Proteome variation among Filifactor alocis strains

Abstract

Filifactor alocis, a Gram-positive anaerobic rod, is now considered one of the marker organisms associated with periodontal disease. Although there was heterogeneity in its virulence potential, this bacterium was shown to have virulence properties that may enhance its ability to survive and persist in the periodontal pocket. To gain further insight into a possible mechanism(s) of pathogenesis, the proteome of F. alocis strains was evaluated. Proteins including several proteases, neutrophil-activating protein A and calcium-binding acid repeat protein, were identified in F. alocis. During the invasion of HeLa cells, there was increased expression of several of the genes encoding these proteins in the potentially more virulent F. alocis D-62D compared to F. alocis ATCC 35896, the type strain. A comparative protein in silico analysis of the proteome revealed more cell wall anchoring proteins in the F. alocis D-62D compared to F. alocis ATCC 35896. Their expression was enhanced by coinfection with Porphyromonas gingivalis. Taken together, the variation in the pathogenic potential of the F. alocis strains may be related to the differential expression of several putative virulence factors.

Figure 1

Quantitative PCR analysis of several putative virulence genes in F alocis. (A) 30-min mono- and coculture. (B) 45-min mono- and coculture. Hela cells were infected with F. alocis ATCC 35896 and D-62D strains [(MOI of 1:100(105 epithelial cells)] in mono or coculture with Porphyromonas gingivalis W83 as previously reported [18]. RNA was isolated at 30- and 45-min post infection using the SV total RNA isolation system (Promega). cDNA was made using the Transcriptor High Fidelity cDNA synthesis kit (Roche). Real-time PCR was performed using the Smart cycler (Cepheid) with gene-specific oligonucleotides. Caax-590 (HMPREF0389_00590); Caax-677 (HMPREF0389_00677); Xaa-1538, Xaa pro dipeptidase (HMPREF0389_01538); NAPA (neutrophil-acitivating factor protein A) (HMPREF0389_01654); Protease-00122 (HMPREF0389_00122); CBARP (calcium-binding acid repeat proteins) (HMPREF0389_01532); Protease La (Pr-La) (HMPREF0389_00279). Fold change was calculated using the formula. Fold change = 2 – ΔΔCt, where ΔΔCt = ΔCt of the sample –ΔCt of reference. (**p < 0.01; *p < 0.05).

Figure 2

PAGE of the F. alocis protein fractions from the type strain and D-62D. All the lanes show protein bands and their corresponding molecular masses (kDa). Each lane was loaded with 35 μg of protein. The prominent protein bands (shown by arrow) were excised and analyzed by MS. Lane 1: F. alocis ATCC 35896—extra cellular fraction. 103 kDa: calcium-binding acid repeat protein (HMPREF0389_01448); 97 kDa: conserved hypothetical protein (HMPREF0389_01431); 34 kDa: cobalt import ATP-binding protein (HMPREF0389_00901). Lane 2: F alocis D-62D-extra cellular fraction. 103 kDa: calcium-binding acid repeat protein (HMPREF0389_01448); 88.5 kDa: protease (HMPREF0389_00122); 67 kDa: S layer Y domain containing protein (HMPREF0389_00223). Lane 3: F alocis ATCC 35896—membrane fraction. 147 kDa: S layer Y domain containing protein (HMPREF0389_01139); 80 kDa: membrane protein (HMPREF0389_000638); 47 kDa: Caax protease (HMPREF0389_00590); 42 kDa: Xaa pro-dipeptidase (HMPREF0389_01538); 14 kDa: neutrophil-activating protein A (HMPREF0389_01654). Lane 4: Falocis D-62D— membrane fraction; 47 kDa: Caax protease (HMPREF0389_00590); 42 kDa: Xaa pro-dipeptidase (HMPREF0389_01538); 14 kDa: neutrophil-activating protein A (HMPREF0389_01654). Lane 5: F alocis ATCC 35896—cytosolic fraction. 68 kDa: fibronectin-binding protein (HMPREF 0389_00575); 42 kDa: Xaa pro-dipeptidase (HMPREF0389_01538); 14 kDa: neutrophil-activating protein A (HMPREF0389_01654). Lane 6: F. alocis D-62D—cytosolic fraction. 262 kDa: cell wall associated serine proteinase (HMPREF0389_01110); 35 kDa: electron transfer flavo-protein alpha (HMPREF0389_00742); 14 kDa: neutrophil-activating protein A (HMPREF0389_01654).

Figure 3

2D-PAGE of the membrane fraction of F. alocis ATCC 35896 strain. 2D page was performed using 7-cm IPG strips of pI 3–10 in Protean IEF cell and 30–50 μg of protein and electrophoresed at 200 V, 0.3 A for 4–5 h, and stained with Coomassie simply blue strain. A total of 50 distinct spots were identified and processed for MS/MS analysis.

Figure 4

2D-PAGE of the membrane fraction of F alocis D-62D strain. 2D page was performed using 7-cm IPG strips of pI 3–10 in Protean IEF cell and 30–50 μg of protein and electrophoresed at 200 V, 0.3 A for 4–5 h and stained with Coomassie simply blue strain. A total of 54 distinct spots were identified and processed for MS/MS analysis.

Figure 5

2D-PAGE of the extra-cellular fraction of F. alocis–ATCC-35896 strain. 2D page was performed using 7 cm IPG strips of pI 3–10 in Protean IEFcell and 30–50 μg of protein and electrophoresed at 200 V, 0.3 A for 4–5 h, and stained with Coomassie simply blue strain. A total of 55 distinct spots were identified and processed for MS/MS analysis.

Figure 6

2D-PAGE of the extra-cellular fraction of F. alocis D-62D strain. 2D page was performed using 7-cm IPG strips of pI 3–10 in Protean IEF cell and 30–50 μg of protein and electrophoresed at 200 V, 0.3 A for 4–5 h, and stained with Coomassie simply blue strain. A total of 60 distinct spots were identified and processed for MS/MS analysis.