Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B

Figures & data

Figure 1. 1D SDS-PAGE of LDP solution (right well), visualized with Coomassie Brilliant Blue stain. Protein bands visible with the Coomassie stain were identified by LC-MS/MS and are listed below. Left well, High-range Rainbow molecular weight marker (bio-rad). 1. Transmembrane secretory component. 2. Serum albumin pre-proprotein, Ig alpha-1 chain C region and alpha-amylase 1 precursor. 3. Alpha-amylase 1 precursor, unnamed protein product with molecular weight 54,127 Da, hypothetical protein with molecular weight 53,228 Da, immunoglobulin alpha heavy chain variable region (partial) and immunoglobulin G heavy chain variable region (partial). 4. Alpha-amylase 1 precursor, Ig alpha-1 chain C region, immunoglobulin alpha-2 heavy chain and bactericidal/permeability-increasing protein 1. 5. Carbonic anhydrase 6 isoform 1 precursor, unnamed protein product with molecular weight 38,775 Da, zinc-alpha-2-glycoprotein precursor, alpha-1-antitrypsin precursor, kallikrein-1 preproprotein, alpha-amylase 1 precursor and protein LEG1 homolog precursor. 6. IgG kappa chain (partial), immunoglobulin light chain (partial), unnamed protein product with molecular weight 18,867 Da, immunoglobulin variable region (partial), immunoglobulin lambda light chain VLJ region (partial), cystatin-SN precursor, unnamed protein product with molecular weight 66,151 Da and parotid secretory protein. 7. Prolactin-inducible protein precursor, unnamed protein product with molecular weight 18,867 Da, cystatin-SN precursor and putative lipocalin 1-like protein 1. 8. Prolactin-inducible protein precursor, cystatin-SN precursor, cystatin-S precursor, cystatin D, cystatin-C precursor, cystatin-SA precursor, thioredoxin isoform 1, cystatin-B and cystatin-A

Table 1. Cellular functions associated with uniquely expressed proteins in planktonic and biofilm cells

Novel biofilm cell proteins, absent in planktonic cells
UniProt Acc No	Protein name	Abbreviation	Protein function
A8AZY0	Phosphoribosylamine–glycine ligase	PurD	Nucleotide metabolism; de novo purine biosynthesis.
A8AUA0	Phosphoribosylglycinamide formyltransferase	PurN	Nucleotide metabolism; de novo purine biosynthesis.
A8AZX7	N5-carboxyaminoimidazole ribonucleotide synthase	PurK	Nucleotide metabolism; de novo purine biosynthesis.
A8AUM3	Membrane dipeptidase (amylase binding protein)	AbpB	Proteolysis.
A8AXS5	Multiple sugar metabolism transmembrane permease F	MsmF	Transmembrane transport permease protein involved in multiple sugar metabolism (msm) mono- and disaccharide transport.
A8AWU6	Uncharacterized protein	SGO_0965	Suggested to be functionally associated with pdxK (SGO_0964) in S. gordonii DL1 based on gene neighborhood [26].
Planktonic cell proteins, absent in all biofilm cells
A8AUP5	Urease cluster protein	Urcl	Urease assembly and activation.
A8AWP5	3-isopropylmalate dehydratase, large subunit	LeuC	Amino acid biosynthesis; Branched amino acid biosynthesis; L-leucine biosynthesis.
A8AY98	Uncharacterized protein	SGO_1479	Suggested to be functionally associated with a GNAT family acetyltransferase (sgo_1481) in S. gordonii DL1 based on gene neighborhood [26].
Proteins uniquely absent in MUC5B biofilm cells
A8AXH7	β-glucoside operon antiterminator, BglG family	LicT	Inhibition of β-glucoside catabolite repression.

