Identification of Proteins Associated with the <i>Pseudomonas aeruginosa</i> Biofilm Extracellular Matrix

Biofilms are surface-associated bacteria that are embedded in a matrix of self-produced polymeric substances (EPSs). The EPS is composed of nucleic acids, polysaccharides, lipids, and proteins. While polysaccharide components have been well studied, the protein content of the matrix is largely unknown. Here we conducted a comprehensive proteomic study to identify proteins associated with the biofilm matrix of <i>Pseudomonas aeruginosa</i> PAO1 (the matrix proteome). This analysis revealed that approximately 30% of the identified matrix proteins were outer membrane proteins, which are also typically found in outer membrane vesicles (OMVs). Electron microscopic inspection confirmed the presence of large amounts of OMVs within the biofilm matrix, supporting previous notions that OMVs are abundant constituents of <i>P. aeruginosa</i> biofilms. Our results demonstrate that while some proteins associated with the <i>P. aeruginosa</i> matrix are derived from secreted proteins and lysed cells, the large majority of the matrix proteins originate from OMVs. Furthermore, we demonstrate that the protein content of planktonic and biofilm OMVs is surprisingly different and may reflect the different physiological states of planktonic and sessile cells

Identification of Proteins Associated with the <i>Pseudomonas aeruginosa</i> Biofilm Extracellular Matrix

Biofilms are surface-associated bacteria that are embedded in a matrix of self-produced polymeric substances (EPSs). The EPS is composed of nucleic acids, polysaccharides, lipids, and proteins. While polysaccharide components have been well studied, the protein content of the matrix is largely unknown. Here we conducted a comprehensive proteomic study to identify proteins associated with the biofilm matrix of <i>Pseudomonas aeruginosa</i> PAO1 (the matrix proteome). This analysis revealed that approximately 30% of the identified matrix proteins were outer membrane proteins, which are also typically found in outer membrane vesicles (OMVs). Electron microscopic inspection confirmed the presence of large amounts of OMVs within the biofilm matrix, supporting previous notions that OMVs are abundant constituents of <i>P. aeruginosa</i> biofilms. Our results demonstrate that while some proteins associated with the <i>P. aeruginosa</i> matrix are derived from secreted proteins and lysed cells, the large majority of the matrix proteins originate from OMVs. Furthermore, we demonstrate that the protein content of planktonic and biofilm OMVs is surprisingly different and may reflect the different physiological states of planktonic and sessile cells

Identification of Proteins Associated with the <i>Pseudomonas aeruginosa</i> Biofilm Extracellular Matrix

Biofilms are surface-associated bacteria that are embedded in a matrix of self-produced polymeric substances (EPSs). The EPS is composed of nucleic acids, polysaccharides, lipids, and proteins. While polysaccharide components have been well studied, the protein content of the matrix is largely unknown. Here we conducted a comprehensive proteomic study to identify proteins associated with the biofilm matrix of <i>Pseudomonas aeruginosa</i> PAO1 (the matrix proteome). This analysis revealed that approximately 30% of the identified matrix proteins were outer membrane proteins, which are also typically found in outer membrane vesicles (OMVs). Electron microscopic inspection confirmed the presence of large amounts of OMVs within the biofilm matrix, supporting previous notions that OMVs are abundant constituents of <i>P. aeruginosa</i> biofilms. Our results demonstrate that while some proteins associated with the <i>P. aeruginosa</i> matrix are derived from secreted proteins and lysed cells, the large majority of the matrix proteins originate from OMVs. Furthermore, we demonstrate that the protein content of planktonic and biofilm OMVs is surprisingly different and may reflect the different physiological states of planktonic and sessile cells

Immunohistochemical localization of ATP9A in non-perfused rat liver 5μm cryosections.

<p>(<b>A</b>) ATP9A. (<b>B</b>) BSEP expressed at the canalicular membrane of rat hepatocytes. (<b>C</b>) Colocalization of ATP9A and BSEP shows ATP9A expression at the canalicular membrane of rat hepatocytes. Scale bar: 20 μm</p

Immunohistochemical localization of ATP11A in perfused rat liver 5μm cryosections.

