Intracellular adaptation of Brucella abortus

Abstract

Macrophages were infected with virulent Brucella abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration; adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain's adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections.

Figure 1. Isolation of intracellular Brucella

(A) Western blot analysis of infected macrophage cell lysates used for intracellular Brucella isolation, and of the intracellular Brucella final products (pellet). The starting cell lysate titration shown was used to estimate the enrichment of the final product. A representative isolation 20 hrs after infection is shown for both bacterial strains (2308 and S19) used. (B) Immunofluorescence analysis of LPS staining (red) on GFP-expressing B. abortus 2308 inside infected macrophages (B1, B2) and following isolation (B3, B4). Bacteria that displayed both green and red fluorescence were considered viable. Bacteria that displayed only red fluorescence were considered non-viable (arrow). Representative staining 3 hrs after infection is shown. Magnification is 40X (B1, B2) and 100X (B3, B4). (C) Transmission electron microscopy of isolated intracellular bacteria at each experimental timepoint. Magnification displayed is 43,000X. A size bar of 0.2μm is indicated.

Figure 2. Global differential expression proteome analysis

Multi-dimensional scaling plot of peptides differentially expressed by B. abortus 2308 (A) and B. abortus S19 (B). Each sphere represents one independent replicate, color coded per timepoint. (C) Venn diagram illustrating the distribution of the differentially expressed proteins identified in the two strains.

Figure 3. Proteins differentially expressed by B. abortus 2308 during an intracellular infection

Intensity values per protein were normalized to time zero and displayed in logarithmic scale per timepoint (y-axis). Protein enumeration follows the numbering indicated in Table 1 (x-axis). The intensity values outside the colored band centered at normalized intensity 1 are considered to be differentially expressed. Median and standard deviations of 6 independent replicates are shown. (A) Proteins that were differentially expressed only by B. abortus 2308. (B) Proteins that were differentially expressed by B. abortus 2308 and S19. The differential intensities for B. abortus 2308 are shown.

Figure 4. Proteins differentially expressed by B. abortus S19 during an intracellular infection

Intensity values per protein were normalized to time zero and displayed in logarithmic scale per timepoint (y-axis). Protein enumeration follows the numbering indicated in Table 1 (x-axis). The intensity values outside the colored band centered at normalized intensity 1 are considered to be differentially expressed. Median and standard deviations of 6 independent replicates are shown. (A) Proteins that were differentially expressed only by B. abortus S19. (B) Proteins that were differentially expressed by B. abortus 2308 and S19. The differential intensities for B. abortus S19 are shown.

Figure 5. Comparison of proteins differentially expressed by both B. abortus 2308 and S19 during an intracellular infection

(A) Shown are the Pearson correlation values for each commonly differentially expressed protein’s expression patterns in both strains (y-axis). Protein enumeration follows the numbering indicated in Table 1 (x-axis). The red line indicates a Pearson correlation value of 0.75. (B) Protein intensities were normalized to the values at time zero for the virulent 2308 strain (y-axis). Protein enumeration follows the numbering indicated in Table 1 (x-axis) . Proteins with values within the colored band centered at normalized intensity ratio of 1 are considered to be equivalently expressed in both strains. Median and standard deviations of 6 independent replicates are shown.

Figure 6. Verification of proteomics results

(A) Bar graph of BvrR peptide intensities measured in B. abortus 2308 were normalized to time zero and compared to western blots of isolated intracellular B. abortus 2308 homogenates. (B) Bar graph of peptide intensities of 4 proteins measured in B. abortus 2308 were normalized to time zero for each protein and compared to MRM results of the same peptides. Median and standard deviations of 6 independent replicates are shown for both the proteomics discovery and the MRM results.