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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Mol Oral Microbiol. 2021 Jun 15;36(4):243–253. doi: 10.1111/omi.12346

Contribution of Adhesion Proteins to Aggregatibacter actinomycetemcomitans Biofilm Formation

David R Danforth 1, Marcella Melloni 1, Jake Tristano 1, Keith P Mintz 1,*
PMCID: PMC8349852  NIHMSID: NIHMS1711553  PMID: 34085776

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 non-fimbrial 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 non-fimbriated 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 non-fimbrial adhesins, which is dependent on the genetic background of the strain investigated.

Keywords: Biofilm, Adhesins, Autotransporter proteins, Periodontitis

Introduction

Bacterial biofilms are initiated by reversible interactions with host surfaces followed by irreversible attachment and subsequent growth (Berne et al., 2015; Kaplan et al., 2003; Tuson & Weibel, 2013). Both types of interactions are typically mediated by surface proteins/structures, which emanate from the outer membrane of Gram-negative organisms. Fimbriae are fiber-like structures associated with adhesion and consist of homopolymers of small protein subunits extending microns from the surface, (Figurski et al., 2013; Giltner et al., 2012). In addition to fimbriae, non-fimbrial adhesins, represented as single proteins or homotrimers radiating hundreds of nanometers from the bacterial surface, are also involved in generalized and specific interactions (Berne et al., 2015). In some instances, sugars are covalently associated with these protein moieties and are directly involved in the interaction (Tang & Mintz, 2010). Alternatively, these post-translational modifications may contribute to the three-dimensional folding of the adhesin to form the specific structures required for binding (Tang et al., 2012). Both fimbriae and non-fimbrial adhesins target either a single substrate or bind to multiple disparate macromolecules, as well as participating in biofilm formation and invasion of mammalian cells (Danforth et al., 2019; Fine et al., 1999; Fine et al., 2005; Mintz, 2004).

The biofilm formed by the oral bacterium Aggregatibacter actinomycetemcomitans is considered to be exclusively directed by the bundle forming fimbriae produced by the bacterium (Figurski et al., 2013; Inoue, 2003; Kachlany, Planet, Desalle, Fine, Figurski, et al., 2001; Schreiner et al., 2003). However, the non-fimbrial adhesins of A. actinomycetemcomitans (EmaA, Aae, and ApiA/Omp100) have also been implicated in biofilm biogenesis (Cugini et al., 2018; Danforth et al., 2019; Nunes et al., 2016). All three of these characterized adhesins are classified as autotransporters, which encode all the information necessary to catalyze the transport of the protein(s) across the outer membrane (Sauri et al., 2009). For this class of secreted proteins, the carboxyl terminus (the translocator domain) integrates into the outer membrane and forms a protein pore which catalyzes the remaining protein (the passenger domain) through the pore; the complete autotransporter protein remains attached to the membrane via the translocator domain (Leyton et al., 2012). The passenger domain forms the functional moiety of the adhesin and can be either monomeric or homotrimeric (Roggenkamp et al., 2003).

The extracellular matrix protein adhesin A (EmaA) is a trimeric autotransporter exclusively associated with A. actinomycetemcomitans and forms antenna-like structures, as observed in whole mount negatively stained bacterial preparations by electron microscopy (Ruiz et al., 2006). EmaA is the largest of the characterized adhesins and extends at least 150 nm from the cell surface (Yu et al., 2009). Two serotype-dependent homologs have been identified= with monomeric molecular masses of 202 or 179 kDa (Tang et al., 2007). Both species form appendages that function as collagen adhesins and participate in biofilm formation (Danforth et al., 2019; Tang & Mintz, 2010; Tang et al., 2012), and the 202 kDa species is glycosylated (Tang et al., 2012).

The A. actinomycetemcomitans epithelial cell adhesin (Aae) is a monomeric autotransporter protein (the structure of which has not been determined) with a mass of 130 kDa. A variable number of 45 amino acid repeats are present in the sequence, which are strain dependent but independent of the serotype (Rose et al., 2003; Tang et al., 2007). The significance of the number of these repeats is unknown. The role of Aae in biofilm formation is disputed (Fine et al., 2005; Nunes et al., 2016).