<p>(<b>A</b>) ATP11A. (<b>B</b>) Expression of RAB7, a late endosome marker. (<b>C</b>) Colocalization of ATP11A and RAB7 shows considerable overlap demonstrating ATP11A in a late endosomal compartment. Scale bar: 20 μm</p

Immunohistochemical localization of ATP11A in perfused rat liver 5μm cryosections.

<p>(<b>A</b>) ATP11A. (<b>B</b>) Expression of EEA1, an early endosome marker. (<b>C</b>) Colocalization of ATP11A and EEA1 shows partial overlap demonstrating ATP11A in an early endosomal compartment. Scale bar: 20 μm</p

Identification of Proteins Associated with the <i>Pseudomonas aeruginosa</i> Biofilm Extracellular Matrix

Biofilms are surface-associated bacteria that are embedded in a matrix of self-produced polymeric substances (EPSs). The EPS is composed of nucleic acids, polysaccharides, lipids, and proteins. While polysaccharide components have been well studied, the protein content of the matrix is largely unknown. Here we conducted a comprehensive proteomic study to identify proteins associated with the biofilm matrix of <i>Pseudomonas aeruginosa</i> PAO1 (the matrix proteome). This analysis revealed that approximately 30% of the identified matrix proteins were outer membrane proteins, which are also typically found in outer membrane vesicles (OMVs). Electron microscopic inspection confirmed the presence of large amounts of OMVs within the biofilm matrix, supporting previous notions that OMVs are abundant constituents of <i>P. aeruginosa</i> biofilms. Our results demonstrate that while some proteins associated with the <i>P. aeruginosa</i> matrix are derived from secreted proteins and lysed cells, the large majority of the matrix proteins originate from OMVs. Furthermore, we demonstrate that the protein content of planktonic and biofilm OMVs is surprisingly different and may reflect the different physiological states of planktonic and sessile cells

RT-PCR and Western blot analysis on P4- and P5-ATPases.

<p>(<b>A</b>) Representation of relative mRNA expression in rat liver of the P4- and P5-ATPases by semi quantitative RT-PCR analysis. The expression is given is relative to the house keeping genes actin and GAPDH as described in Materials and Methods. Data are the mean of two independent determinations (biological replicates) with 3 technical replicates each. (<b>B</b>) Western blot analysis of P4-ATPases in different liver subcellular fractions. The lanes were loaded with 1. homogenate 2. microsomes, 3. cLPM 4. bLPM, 5.mitochondria. For detecting the P4-ATPases and the canalicular marker APN, the following amount of protein was loaded for each lane: ATP8B1: 100 μg; ATP9A: 150 μg; ATP11A: 100 μg, ATP11C: 50 μg; APN: 150 μg</p

Immunohistochemical localization of ATP8A1 in non-perfused rat liver 5μm cryosections.

<p>(<b>A</b>) ATP8A1. (<b>B</b>) Aminopeptidase-N localized on the canalicular membrane (<b>C</b>) Colocalization of ATP8A1 and aminopeptidase-N illustrates the expression of ATP8A1 at canalicular membrane of rat hepatocytes. Scale bar: 20 μm</p

Comparison of rat cLPM proteomics with previous rat liver and brush border membrane proteomic studies.

<p>A: Representation of unique and overlapping proteins between rat cLPM compared with previous proteomics studies conducted on rat liver: cLPM this study, total [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158033#pone.0158033.ref024" target="_blank">24</a>], microsomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158033#pone.0158033.ref025" target="_blank">25</a>], plasma membrane [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158033#pone.0158033.ref026" target="_blank">26</a>] B: Representation of unique and overlapping proteins between rat cLPM and brush broader membrane proteome: cLPMthis study, kidney brush border membrane [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158033#pone.0158033.ref027" target="_blank">27</a>], total [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158033#pone.0158033.ref024" target="_blank">24</a>], plasma membrane[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158033#pone.0158033.ref026" target="_blank">26</a>].</p