Contribution of adhesion proteins to Aggregatibacter actinomycetemcomitans biofilm formation

Abstract

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with periodontal disease and multiple disseminated extra-oral infections. Colonization of these distinct physiological niches is contingent on the expression of specific surface proteins during the initiation of developing biofilms. In this investigation, we studied fimbriae and three well-characterized nonfimbrial surface proteins (EmaA, Aae, and ApiA/Omp100) for their contribution to biofilm formation. Mutations of these proteins in multiple strains covering four different serotypes demonstrated variance in biofilm development that was strain dependent but independent of serotype. In a fimbriated background, only inactivation of emaA impacted biofilm mass. In contrast, inactivation of emaA and/or aae affected biofilm formation in nonfimbriated A. actinomycetemcomitans strains, whereas inactivation of apiA/omp100 had little effect on biofilm formation. When these genes were expressed individually in Escherichia coli, all transformed strains demonstrated an increase in biofilm mass compared to the parent strain. The strain expressing emaA generated the greatest mass of biofilm, whereas the strains expressing either aae or apiA/omp100 were greatly reduced and similar in mass. These data suggest a redundancy in function of these nonfimbrial adhesins, which is dependent on the genetic background of the strain investigated.

Keywords: adhesins; autotransporter proteins; biofilm; periodontitis.

Figure 1.

Inactivation of aae and biofilm formation of non-fimbriated strains. A. Immunoblot analysis of outer membrane proteins for the presence or absence of Aae in serotype a strain ATCC 25923 (wild type), aae minus strain (mutant), and in trans complemented strain (complement). B. Impact of Aae inactivation or complementation on biofilm formation across multiple strains and serotypes. Wild type (black), aae minus (light gray), and aae in trans complement (dark gray). ATCC 33384, serotype c; HK1651, serotype b; ATCC 29523, serotype a; CU1000N, serotype f. Representative standard static biofilm assay of non-fimbriated strains, in triplicate. A minimum of three biological replicates were performed for each strain. The statistical significance, as compared to the wild type, is indicated (*, P < 0.05).

Figure 2.

Inactivation of apiA/omp100 and biofilm formation of non-fimbriated strains. A. Immunoblot of outer membrane proteins for presence or absence of ApiA/Omp100 in a serotype a (ATCC 29523) strain. ATCC 25923 (wild type), apiA/omp100 minus strain (mutant), and in trans complemented strain (complement). B. Impact on biofilm formation of apiA/omp100 inactivation in strains with different serotypes. Wild type (serotype a (ATCC 29523) and serotype b (HK1651)); strains with inactivated gene (apiA/omp100^-). Representative standard static biofilm assay of non-fimbriated strains, in triplicate. A minimum of three biological replicates were performed for each strain. No statistical significance determined (P < 0.05).

Figure 3.

Impact of inactivation of emaA and aae on biofilm formation of a fimbriated strain. Wild type (VT1257), emaA mutant (emaA⁻), aae mutant (aae⁻). Representative 72-hour static biofilm assay performed in triplicate. The statistical significance, as compared to the wild type, is indicated (*, P < 0.05).

Figure 4.

Impact of single and emaA and aae double mutants on biofilm formation by serotype b strain HK1651. A. Comparison of biofilm formation between wild type strain (fimbriated) and strain with a spontaneous mutation in the flp operon (non-fimbriated) after 24 hours. B. Representative assay comparing the biofilm formation between strains: flp mutant (non-fimbriated), emaA mutant (emaA⁻), aae mutant (aae⁻), and inactivation of both genes (emaA⁻/aae⁻). A minimum of three biological replicates were performed. The statistical significance, as compared to the wild type, is indicated (*, P < 0.05).

Figure 5.

Expression and biofilm formation of E. coli strain expressing emaA, aae and apiA/omp100. A. Outer membrane proteins from E. coli DH5α transformed with either emaA, aae, or apiA/omp100 were isolated and detected for expression using antibodies developed to the individual proteins. B. Representative static biofilm assay of the individual strains transformed with emaA, aae, or apiA/omp100 in triplicate. A minimum of three biological replicates were performed. The statistical significance, as compared to the wild type, is indicated (*, P < 0.05).