Transcriptional and translational analysis of biofilm determinants of Aggregatibacter actinomycetemcomitans in response to environmental perturbation

Abstract

Fimbriae, lipopolysaccharide (LPS), and extracellular polymeric substance (EPS) all contribute to biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans. To understand how individual biofilm determinants respond to changing environmental conditions, the transcription of genes responsible for fimbria, LPS, and EPS production, as well as the translation of these components, was determined in rough (Rv) and isogenic smooth (Sv) variants of A. actinomycetemcomitans cultured in half-strength and full-strength culture medium under anaerobic or aerobic conditions, and in iron-supplemented and iron-chelated medium. The transcription of tadV (fimbrial assembly), pgaC (extracellular polysaccharide synthesis), and orf8 or rmlB (lipopolysaccharide synthesis) was measured by real-time PCR. The amounts of fimbriae, LPS, and EPS were also estimated from stained sodium dodecyl sulfate-polyacrylamide gels and verified by Western blotting and enzyme-linked immunoadsorbent assay using specific antibodies. Each gene was significantly upregulated in the Rv compared to in the Sv. The transcription of fimbrial, LPS, and EPS genes in the Rv was increased approximately twofold in cells cultured in full-strength medium under anaerobic conditions compared to that in cells cultured under aerobic conditions. Under anaerobic conditions, the transcription of fimbrial and EPS enzymes was elevated in both Rv and Sv cells cultured in half-strength medium, compared to that in full-strength medium. Iron chelation also increased the transcription and translation of all biofilm determinants compared to their expression with iron supplementation, yet the quantity of biofilm was not significantly changed by any environmental perturbation except iron limitation. Thus, anaerobic conditions, nutrient stress, and iron limitation each upregulate known biofilm determinants of A. actinomycetemcomitans to contribute to biofilm formation.

FIG. 1.

Phenotype comparison. (I) Fimbrial expression. Standardized fimbrial preparations from the Rv (D7R) and the Sv (D7S) of strain D7 grown anaerobically in 1× TSBY and separated by SDS-PAGE on 14% gels. A, silver stain; B, Western blot probed with anti-D7R-flp1 peptide antibody; and C, ELISA with anti-D7R-flp1 peptide antibody. (II) EPS expression. Standardized EPS preparations from the Rv and Sv of strain D7 grown anaerobically in 1× TSBY and separated by SDS-PAGE on 12.5% gels. A, silver stain; B, Western blot probed with anti-CifA antibody; and C, ELISA with anti-CifA antibody.

FIG. 2.

Effect of atmosphere conditions on fimbrial and EPS expression. (I) Fimbrial expression. Standardized fimbrial preparations from the Rv and Sv of strain D7 grown in 1× TBSY, either anaerobically (ana) or aerobically (aero), and separated by SDS-PAGE on 14% gels. A, silver stain; B, Western blot probed with anti-D7R-flp1 peptide antibody; and C, ELISA with anti-D7R-flp1 peptide antibody. (II) EPS expression. Standardized EPS preparations from the Rv and Sv of strain D7 grown in 1× TSBY, either anaerobically or aerobically, and separated by SDS-PAGE on 12.5% gels. A, silver stain; B, Western blot probed with anti-CifA antibody; and C, ELISA with anti-CifA antibody.

FIG. 3.

Effect of medium on fimbriae and EPS expression. (I) Fimbrial expression. Standardized fimbrial preparations from the Rv and Sv of strain D7 grown anaerobically, either in 1× or 0.5× TSBY, and separated by SDS-PAGE on 14% gels. A, silver stain; B, Western blot probed with anti-D7R-flp1 peptide antibody; and C, ELISA with anti-D7R-flp1 peptide antibody. (II) EPS expression. Standardized EPS preparations from the Rv and Sv of strain D7 grown anaerobically, either in 1× or 0.5× TSBY, and separated by SDS-PAGE on 12.5% gels. A, silver stain; B, Western blot probed with anti-CifA antibody; C, dot assay; and D, ELISA with anti-CifA antibody.

FIG. 4.

Effect of iron concentration on fimbrial and EPS expression. Standardized fimbrial and EPS preparations from the Rv of strain HK1651 grown anaerobically in iron-chelated, iron-normal, and iron-supplemented TSBY. EPS from S. aureus was used as a positive control. (I) RcpB (fimbria) expression. Fimbrial preparation separated by SDS-PAGE on a 14% gel. A, silver stain; B, Western blot probed with anti-RcpB antibody. (II) EPS expression. EPS preparation separated by SDS-PAGE on a 12.5% gel. A, silver stain; B, Western blot with anti-CifA antibody.

FIG. 5.

Effect of oxygen tension and nutrient stress on biofilm formation. A. actinomycetemcomitans D7 Rv (D7R) and Sv (D7S) strains grown under aerobic (Aer; microaerophilic) or anaerobic (Ana) conditions in 1× and 0.5× BHI media. Growth (A), biofilm (B), and biofilm/growth ratio (C). The biofilm assay was performed in triplicate.

FIG. 6.

Effect of iron on biofilm formation. A. actinomycetemcomitans strains D7 and HK1651 grown in BHI medium, BHI with iron supplement (Fe⁺), and BHI with iron chelation (Fe⁻). Growth (A), biofilm (B), and biofilm/growth ratio (C). Biofilm assay was performed in triplicate. *, significance (P < 0.001).