ApiA /Omp100, another trimeric autotransporter with a monomeric molecular mass of 37,000 kDa, is predicted to form structures similar to the YadA protein of Yersinia entercolitica, the prototype of this family of proteins (Asakawa et al., 2003; Komatsuzawa et al., 2002). ApiA/Omp100 is a multifunctional protein associated with collagen binding, epithelial cell invasion and resistance to serum killing (Asakawa et al., 2003; Yue et al., 2007).

We have observed residual biofilm formation in strains lacking both fimbriae and EmaA in our recent study (Danforth et al., 2019). An overlap or redundancy in function of either Aae or ApiA/Omp100 may explain these results. To address this hypothesis, we generated single and double mutant strains to investigate the contribution of EmaA, ApiA/Omp100 and Aae to biofilm formation. In strains expressing fimbriae, the absence of ApiA/Omp100 and/or Aae did not impact biofilm formation. In the absence of fimbriae and EmaA, only Aae mediated biofilm formation. ApiA/Omp100 does not appear to contribute to biofilm formation in A. actinomycetemcomitans. However, when aae and apiA/omp100 were expressed in E. coli, both strains demonstrated comparable biofilm formation, but to a lesser amount compared with the strain expressing emaA. The data suggest that the contribution of EmaA and Aae to biofilm formation is highly dependent on the genetic background of the strains expressing these adhesins.

Materials and Methods

Bacterial strains and growth conditions.

Bacterial strains and plasmids used in this study are listed in Table 1. All A. actinomycetemcomitans strains were grown from frozen stocks on solid TSBYE medium (3% tryptic soy broth, 0.6% yeast extract, 1.5% agar; Beckton Dickinson, Franklin Lakes, NJ) in a humidified 10% CO2 atmosphere at 37°C. A single colony was used as the inoculum for all experiments. Fimbriated strains were grown on solid medium, and non-fimbriated strains were grown statically in TSBYE broth. Plasmids were maintained in A. actinomycetemcomitans strains by incorporation of 1.0 μg/ml chloramphenicol in the growth medium. Escherichia coli strains were grown in LB medium (1.0% tryptone, 0.5% yeast extract, 0.5% NaCl; Beckton Dickinson) at 37°C in ambient air with agitation. Strains containing plasmids were maintained at the following antibiotic concentrations: 100 μg/ml ampicillin; 20 μg/ml chloramphenicol; 50 μg/ml spectinomycin; or 50 μg/ml kanamycin.

Table 1.

Bacterial Strain or Plasmid

Name Description Reference or Source
E. coli
DH5αλpir endA1 hadR17(r− m+) supE44 thi-1 recA gyrA1(Nalr) relA1 Δ(lacIZYA-argF) U169 deoR (φ80dlacΔ(lacZ)M15) λ pir, for shuttle plasmids (Babic et al., 2008)
XL10G endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 tetR F’[proAB lacIqZΔM15 Tn10(TetR Amy CmR)] Agilent Technologies
KM482 DAP auxotroph strain with chromosomal λ pir, for conjugation Andrew Goodman, Yale
BL21-DE3 F– ompT gal dcm lon hsdSB(rB–mB–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λS) lab strain
A. actinomycetemcomitans
VT1257 Fimbriated clinical isolate, serotype b Maria Saarela, IDH, Finland
KM667 Isogenic emaA mutant of VT1257, Specr (Danforth et al., 2019)
KM871 Isogenic aae mutant of VT1257, Kanr This study
VT1518 Fimbriated clinical isolate strain HK1651, serotype b lab strain
VT1519 Non-fimbriated variant of VT1518 (Danforth et al., 2019)
KM872 Isogenic aae mutant of VT1519, Kanr This study
KM873 Isogenic apiA mutant of VT1519, Kanr This study
VT1281 Non-fimbriated laboratory strain, serotype a ATCC 29523
VT1568 Isogenic aae mutant of VT1281, Kanr (Rose et al., 2003)
KM874 Isogenic apiA mutant of VT1281, Kanr This study
KM397 Non-fimbriated laboratory strain, serotype c ATCC 33384
KM875 Isogenic aae mutant of KM397, Kanr This study
KM876 Isogenic apiA mutant of KM397, Kanr This study
KM662 Fimbriated clinical isolate strain CU1000N, serotype f (Fine et al., 2005)
KM847 Non-fimbriated variant of CU1000N This study
KM870 Isogenic aae mutant of NFC, Kanr This study
Plasmids
pGEM-T Easy TA cloning vector, Ampr Promega
pET28a N terminal 6xHIS protein expression vector. Novagen
pKM02 E. coli and A. actinomycetemcomitans shuttle vector, Cmr (Gallant et al., 2008)
pKM11 pKM2 containing full-length serotype b emaA (Tang & Mintz, 2010)
pKM811 pKM2 containing serotype b aae This study
pKM813 pKM2 containing serotype b apiA This study
pVT1460 mobilizable plasmid, Kanr, requires chromosomal pir (Mintz et al., 2002)
pVT1461 mobilizable plasmid, Specr, requires chromosomal pir (Mintz et al., 2002)
pKM827 apiA mutant construct on mobilizable plasmid, Kanr This study
pKM848 aae mutant construct on mobilizable plasmid, Kanr This study
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Generation of mutant strains.

