Utilization of Variant and Fusion Proteins To Functionally Map the Aggregatibacter actinomycetemcomitans Trimeric Autotransporter Protein ApiA

Abstract

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a causative agent of localized aggressive periodontitis. Critical to its infection process is the first and essential step of attachment, which is related to the coordinated functions of surface components comprised of proteins and extracellular polysaccharides. One such protein is the outer membrane trimeric autotransporter protein ApiA, a versatile virulence factor with numerous functions, including cell binding, invasion, serum resistance, autoaggregation, and induction of cytokine release. Here we report on the use of Escherichia coli strains expressing protein variants to define the separate functions ascribed to the N terminus and those related to the C terminus. Importantly, a hybrid protein that comprised the N terminus of trimeric ApiA and the β-barrel domain of monomeric autotransporter Aae was constructed, which allowed the expression of a monomer surface-exposed domain of ApiA. Functional and phenotypic analyses demonstrated that the C terminus of ApiA forms an independent domain that is crucial for general stability and trimer formation, which appears to be associated with autoaggregation, biofilm formation, and surface expression. Importantly, the results show that the monomeric form of the N-terminal passenger domain of ApiA, while surface exposed, is sufficient for binding to buccal epithelial cells; however, it is not sufficient to allow aggregation and biofilm formation, strengthening the importance of the role of trimerization in these phenotypes.

Keywords: autoaggregation; autotransporter; autotransporter proteins; biofilm; biofilms; buccal epithelial cell binding.

FIG 1

Schematic representation of ApiA and protein variants. The putative signal (white), passenger (black), and C-terminal (Cter) β-barrel (gray) domains of ApiA were identified using a number of sequence analysis and protein prediction algorithms. Plasmids were constructed to express proteins with a deletion in each region. WT, wild type; MM, molecular mass.

FIG 2

Autoaggregation assay of wild-type ApiA and protein variants. (A) Quantitative assessments of autoaggregation were made over a time course of 50 min. Strains with a defect in the C-terminal domain, ApiAΔ284-295 and ApiAΔ186-240, failed to autoaggregate and were indistinguishable from BL21(DE3) containing pET29a+ control strains. Variants with deletions in the passenger domain, ApiAΔ186-217, ApiAΔ186-229, and ApiAΔ28-185, retained the autoaggregation phenotype, similar to the autoaggregation displayed by the strain expressing wild-type ApiA. (B) Hybrid ApiA-Aae proteins were not capable of autoaggregation, as their aggregation ability was indistinguishable from that of the vector-only control. Represented here are the results for one biological replicate, with error bars representing the standard deviations for technical replicates.

FIG 3

Microscopic evaluation of aggregation. Wild-type ApiA and a variant with a deletion in the passenger domain (ApiAΔ186-217) formed tightly adherent clumps. The negative control (pET29b+, empty vector control) and a variant with a deletion in the C-terminal domain (ApiAΔ284-295) displayed no observable clumping. Magnifications, ×40. Bars, 10 μm.

FIG 4

Western blot analysis of ApiA and protein variants. To observe the potential formation of multimers by ApiA and the variant proteins, cell lysates treated under mild reducing conditions were run on SDS-PAGE gels, transferred to a PVDF membrane, and probed with an anti-ApiA monoclonal antibody directed against amino acids 55 to 69. (A) Wild-type ApiA and ApiAΔ186-217, with a deletion involving the passenger domain, retained the ability to form trimers, dimers, and monomers. The variant ApiAΔ284-295, with a deletion involving the C-terminal domain, could not form multimeric structures, as indicated by the presence of a single monomer band. (B) ApiAΔ186-229, with a deletion involving the passenger domain, retained the ability to form trimers, dimers, and monomers. ApiAΔ186-240, with a deletion involving the passenger domain and a portion of the C-terminal domain, as well as hybrid proteins ApiAΔ218-295+Aae-Cter and ApiAΔ186-295+Aae-Cter all lost the ability to multimerize. The unmarked lanes on the left contain molecular mass markers.

FIG 5

Immunofluorescence analysis of ApiA and protein variants to observe surface display. To observe if the recombinant proteins were capable of being expressed on the surface of E. coli, strains were probed with an anti-ApiA monoclonal antibody to detect surface-exposed ApiA and variant proteins. Wild-type ApiA was detected on the E. coli cell surface. Variant proteins with a deletion in the passenger domain (ApiAΔ186-217 and ApiAΔ186-229) and the hybrid protein ApiAΔ218-295+Aae-Cter could be presented on the cell surface. The ApiAΔ284-295 variant had lower immunofluorescence, indicative of less surface-displayed protein. The negative control consisting of E. coli with vector plasmid pET29a+ showed no fluorescence, indicating no protein surface display. (Insets) Enlargements of select cells from the images.

FIG 6

Biofilm formation by wild-type ApiA and protein variants. Biofilm formation was assayed in 96-well plates and observed macroscopically as well as quantitatively. (A) Biofilm formation was quantified with crystal violet staining and is represented as a percentage of ApiA biofilm formation. Error bars represent the standard deviations of the average biofilm formation from serially diluted cells. (B) Statistical analysis was performed using one-way ANOVA for statistical significance with a confidence interval of 5% (P < 0.05) and the Tukey-Kramer HSD test for pairwise comparisons. Taken together, the data indicate that the C-terminal domain from amino acids 230 to 295 and the ability to form a trimeric protein structure are involved in biofilm formation.

FIG 7

Buccal epithelial cell binding. Strains expressing either wild-type ApiA or the plasmid control (pET29b+) were incubated with freshly scraped human buccal epithelial cells. The protein variant ApiAΔ218-295+Aae-Cter displayed binding enhanced over that of the vector control (P = 0.005). Data are from experiments that were conducted three times in duplicate. Error bars represent standard deviations. Student's t test was performed (P = 0.005).