Proteomic Profile of <i>Brucella abortus</i>-Infected Bovine Chorioallantoic Membrane Explants

<div><p><i>Brucella abortus</i> is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with <i>B</i>. <i>abortus</i> (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with <i>B</i>. <i>abortus</i> were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation.</p></div

Functional classification, subcellular localization and the experimental group which were identified the proteins expressed in uninfected CAM explants or CAM explants infected with <i>Brucella abortus</i> 2308 at 0.5 and 4 h post inoculation.

<p>Functional classification, subcellular localization and the experimental group which were identified the proteins expressed in uninfected CAM explants or CAM explants infected with <i>Brucella abortus</i> 2308 at 0.5 and 4 h post inoculation.</p

Two-dimensional gels stained with Coomassie Blue with samples from bovine CAM explants uninfected or infected with <i>B</i>. <i>abortus</i> at 0.5 or 4 h post infection.

<p>A. Number of spots selected for identification by mass spectrometry after DIGE analysis in infected and uninfected CAM explants at different time intervals after <i>B</i>. <i>abortus</i> 2308 infection. Differentially expressed spots were selected (i.e. spots that were present in one experimental group—infected or uninfected controls—and absent in the other). B. After analysis of DIGE gels using DeCyder^™2-D Differential Analysis v7.0 software (GE Healthcare, UK) for determination of protein expression levels, new two-dimensional gels were prepared, stained with Coomassie Brilliant Blue G-250, and scanned for selecting spots of interest. Numbers refer to the spot identification used in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154209#pone.0154209.s002" target="_blank">S1 Table</a>.</p

Number of <i>Brucella abortus</i> internalized (Log CFU/mL) in bovine CAM explants.

<p>Chorioallantoic membrane (CAM) explants were inoculated with <i>Brucella abortus</i> 2308, centrifuged for 15 min at 1000 xg and maintained at 37°C in 5% CO₂ for 30 min to allow internalization of bacteria, followed by 1 h of incubation with gentamicin, and then lysed for intracellular CFU counting. Quantification of <i>B</i>. <i>abortus</i> internalized was performed using the drop count method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154209#pone.0154209.ref019" target="_blank">19</a>] and data represent mean and standard error of three explants for each fetuses (n = 3), after logarithmic transformation. Analysis of variance (ANOVA) and Tukey's multiple comparison test were performed and no statistically significant differences were observed (P > 0.05).</p

Functional classification and Venn diagram of differentially expressed proteins identified by mass spectrometry (MALDI TOF/TOF).

<p>A. Distribution of functional classification of proteins selected by DIGE analysis and identified by mass spectrometry in extracts of bovine CAM explants uninfected or infected with <i>Brucella abortus</i> at 0.5 h and 4 h post inoculation. B. Venn diagram showing differentially expressed proteins in each experimental group. Abbreviations: 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1), 3-hydroxyisobutyrate dehydrogenase, mitochondrial precursor (HIBADH), aldose 1-epimerase (GALM); abhydrolase domain-containing protein 14B (ABHD14B), activator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1), adenosylhomocysteinase (AHCY), aldose reductase (AKR1B1), alpha-actinin-4 (ACTN4), alpha-fetoprotein precursor (AFP), beta-actin (ACTB), beta-hexosaminidase subunit beta preproprotein (HEXB), biliverdin reductase A (BLVRA), calmodulin (CALM1), cathepsin D (CTSD), complement component 1 Q subcomponent-binding protein, mitochondrial precursor (C1QBP), creatine kinase B-type (CKB), cytokeratin 8 (KRT8), dynein light chain roadblock-type 1 (DYNLRB1), endoplasmin precursor (HSP90B1), F-actin-capping protein subunit beta (CAPZB), galectin-7-like (LGALS7), gelsolin isoform a (GSNA), gelsolin isoform b (GSNB) heat shock cognate 71 kDa protein (HSC70), heat shock protein beta-1 (HSPB1), hemoglobin subunit beta (HBB), high-mobility group box 1-like (HMGB1), inositol-3-phosphate synthase 1 (ISYNA1), keratin 14-like (KRT14), keratin, type II cytoskeletal 7 (KRT7), LDLR chaperone MESD (MESDC2), malate dehydrogenase, cytoplasmic (MDH1), NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (NDUFS8), ornithine aminotransferase, mitochondrial precursor (OAT), placental prolactin (PRCII), proactivator polypeptide/prosaposin (PSAPL1/PSAP), protein disulfide-isomerase A3 precursor (PDIA3), prostaglandin reductase 2 (PTGR2), ras-related protein Rab-11A (RAB11A), ribosomal protein P1-like isoform 1 (RPLP1), secretory carrier-associated membrane protein 2 (SCAMP2), serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 5 (<b>S</b>ERPINB5), similar to BolA-like protein 2 isoform 1 (BOLA2), thioredoxin-dependent peroxide reductase, mitochondrial precursor (PRDX3), toll-interacting protein (TOLLIP), transgelin-2 (TAGLN), transitional endoplasmic reticulum ATPase (VCP), transthyretin precursor (TTR), tropomyosin 4 (TPM4).</p