frr). Monoclonal antibody against anti-Omp10 was used as a normalizing control. Blots are representative of two independent experiments. Displayed Western blots bands were cropped from the original blot for clarity. Corresponding full-length blots are shown in Supplementary Figure S3. Unpaired two-tailed Student’s t-test (P < 0.05) was performed. (B) The table shows the comparison of the results of the mean ratio (I/C) obtained for iTRAQ or Western blot analysis.

Figure 3

Global analysis of differentially expressed proteins. (A) Mean of log₂ I/C ratio distribution of intracellular modulated proteins. The red and blue dots represent up-regulated and down-regulated proteins, respectively. Shade gray area represent proteins involved in iron transport and metabolism. Dash lines indicate the upper and lower log2 ratio cutoff (0.24, −0.26) used to consider proteins as being modulated. Numbers located at the right, indicate the total number of up, down and non-modulated proteins identified in this study. (B) Distribution of the identified modulated proteins accordingly to their assigned functional category (COGs).

Figure 4

Protein expression changes of metabolic pathways involved in (A) central carbon metabolism or (B) oxidative phosphorylation in B. abortus during its intracellular life. Locus Tags depicted either in red or blue indicate up or down-regulated proteins respectively.

Figure 5

Comparison of the results of previous intracellular proteomic report in B. abortus 2308 with our study. Intersection of the Venn diagram shows common differentially expressed proteins identified in both studies.