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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

SILAC mixing scheme.The three protein extracts were prepared separately from the biofilm and planktonic organisms, and then mixed 1∶1 by protein concentration. The extraction conditions were as follows: Extract 1, sonication in 40 mM Tris; Extract 2, extraction in 8 M urea, 4% Chaps, 40 mM Tris, and 0.2% Bio-Lyte 3/10 ampholyte; and Extract 3, boiling for 10 min in 2% SDS.
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pone-0038303-g002: SILAC mixing scheme.The three protein extracts were prepared separately from the biofilm and planktonic organisms, and then mixed 1∶1 by protein concentration. The extraction conditions were as follows: Extract 1, sonication in 40 mM Tris; Extract 2, extraction in 8 M urea, 4% Chaps, 40 mM Tris, and 0.2% Bio-Lyte 3/10 ampholyte; and Extract 3, boiling for 10 min in 2% SDS.

Mentions: The biofilm and planktonic cells were then independently taken through three extraction steps to generate three separate protein extracts (Extracts 1–3) to both decrease the sample complexity for MS analysis and ensure coverage of even the most hydrophobic proteins (see Figure 2). The first extraction condition (sonication in 40 mM Tris buffer) released soluble proteins to give Extract 1. The subsequent extraction conditions were designed to solubilize membrane proteins (Extracts 2 and 3). On the basis of measured protein concentrations, the corresponding unlabeled (L) biofilm and 13C6-Arg-labeled (H) planktonic Extracts were mixed 1∶1 as shown in Figure 2. Thus, three mixed protein Extracts (L + H) were generated from one biological experiment. The entire protocol was repeated on three different occasions to give three biological replicates.


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)

SILAC mixing scheme.The three protein extracts were prepared separately from the biofilm and planktonic organisms, and then mixed 1∶1 by protein concentration. The extraction conditions were as follows: Extract 1, sonication in 40 mM Tris; Extract 2, extraction in 8 M urea, 4% Chaps, 40 mM Tris, and 0.2% Bio-Lyte 3/10 ampholyte; and Extract 3, boiling for 10 min in 2% SDS.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038303-g002: SILAC mixing scheme.The three protein extracts were prepared separately from the biofilm and planktonic organisms, and then mixed 1∶1 by protein concentration. The extraction conditions were as follows: Extract 1, sonication in 40 mM Tris; Extract 2, extraction in 8 M urea, 4% Chaps, 40 mM Tris, and 0.2% Bio-Lyte 3/10 ampholyte; and Extract 3, boiling for 10 min in 2% SDS.
Mentions: The biofilm and planktonic cells were then independently taken through three extraction steps to generate three separate protein extracts (Extracts 1–3) to both decrease the sample complexity for MS analysis and ensure coverage of even the most hydrophobic proteins (see Figure 2). The first extraction condition (sonication in 40 mM Tris buffer) released soluble proteins to give Extract 1. The subsequent extraction conditions were designed to solubilize membrane proteins (Extracts 2 and 3). On the basis of measured protein concentrations, the corresponding unlabeled (L) biofilm and 13C6-Arg-labeled (H) planktonic Extracts were mixed 1∶1 as shown in Figure 2. Thus, three mixed protein Extracts (L + H) were generated from one biological experiment. The entire protocol was repeated on three different occasions to give three biological replicates.

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