Non-fimbriated A. actinomycetemcomitans mutant strains were obtained by serial passaging as described (Danforth et al., 2019).

Generation of aae mutants were designed as follows. First, the aae gene was PCR amplified from serotype b genomic DNA using the primers aae_insert_fwd (5`-ACTGAAACCTTTCCGCTATTCTG-3`) and aae_insert_rev (5`-GGACCAGTAGTAATTCAGTTTTACACC-3`). The 2,654 bp nucleic acid fragment was gel purified, cloned into pGEM T-Easy (Promega, Madison, WI) and verified by sequencing at the Advanced Genome Technologies Core facility at the University of Vermont. To create insertional aae mutants, the DNA was restriction endonuclease digested at two internal BglII sites (New England Biolabs) and the spectinomycin aad9 resistance gene (Lukomski et al., 2000) was inserted into the aae fragment, resulting in a 474-bp internal deletion. The spectinomycin-resistant gene fragment was excised with EcoRI, gel purified, and ligated with the mobilization plasmid pVT1460, as described previously (Mintz, 2004). This plasmid was transformed into a diaminopimelic acid-deficient (DAP) strain of E. coli (Babic et al., 2008), which was used as the donor strain for conjugation with A. actinomycetemcomitans. Transconjugants were selected on TSBYE-spectinomycin agar plates, patched to TSBYE-kanamycin to determine sensitivity, and verified by colony PCR and immunoblot analyses (Rose et al., 2003).

An aae/emaA double mutant was developed by inactivation of aae in the emaA mutant strain by integration of the plasmid into the chromosome via homologous recombination (aae is transcribed as a single gene (Rose et al., 2003)). A 477 bp fragment of aae was isolated by EcoRI digestion of the pGEM plasmid and ligated into a kanamycin-resistant modification of the mobilization plasmid described above. To enable selection in the DAP E. coli strain, the spectinomycin aad9 resistance gene was blunt-end cloned into the BstZ17I site on the backbone of the mobilization plasmid containing the aae fragment. The plasmid was transformed into the DAP-deficient donor strain and conjugated with A. actinomycetemcomitans. The transconjugants were verified to be aae mutants via colony PCR and immunoblot analysis, as described above. Both aae mutant variants behaved similarly and displayed no observable differences in growth characteristics or phenotype.

apiA/omp100 mutants were generated by integration of the plasmid by homologous recombination of an internal fragment of the gene, since apiA/omp100 is not part of an operon. A 342 bp internal fragment of the gene was PCR amplified using the primers apiAinsert_revEcoRI (5`-TAAGAATTCCGGTAGATCTCACATCTACAT-3`) and apiAinsert_fwdEcoRI (5`-CGGGAATTCGACTCTTTTCTGG-3`), corresponding to nucleic acids 79 to 279. The fragment was TA cloned into pGEM T-Easy prior to processing as described above for the aae/emaA double mutant construct. No observable growth defects or unexpected phenotypic changes from the parental strains were observed for any of the mutant strains.

Construction of adhesin expression plasmids.

The emaA gene complementation plasmid was generated as described previously (Tang & Mintz, 2010).

The aae expression plasmid was accomplished by PCR amplification of the gene and its endogenous promoter from serotype b (VT1169) genomic DNA using the primers aae5 (5`-CAGAACCACAACCAGTACCAGCACAC-3`) and aae3 (5`- GCAGAAGTGAGTTATTCATCG-3`). The PCR product was ligated into pGEM-T Easy, digested with NotI, and subsequently gel purified. The fragment was ligated into the shuttle plasmid pKM02 treated with the same enzyme (Gallant et al., 2008). After transformation into E. coli or A. actinomycetemcomitans, Aae expression was detected by immunoblotting (see below) .

