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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

Plot of the functional classifications of all upregulated and downregulated proteins in the biofilm compared to the planktonic growth form.All of the tabulated proteins met a 1.5-fold cutoff threshold for differential expression, with quantitation p-values <0.05. Functional role assignments for the N. gonorrhoeae proteins were downloaded from the JCVI-CMR website (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi). In a few cases where functional role assignments were not available, the N. gonorrhoeae proteins were given tentative assignments in the present study after conducting BLAST and KEGG orthology searches (see Supplementary Tables S5 and S6 for details).
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pone-0038303-g005: Plot of the functional classifications of all upregulated and downregulated proteins in the biofilm compared to the planktonic growth form.All of the tabulated proteins met a 1.5-fold cutoff threshold for differential expression, with quantitation p-values <0.05. Functional role assignments for the N. gonorrhoeae proteins were downloaded from the JCVI-CMR website (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi). In a few cases where functional role assignments were not available, the N. gonorrhoeae proteins were given tentative assignments in the present study after conducting BLAST and KEGG orthology searches (see Supplementary Tables S5 and S6 for details).

Mentions: Known functional roles (both main and sub-roles) for most of the N. gonorrhoeae proteins were available from the JCVI-CMR website (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi), and this information is given in Supplementary Tables S5, S6, and S7. Figure 5 shows a plot of the main functional roles of all of the upregulated and downregulated proteins that met an average fold change cutoff threshold of ≥1.5. As indicated, the major functional categories of upregulated proteins included cell envelope, cellular processes, energy metabolism, protein synthesis, and transport and binding proteins. The main functional categories of downregulated proteins were energy metabolism, protein fate, protein synthesis, and transport and binding proteins.


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)

Plot of the functional classifications of all upregulated and downregulated proteins in the biofilm compared to the planktonic growth form.All of the tabulated proteins met a 1.5-fold cutoff threshold for differential expression, with quantitation p-values <0.05. Functional role assignments for the N. gonorrhoeae proteins were downloaded from the JCVI-CMR website (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi). In a few cases where functional role assignments were not available, the N. gonorrhoeae proteins were given tentative assignments in the present study after conducting BLAST and KEGG orthology searches (see Supplementary Tables S5 and S6 for details).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038303-g005: Plot of the functional classifications of all upregulated and downregulated proteins in the biofilm compared to the planktonic growth form.All of the tabulated proteins met a 1.5-fold cutoff threshold for differential expression, with quantitation p-values <0.05. Functional role assignments for the N. gonorrhoeae proteins were downloaded from the JCVI-CMR website (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi). In a few cases where functional role assignments were not available, the N. gonorrhoeae proteins were given tentative assignments in the present study after conducting BLAST and KEGG orthology searches (see Supplementary Tables S5 and S6 for details).
Mentions: Known functional roles (both main and sub-roles) for most of the N. gonorrhoeae proteins were available from the JCVI-CMR website (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi), and this information is given in Supplementary Tables S5, S6, and S7. Figure 5 shows a plot of the main functional roles of all of the upregulated and downregulated proteins that met an average fold change cutoff threshold of ≥1.5. As indicated, the major functional categories of upregulated proteins included cell envelope, cellular processes, energy metabolism, protein synthesis, and transport and binding proteins. The main functional categories of downregulated proteins were energy metabolism, protein fate, protein synthesis, and transport and binding proteins.

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