Comparative proteomic and genomic analyses of Brucella abortus biofilm and planktonic cells

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.

Proteomics of Brucella

“…Since 2012, some of these technological advances have been implemented, yet traditional methods have remained popular in Brucella proteomics. There have been frequent uses of 2D-PAGE and MALDI-TOF (-TOF) [ 25 , 26 , 27 , 28 , 29 , 30 ], sometimes utilizing DIGE to minimize gel-to-gel variation [ 31 , 32 ] or other fluorescent dyes for extended dynamic range [ 33 ]. Additionally, the unusual combination of 2D-PAGE with LC-MS/MS has been reported [ 34 ].…”

The Flagellar Transcriptional Regulator FtcR Controls Brucella melitensis 16M Biofilm Formation via a betI-Mediated Pathway in Response to Hyperosmotic Stress

“…We investigated the role of FtcR in the response of B. melitensis 16M biofilms to various conditions of environmental stress. Inconsistent with B. melitensis 16M in a planktonic state, the ftcR mutant showed a significant biofilm growth defect compared with that of the WT and complemented strains only under hyperosmotic stress; B. melitensis 16M did not show the same susceptibility to other stressors as the planktonic bacteria of the Δ ftcR strain, possibly highlighting the difference in the protective effects of the biofilm structure on bacteria and the bacteria in the planktonic state [ 11 , 14 , 15 ]. This biofilm phenotype was not a response to high concentrations of NaCl alone, because it was also observed when the biofilms were treated with other types of osmotic solutes.…”

“…Biofilms are formed by a complex process that initiates when microorganisms attach to a surface via an extracellular matrix and other adhesins that allow them to develop into large communities [ 13 ]. Compared to planktonic bacteria, biofilms have unique transcriptional and expressive programs [ 14 , 15 ]. Bacteria form a niche within these biofilms, exhibiting increased virulence and resistance to various hostile microenvironments [ 16 , 17 ].…”

How to study biofilms: technological advancements in clinical biofilm research

“…Proteomic analysis of biofilms has been used to identify key proteins that are essential in complex biofilm networks, including; in the EPS ( Egorova et al., 2022 ) , required for temperature adaptation stress ( Lee and Wang, 2020 ) , required for adaptation to antimicrobial treatment ( MaChado and Coquet, 2016 ) , and that change with biofilm development ( Serra et al., 2008 ; Suriyanarayanan et al., 2018 ) and aging ( Rahman et al., 2022 ), and between planktonic and biofilm conditions ( Khan et al., 2019 ; Suryaletha et al., 2019 ; Llama-Palacios et al., 2020 ; Tang et al., 2020 ). These studies use a range of different mass spectrometry methodologies, including tandem liquid chromatography (LC-MS/MS) alone ( Lee and Wang, 2020 ) or coupled with nano-high performance liquid chromatography (HPLC) ( Egorova et al., 2022 ), lab-on-a-chip and XCT mass spectrometry ( MaChado and Coquet, 2016 ), tandem mass tag (TMT) mass spectrometry ( Rahman et al., 2022 ), fourier transform infrared spectroscopy (FT-IR) ( Serra et al., 2008 ), nano-LC/MS coupled with quadrupole-ToF (Q-ToF) ( Suryaletha et al., 2019 ) , iTRAQ labelling plus LC-MS/MS for quantitative proteomics, and matrix-assisted laser desorption ionisation time-of-flight (MALDI-ToF) mass spectrometry ( Llama-Palacios et al., 2020 ; Tang et al., 2020 ). MALDI-ToF analysis of bacteria is routinely used in clinical diagnostic laboratories to identify bacterial species, and this method is increasingly being developed to discriminate between isolates and their status as biofilm-producers or non-producers ( Caputo et al., 2018 ), which will aid in establishing effective antimicrobial therapy.…”