The apiA/omp100 expression plasmid was generated by amplification of VT1257 genomic DNA using the primers omp100-fp (5`-GTCGTTTTGGCAGGTGTTTT-3`) and omp100-rp (5`-CCTTAAACCGCACCGAATTA-3`) and the product was ligated into pGEM-T Easy. The endogenous promoter sequence and apiA/omp100 gene was digested from the plasmid using EcoRI and gel purified before ligation into pKM02. Protein expression was detected by immunoblotting as described below.

Biofilm assay.

Biofilm assays were based on the method of Merritt et al. (Merritt et al., 2005). A. actinomycetemcomitans strains were initially grown from frozen stocks on TSBYE agar. For fimbriated strains, bacteria were collected by scraping an agar plate in the presence of 1.5 ml of TSBYE. The cell suspension was vortexed rigorously for 30 seconds and the tube incubated for 10 minutes to allow large clumps to settle to the bottom. A portion of the suspension was removed, and the concentration adjusted to absorbance of 0.05 at a wavelength of 495 nm. Two hundred microliters of each strain were inoculated into sterile 96-well microtiter plates (Nunc, Roskilde, Denmark) and grown for 72 hours. After 24 and 48 hours 100 μl of spent media was removed and replaced with fresh media to allow for continuous growth.

For non-fimbriated strains, single colonies were inoculated into 5 ml of TSBYE and grown overnight. The cultures were diluted 1:10 and grown to an absorbance of 0.3 at a wavelength of 495 nm. A 100 μl aliquot of a 1:1000 dilution was added to the 96-well plates and the cultures were grown for 24 hours.

Following the growth interval, supernatants were aspirated, and the remaining non-adherent cells were removed by three consecutive washes with phosphate buffered saline (PBS, 136.9 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.46 mM KH2PO4, 0.46 mM MgCl2, pH 7.4). Biofilms were stained with 0.1% crystal violet in water for 20 minutes, washed three times with PBS and solubilized using a 2:1 solution of water:glacial acetic acid. The relative biofilm mass of each strain was quantified by absorbance at 562 nm using an ELx800 plate reader (Biotek, Winooski, VT). A two-tailed Student’s t-test was used to identify significant differences (P < 0.05). A minimum of three independent experiments was performed for each strain.

Confocal microscopy.

Fimbriated A. actinomycetemcomitans biofilms were grown in glass-bottom petri dishes (MatTek, Ashland, MA) as described above for 24 hours. After growth, the supernatants were removed by aspiration, and nonadherent cells were removed by three washes with Tris-buffered saline (TBS; 20 mM Tris, 150 mM NaCl [pH 7.4]; Sigma). Biofilms were stained with SYTO9 (Invitrogen, Carlsbad, CA) in TBS at 5 μM for 30 min. The staining solution was aspirated, and any unbound stain was removed by four washes with TBS. Images were recorded at the University of Vermont Microscopy Imaging Center using a Nikon A1R-ER laser scanning confocal microscope (Nikon, Tokyo, Japan) with a plan-apochromat 60x objective and an excitation wavelength of 488 nm. Random fields were selected, Z-slices were acquired at increments of 0.37 μm and Z-stacks were generated. Stack sizes depended on biofilm depth and ranged from 5.2 to 40 μm. Surface coverage of the biofilm was determined using the particle analysis function of the ImageJ program (Schneider et al., 2012). Shape, volume, and surface area of individual microcolonies were quantified using the Volocity software package (PerkinElmer, Waltham, MA). All experiments were performed in triplicate. For comparisons between strains, a Student’s t-test was used, with significance defined as P < 0.1.

Antibody development.

Immunological reagents for the detection of Aae (Rose et al., 2003) and EmaA (Tang et al., 2007) were developed as described previously.

Antibodies specific for ApiA/Omp100 were designed corresponding to amino acids 27–214 (nucleotides 81–645, GenBank accession no. AB064943). The predicted protein sequence does not contain the putative amino terminal signal peptide or the carboxyl translocator domain. The replicon was amplified with ApiAFwd (5`-GGATCCGACTCTTTTCTGG-3`) and ApiARvr (5`-AAGCT TGACTCGGCTATCTAATCG-3`) using serotype b genomic DNA as the template and directionally cloned into the pET28-A expression plasmid (Novagen); the fidelity of the sequence was verified by sequencing at the Advanced Genome Technologies Core facility at the University of Vermont. The plasmid was transformed into the E. coli expression strain BL21-DE3 for auto-induction and peptide purification (Smith et al., 2015). ApiA/Omp100 antisera was subsequently developed in rabbits (Cocalico Biologics) by immunization with the purified protein. The immunoglobulin fraction was concentrated by precipitation using 50% ammonium sulphate and stored in 50% glycerol at −20°C. The antibodies were affinity purified (Robinson et al., 1988).

