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. 2020 Feb;21(2):731-743.
doi: 10.3892/mmr.2019.10888. Epub 2019 Dec 17.

Comparative Proteomic and Genomic Analyses of Brucella Abortus Biofilm and Planktonic Cells

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Free PMC article

Comparative Proteomic and Genomic Analyses of Brucella Abortus Biofilm and Planktonic Cells

Taishan Tang et al. Mol Med Rep. .
Free PMC article

Abstract

The present study aimed to explore the differences in protein and gene expression of Brucella abortus cultured under biofilm and planktonic conditions. The proteins unique to biofilms and planktonic B. abortus were separated by two‑dimensional (2‑D) electrophoresis and then identified by matrix‑assisted laser desorption/ionization‑tandem time of flight‑mass spectrometry (MALDI‑TOF/TOF‑MS). High‑throughput sequencing and bioinformatic analyses were performed to identify differentially expressed genes between B. abortus cultured under biofilm and planktonic conditions. The proteins and genes identified by proteomic and genomic analyses were further evaluated via western blot and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analyses. 2‑D electrophoresis identified 20 differentially expressed protein spots between biofilms and planktonic cells, which corresponded to 18 individual proteins (12 downregulated and 6 upregulated) after MALDI‑TOF/TOF‑MS analysis, including elongation factor Tu and enolase. RT‑qPCR analysis revealed that all of the 18 genes were downregulated in biofilms compared with planktonic cells. Western blot analysis identified 9 downregulated and 3 upregulated proteins. High‑throughput sequencing and bioinformatic analyses identified 14 function and pathway‑associated genes (e.g., BAbS19_I14970). RT‑qPCR analysis of the 14 genes showed that they were upregulated in biofilm compared with in planktonic state. In conclusion, these differentially expressed genes may play important roles in bacterial defense, colonization, invasion, and virulence.

Figures

Figure 1.
Figure 1.
Observation of Brucella abortus strain isolate A3313 biofilm by crystal violet staining under a phase-contrast light microscopy. Magnification, ×200.
Figure 2.
Figure 2.
Scanning electron microscope images of Brucella abortus strain isolate A3313 biofilm. Magnifications, (A) ×1,500 and (B) ×4,000.
Figure 3.
Figure 3.
2-D electrophoresis patterns of Brucella abortus A3313 from whole cell lysate proteins. B. abortus was cultured in biofilm or planktonic conditions, and the proteins were separated via 2-D electrophoresis. The proteins were separated in the first dimension by IEF and in the second dimension by SDS-PAGE. Molecular weight markers are shown in the left lane (kDa). (A) Protein pattern in the biofilm culture. (B) Protein pattern in the planktonic culture. 2-D, two-dimensional; IEF, isoelectric focusing; pI, isoelectric point.
Figure 4.
Figure 4.
Differential expression of mRNAs between Brucella abortus cultured under planktonic or biofilm conditions. The relative expression levels of (A) 18 genes encoding proteins identified by 2-D electrophoresis and (B) 14 genes identified via high-throughput sequencing. 2-D, two-dimensional. *P<0.05 vs. control (planktonic cells). E11-22, hypothetical protein BAbS19_I16470; E15-1, polyprenyl synthetase; E21, ExsB protein; J12, Chaperonin Cpn60TCP-1; E13-1, elongation factor Tu; E20-2, aspartate-semialdehyde dehydrogenase; C24-1, enoyl-(acyl carrier protein) reductase; E12-1, DnaJ, chaperone protein DnaJ; E12-3, isocitrate dehydrogenase; E17, Enolase; E18, Acetyl-CoA carboxylase, alpha subunit; C24-2, putative sulfite oxidase subunit YedY; E11-1, Bacterial protein export chaperone SecB; E13-2, Antifreeze protein, type I; E15-2, Lactatemalate dehydrogenase; E15-5, Phosphoribosylformylglycinamidinecyclo-ligase; E16-3, Periplasmic binding protein; E19-2, tryptophanyl-tRNAsynthetase.
Figure 5.
Figure 5.
Analysis of differential protein expression in Brucella abortus cultured under planktonic or biofilm conditions. Western blot analysis of 12 proteins showed that 9 proteins were downregulated and 3 were upregulated between biofilms and planktonic cells. Omp16, outer membrane protein 16. *P<0.05 vs. control (planktonic cells).
Figure 6.
Figure 6.
Functional and pathway analysis of differentially expressed genes in Brucella abortus biofilm. The significant (A) GO terms (BP, CC and MF) and (B) pathways [significant (red); not significant (blue)] enriched by differentially expressed genes. The horizontal axis represents significant GO terms and pathways, and the vertical axis represents the percentage of genes. GO, Gene Ontology; BP, biological process; MF, molecular function; CC, cellular compartment; KS, Kolmogorov-Smirnov.

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