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Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins.

Phillips NJ, Steichen CT, Schilling B, Post DM, Niles RK, Bair TB, Falsetta ML, Apicella MA, Gibson BW - PLoS ONE (2012)

Bottom Line: Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group.Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism.Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with (13)C(6)-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.

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Related in: MedlinePlus

Venn diagrams showing the numbers of differentially expressed proteins identified in Extracts 1–3 over the three biological replicates of the experiment, comparing biofilm to planktonic organisms.The three extraction conditions progressed from the least stringent (Extract 1, 30 mM Tris buffer) to the most stringent (Extract 3, 2% SDS) so as to capture as many different classes of proteins as possible. All proteins represented had quantitation p-values <0.05 and met the 1.5-fold cutoff threshold for differential expression. The diagrams show (A) all differentially expressed proteins, (B) upregulated proteins, and (C) downregulated proteins. Twenty-five of the 152 proteins represented in panel A exhibited variable expression trends across Extracts (see text).
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pone-0038303-g004: Venn diagrams showing the numbers of differentially expressed proteins identified in Extracts 1–3 over the three biological replicates of the experiment, comparing biofilm to planktonic organisms.The three extraction conditions progressed from the least stringent (Extract 1, 30 mM Tris buffer) to the most stringent (Extract 3, 2% SDS) so as to capture as many different classes of proteins as possible. All proteins represented had quantitation p-values <0.05 and met the 1.5-fold cutoff threshold for differential expression. The diagrams show (A) all differentially expressed proteins, (B) upregulated proteins, and (C) downregulated proteins. Twenty-five of the 152 proteins represented in panel A exhibited variable expression trends across Extracts (see text).

Mentions: The Venn diagram in Figure 4A shows the distribution of all 152 differentially expressed proteins according to the Extract(s) in which they were detected. The diagrams in Figures 4B and 4C separate out the 73 upregulated and 54 downregulated proteins, respectively. As indicated, a significant number of differentially expressed proteins (102 of 152) were detected in single extracts, whereas only 50 proteins were detected in multiple extracts. Of those proteins found in multiple extracts, 25 showed different expression trends in different extracts and were classified as variable. Complete listings of the upregulated, downregulated, and variable proteins are given in Supplementary Tables S5, S6, and S7, respectively.


Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins.

Phillips NJ, Steichen CT, Schilling B, Post DM, Niles RK, Bair TB, Falsetta ML, Apicella MA, Gibson BW - PLoS ONE (2012)

Venn diagrams showing the numbers of differentially expressed proteins identified in Extracts 1–3 over the three biological replicates of the experiment, comparing biofilm to planktonic organisms.The three extraction conditions progressed from the least stringent (Extract 1, 30 mM Tris buffer) to the most stringent (Extract 3, 2% SDS) so as to capture as many different classes of proteins as possible. All proteins represented had quantitation p-values <0.05 and met the 1.5-fold cutoff threshold for differential expression. The diagrams show (A) all differentially expressed proteins, (B) upregulated proteins, and (C) downregulated proteins. Twenty-five of the 152 proteins represented in panel A exhibited variable expression trends across Extracts (see text).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368942&req=5

pone-0038303-g004: Venn diagrams showing the numbers of differentially expressed proteins identified in Extracts 1–3 over the three biological replicates of the experiment, comparing biofilm to planktonic organisms.The three extraction conditions progressed from the least stringent (Extract 1, 30 mM Tris buffer) to the most stringent (Extract 3, 2% SDS) so as to capture as many different classes of proteins as possible. All proteins represented had quantitation p-values <0.05 and met the 1.5-fold cutoff threshold for differential expression. The diagrams show (A) all differentially expressed proteins, (B) upregulated proteins, and (C) downregulated proteins. Twenty-five of the 152 proteins represented in panel A exhibited variable expression trends across Extracts (see text).
Mentions: The Venn diagram in Figure 4A shows the distribution of all 152 differentially expressed proteins according to the Extract(s) in which they were detected. The diagrams in Figures 4B and 4C separate out the 73 upregulated and 54 downregulated proteins, respectively. As indicated, a significant number of differentially expressed proteins (102 of 152) were detected in single extracts, whereas only 50 proteins were detected in multiple extracts. Of those proteins found in multiple extracts, 25 showed different expression trends in different extracts and were classified as variable. Complete listings of the upregulated, downregulated, and variable proteins are given in Supplementary Tables S5, S6, and S7, respectively.

Bottom Line: Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group.Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism.Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with (13)C(6)-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.

Show MeSH
Related in: MedlinePlus