Figure 2. Comparison of proteomes in planktonic and biofilm cells (grown on uncoated and MUC5B- or LDP-coated surface). Urcl: Urease cluster protein, LeuC: 3-isopropylmalate dehydratase (large subunit), SGO_1479: Uncharacterized protein, PurD: Phosphoribosylamine–glycine ligase, PurN: Phosphoribosylglycinamide formyltransferase, PurK: N5-carboxyaminoimidazole ribonucleotide synthase, AbpB: Membrane dipeptidase (amylase binding protein), MsmF: Multiple sugar metabolism transmembrane permease, SGO_0965: Uncharacterized protein

Figure 3. Clustering analysis. Hierarchical cluster of z-scored LFQ intensities for proteins with significant difference in abundance (ANOVA, p < 0.05; Benjamini–Hochberg FDR) between the different growth conditions

Table 2. Protein abundances in MUC5B biofilms compared to LDP biofilms (p < 0.05, FC > 2)

Proteins more abundant in MUC5B biofilm cells compared to LDP biofilm cells
UniProt Acc No	Protein name	Abbreviation	Protein function	Fold change
A8AUR8	Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase	Endo	Hydrolysis of high-mannose glycopeptides and glycoproteins containing the -(Man(GlcNAc) [2])Asn-structure. Putative role in immune evasion.	2.7
A8AVM2	GCN5-related N-acetyltransferase (GNAT family)	GNAT	Protein regulation. Putative role in immune evasion.	2.2
A8AW57	Dephospho-CoA kinase	CoaE	Coenzyme A biosynthesis.	2.7
A8AUF3	Uncharacterized protein	SGO_0091	Putative ABC transporter, suggested to be functionally associated with transcriptional regulator tetracycline repressor (TetR) family (SGO_0090) in S. gordonii DL1 based on gene neighborhood and gene co-occurrence [26].	2.5
Proteins less abundant in MUC5B biofilm cells compared to LDP biofilm cells
A8AUC8	D-3-phosphoglycerate dehydrogenase, putative	Phgdh	Amino acid biosynthesis, L-serine biosynthesis.	−2.6
A8AYY1	SUF system FeS assembly protein, NifU family	NifU	Iron-sulfur cluster assembly.	−2.9
A8AZ18	Putative transcriptional regulator	SGO_1758	HTH-type transcriptional regulator.	−7.1
A8AZ65	Glutamate formimidoyltransferase	FtcD	Amino acid metabolism; Histidine catabolic process to glutamate and formamide.	−6.0
A8AZR0	UDP-glucose 4-epimerase BH3715	GALE	Carbohydrate metabolism; Galactose catabolism.	−3.0
A8AUX3	Uncharacterized protein	SGO_0264	Suggested to be functionally associated with Nac (SGO_0265) in S. gordonii DL1 based on gene neighborhood [26].	−2.2
A8AV65	Uncharacterized protein	SGO_0356	Suggested to be functionally associated with DegV (SGO_0357) in S. gordonii DL1 based on gene neighborhood and co-expression [26].	−2.6
A8AVX9	Uncharacterized protein	SGO_0636	-	−2.1
A8AWH3	Uncharacterized protein	SGO_0834	Suggested to be functionally associated with Nrd (SGO_0835) and PepV (SGO_0836) in S. gordonii DL1 based on gene neighborhood [26].	−3.2

Figure 4. Volcano plot representation of proteins in S. gordonii DL1 biofilm cells grown on salivary MUC5B-coated surface versus LDP-coated surface from LC-MS/MS data. The lines indicate arbitrary cut-off values for enrichment (fold change > 2) and significance (p < 0.05). Proteins with significant difference in abundance are indicated in the figure and listed in Table 2

Figure 5. S. gordonii DL1 and S. mutans UA159 biofilms on LDP- and MUC5B-coated Ibidi VI flow cell IbiTreat surfaces after 2 h of flow. Differences in % coverage were calculated between all four groups using one-way ANOVA, * = p < 0.05. The experiment was performed in triplicate using independent bacterial cultures

Willforss J, Chawade A, Levander F, et al. NormalyzerDE: online tool for improved normalization of omics expression data and high-sensitivity differential expression analysis. J Proteome Res. 2019 Feb 1;18(2):732–740.

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