Limits...
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.

Show MeSH

Related in: MedlinePlus

1D SDS-PAGE gels of mixed protein extracts (biofilm + planktonic) from one representative biological replicate.The four gels show (A) molecular weight markers, with molecular weights given in KDa, (B) the Extract 1 protein mixture, 40 µg loaded, (C) the Extract 2 protein mixture, 20 µg loaded, and (D) the Extract 3 protein mixture, 40 µg loaded. Bands were excised from the gel lanes as indicated by the numbered red circles.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368942&req=5

pone-0038303-g003: 1D SDS-PAGE gels of mixed protein extracts (biofilm + planktonic) from one representative biological replicate.The four gels show (A) molecular weight markers, with molecular weights given in KDa, (B) the Extract 1 protein mixture, 40 µg loaded, (C) the Extract 2 protein mixture, 20 µg loaded, and (D) the Extract 3 protein mixture, 40 µg loaded. Bands were excised from the gel lanes as indicated by the numbered red circles.

Mentions: While the 2D SDS-PAGE gel approach allowed us to initially assess overall protein expression changes and to establish incorporation efficiency of the heavy labeled 13C6-Arg, for a more comprehensive, in-depth investigation of the N. gonorrhoeae proteome, we subsequently chose a 1D SDS-PAGE approach. This approach also allowed better separation and proteomic coverage of membrane proteins, as 2D gels are known to be problematic with regard to hydrophobic proteins. As shown in Figure 3, Extracts 1, 2, and 3 from each biological replicate were separated on 1D SDS-PAGE gels and ∼40–45 bands were excised contiguously from each gel lane. Following in-gel digestion with trypsin, proteolytic peptide mixtures obtained from each band were subjected to HPLC-MS/MS analysis.


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)

1D SDS-PAGE gels of mixed protein extracts (biofilm + planktonic) from one representative biological replicate.The four gels show (A) molecular weight markers, with molecular weights given in KDa, (B) the Extract 1 protein mixture, 40 µg loaded, (C) the Extract 2 protein mixture, 20 µg loaded, and (D) the Extract 3 protein mixture, 40 µg loaded. Bands were excised from the gel lanes as indicated by the numbered red circles.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038303-g003: 1D SDS-PAGE gels of mixed protein extracts (biofilm + planktonic) from one representative biological replicate.The four gels show (A) molecular weight markers, with molecular weights given in KDa, (B) the Extract 1 protein mixture, 40 µg loaded, (C) the Extract 2 protein mixture, 20 µg loaded, and (D) the Extract 3 protein mixture, 40 µg loaded. Bands were excised from the gel lanes as indicated by the numbered red circles.
Mentions: While the 2D SDS-PAGE gel approach allowed us to initially assess overall protein expression changes and to establish incorporation efficiency of the heavy labeled 13C6-Arg, for a more comprehensive, in-depth investigation of the N. gonorrhoeae proteome, we subsequently chose a 1D SDS-PAGE approach. This approach also allowed better separation and proteomic coverage of membrane proteins, as 2D gels are known to be problematic with regard to hydrophobic proteins. As shown in Figure 3, Extracts 1, 2, and 3 from each biological replicate were separated on 1D SDS-PAGE gels and ∼40–45 bands were excised contiguously from each gel lane. Following in-gel digestion with trypsin, proteolytic peptide mixtures obtained from each band were subjected to HPLC-MS/MS analysis.

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