Detection of autotransporter adhesins.

Bacterial membranes were isolated via a modified protocol from (Tang et al., 2007). Harvested cells were grown to mid-log phase (OD495 = 0.3 for A. actinomycetemcomitans and OD600 = 0.3 for E. coli), washed with TBS and pelleted. The pellet was resuspended in 10 mM HEPES pH7.4 containing 10 mM EDTA, 0.1 mM PMSF and additional protease inhibitors (Thermo Fisher). Cells were lysed by three freeze/thaw cycles followed by centrifugation at 8,000 x g and the outer membrane fragments were collected by centrifugation at 100,000 x g for 30 minutes. The membranes were resuspended in 2% sarcosine in 10 mM HEPES and centrifuged to separate the inner from outer membrane proteins (Mintz, 2004).

Protein concentrations were spectrophotometrically determined by absorbance at 280 nm, and equivalent amounts of protein were applied to 4–15% polyacrylamide-SDS gels. Separated proteins were transferred to nitrocellulose under electrical current (120 mA, 160 min, 4°C).

Membranes were probed with primary antibodies at a concentration of 1.0 μg/ml (EmaA), or a dilution of 1:20,000 (Aae) or 1:10,000 (ApiA/Omp100). The immune complexes were detected with a dilution of either horseradish peroxidase-conjugated goat anti-mouse IgG for EmaA detection, or goat anti-rabbit IgG for ApiA/Omp100 and Aae detection (Jackson Laboratory, Bar Harbor, ME). Signal was visualized on film (USA Scientific, Ocala, FL) after incubation with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL).

Results

Information regarding the involvement of non-fimbrial adhesins in A. actinomycetemcomitans biofilm formation is scant and in some cases contradictory. Our previously published data suggests a role for EmaA in biofilm formation (Danforth et al., 2019). However, there are contradicting reports in the literature of the involvement of Aae in biofilm development (Fine et al., 2005; Nunes et al., 2016), while little information is available about the role of ApiA/Omp100 in this process. Some data indicates that ApiA/Omp100 contributes to biofilm formation when expressed in an E. coli background (Cugini et al., 2018), but no information is available about the role of this adhesin when expressed in A. actinomycetemcomitans. Therefore, the contribution of these three outer membrane autotransporter proteins to biofilm formation was investigated in this study.

The contribution to biofilm development of Aae and ApiA/Omp100 were assessed by selective inactivation via targeted mutagenesis across multiple strains and serotypes. All mutations were confirmed at both the genomic and protein level by colony PCR and immunological staining, respectively (Figs. 1A and 2A).

Figure 1.

Figure 1.

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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.

Figure 2.

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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).

The effect of EmaA inactivation on biofilm formation is much more pronounced in non-fimbriated strains (Danforth et al., 2019), therefore this strain type was first investigated for any potential defects in biofilm formation. Inactivation of aae resulted in a significant reduction in the mass of the biofilm formed in all tested strains (Fig. 1B). However, we observed variations in the amount of residual biofilm remaining after aae inactivation. Strains ATCC33384 (serotype c, 40.2 ± 3.5%) and HK1651 (serotype b, 47.7 ± 3.8%) demonstrated an approximately 40% decrease in the amount of biofilm formed when compared to the non-fimbriated parent strain. This is in comparison with strains ATCC 29523 (serotype a, 98.7 ± 1.0%) and CU1000N (serotype f, 95.3 ± 2.0%), which formed very little biofilm. Near-wild type levels of biofilm mass was restored following complementation of these strains with a plasmid expressing aae under the control of the heterologous leukotoxin promoter (Fig. 1B). In contrast, inactivation of apiA/omp100 had little if any impact on biofilm formation when assayed in two different serotypes (a and b) (Fig. 2B). The data suggests that in non-fimbriated strains Aae, but not ApiA/Omp100, contributes to biofilm formation.

In fimbriated A. actinomycetemcomitans strains a different pattern of contribution to biofilm formation for the Aae outer membrane protein was observed. In a serotype b fimbriated strain (VT1257) it was previously determined that the inactivation of emaA resulted in a 15 ± 4.8% decrease in the amount of biofilm formed and a change in microcolony formation as determined by confocal microscopy ((Danforth et al., 2019), Fig. 3). In contrast, mutation of aae in the same fimbriated strain showed little if any change in the mass of the biofilm formed when compared with the parent strain (Fig. 3). Confocal microscopy of the microcolonies formed by the aae mutant strain did not show any difference in the volume or surface area of the microcolonies formed when compared with the fimbriated parent strain (Table 2), unlike that observed with EmaA (Danforth et al., 2019). The data suggests that EmaA has a more impactful contribution to the ability of fimbriated strains to form a biofilm than does Aae. ApiA/Omp100 mutants were not examined due to the lack of effect on biofilm formation as seen in non-fimbriated strains (Fig. 2B).

Figure 3.

Figure 3.

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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).

Table 2.

Quantification of biofilm architecture.

Avg ± SDa
Strain Biofilm Volume (μm3) Biofilm Surface Area (μm2) Surface / Volume (μm2 μm−3)
wild type 162591.03 ± 2841.01 598352.64 ± 11289.97 3.68
aae−b 151307.56 ± 103311.58 244688.95 ± 105473.46 1.62
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a:

The results are an average of three (3) separate experiments.

b:

No statistical significance, P < 0.1

The contradictory observation of the contribution of EmaA and/or Aae in biofilm formation in fimbriated versus non-fimbriated strains was investigated in a single serotype b (HK1651) strain. The loss of fimbriation results in 86.2 ± 0.8% reduction in 24-hour biofilm formation when compared with the wildtype strain; a similar reduction was observed using the fimbriated strain VT1257 (90.0 ± 1.5%). A spontaneous non-fimbriated variant was used to generate single and double mutants in emaA and aae. In this strain, inactivation of emaA resulted in approximately a 50% decrease in biofilm formation (Fig. 4) and a similar decrease was observed when aae was disrupted (47.7 ± 3.8%). The emaA/aae double mutant strain displayed a 95.5 ± 0.7% decrease in biofilm mass formed when compared with the control strain (Fig. 4).

Figure 4.

Figure 4.

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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).

The relative contribution of ApiA, EmaA, and Aae in biofilm formation were individually examined by expression in a laboratory strain of E. coli DH5α and assessed for the ability of this typically non-biofilm forming strain to form a biofilm. All three proteins were shown to be associated with the outer membrane fraction of the bacterium (Fig. 5A). Transformation of each individual adhesin-expressing plasmid resulted in E. coli strains that formed superior biofilms compared with the parent E. coli strain (Fig. 5B). Strikingly, the strain transformed with emaA formed the greatest amount of biofilm compared to the laboratory E. coli strain, (68 ± 9.0 fold greater), while the strains transformed with aae or apiA were 12.0 ± 1.8 and 13.8 ± 1.0 fold greater, respectively.

Figure 5.

Figure 5.

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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).

Discussion

The contribution of the individual non-fimbrial adhesins to A. actinomycetemcomitans biofilm formation was investigated in both fimbriated and non-fimbriated strains. Fimbriated strains are typically isolated from the oral cavity of individuals harboring A. actinomycetemcomitans (Figurski et al., 2013; Fine et al., 2019), where the fimbriae play the predominant role in adhering to abiotic surfaces (Figurski et al., 2013; Kachlany, Planet, DeSalle, Fine, & Figurski, 2001). The non-fimbrial adhesins play a role in adhesion to specific substrates, such as epithelial cells (Aae, ApiA/Omp100) (Asakawa et al., 2003; Fine et al., 2005) or extracellular matrix proteins (e.g., collagen (EmaA, ApiA/Omp100) (Asakawa et al., 2003; Tang & Mintz, 2010) or laminin (Alugupalli et al., 1996)). However, as we have shown previously (Danforth et al., 2019) and demonstrated in this study, EmaA contributed to biofilm formation in serotype b (VT1257) fimbriated cells, whereas the two other adhesins did not participate in this process. This suggests that in this specific genetic background, neither Aae nor ApiA/Omp100 are relevant in generalized biofilm formation. Our results support the observation by Fine et al. (Fine et al., 2005), which indicate that Aae has little to no role in biofilm formation of a fimbriated serotype f strain (CU1000N), which is different from the strain used in this study.

In the absence of fimbriae, biofilm formation is dependent on the expression of two of the three adhesins investigated in this study, EmaA and Aae. The relative contribution of these two adhesins is strain dependent. Inactivation of aae in some strains demonstrated a partial reduction in biofilm mass, whereas other strains demonstrated very little biofilm formation. The variability in biofilm formation in these strains after inactivation of aae is dependent on the expression of emaA. The strains that demonstrated only partial reduction express emaA, whereas the low biofilm producing aae mutant strains are identified as having an inactivated emaA and do not make a functional protein (Tang et al., 2007). Therefore, we conclude that Aae plays a role in biofilm formation in strains that do not express fimbriae, independent of EmaA expression. The data presented in this study support the observation of Nunes et al. (Nunes et al., 2016), which demonstrated a reduction of biofilm formation in a non-fimbriated background; we have also observed a decrease in biofilm formation of the same strain following inactivation of aae (data not shown). Additionally, we demonstrated that Aae does indeed play a role in the biofilm development of a non-fimbriated CU1000N variant.

Strains with an inactivation of either emaA or aae in the non-fimbriated background still maintain the ability to form a biofilm, although substantially reduced compared to the fimbriated parent strain. This suggests a redundancy in function between these two adhesins, and each appears to contribute equally to biofilm formation. Yet, it is interesting that the inactivation of aae in the fimbriated strains does not influence biofilm development when compared with inactivation of emaA. Additionally, confocal microscopy revealed alterations to microcolony formation in fimbriated strains lacking EmaA when compared to the parent strain (Danforth et al., 2019), whereas no changes were apparent in the strains lacking Aae. These observations may be attributed to the relative size and conformation of the adhesins in relation to fimbriae. The EmaA adhesin extends up to 150 nm from the surface of the bacteria and forms antennae-like structures (Ruiz et al., 2006), whereas Aae is closer to the bacterial surface and most likely forms a less linear, shorter, globular 3-dimensional structure (the structure of Aae has not yet been described). Therefore, Aae may have less access to interact with the surface in the presence of fimbriae.

The relative differences between EmaA and Aae in biofilm formation suggest a hierarchical order to the function of non-fimbrial adhesive molecules in A. actinomycetemcomitans. The principal difference between the adhesins is that the absence of EmaA structures reduces the biofilm mass of fimbriated strains, whereas the loss of Aae has no effect. Second, the loss of EmaA changes microcolony formation, whereas the absence of Aae does not. Third, EmaA potentiates a more robust biofilm formed by E. coli cells expressing EmaA compared with cells expressing Aae. This implies that EmaA, isolated from other A. actinomycetemcomitans proteins, has a stronger biofilm producing potential than Aae or ApiA/Omp100. The relative inability of Aae to form an EmaA-equivalent level biofilm when expressed in E. coli suggests that Aae may require a A. actinomycetemcomitans-specific surface factor(s) or post-translational modification of this adhesin to maximize biofilm potential.

Non-fimbrial adhesins play a role in A. actinomycetemcomitans biofilm formation. However, the relative contribution of these adhesins varies among strains and may diverge among strains of the same serotype, due to differences in cultivars and/or propagation conditions. Since the non-fimbriated strains in this study were generated by spontaneous mutation(s) in the flp operon over extended periods of time, changes at either the DNA and/or transcriptomic level may lead to alterations in other protein or carbohydrate components of the membrane. Taken together, the data presented in this study suggests that a hierarchical order of function of these protein adhesins in biofilm formation exists: fimbriae (the longest of the adhesins) makes primary contact with the surface, followed by increased aggregation of bacterial cells mediated by EmaA, followed by more efficient adherence to the surface by Aae and to a lesser (if any) extent by ApiA/Omp100.

Acknowledgements

We thank Teresa Ruiz for editorial and writing assistance. The research was supported by grant 5R01DE024554 from National Institutes of Health/National Institutes for Dental Craniofacial Research (NIH/NIDCR). Confocal microscopy was performed on a Nikon A1R-ER point scanning confocal supported by NIH award number 1S10OD025030–01 from the National Center for Research Resources.

This study was supported by a grant from the National Institutes of Health/National Institutes for Dental Craniofacial Research (5R01DE024554).

All authors listed contributed to this research and report no conflicts of interest.

This study is original and reflects the authors’ own research and analysis in a truthful and complete manner. Data available upon request.

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