Periodontitis is an inflammatory disease caused by polymicrobial biofilms. The periodontal pathogen Aggregatibacter actinomycetemcomitans displays two proteinaceous surface structures, the fimbriae and the nonfimbrial extracellular matrix binding protein A (EmaA), as observed by electron microscopy. Fimbriae participate in biofilm biogenesis and the EmaA adhesins mediate collagen binding. However, in the absence of fimbriae, A. actinomycetemcomitans still retains the potential to form robust biofilms, suggesting that other surface macromolecules participate in biofilm development. Here, isogenic mutant strains lacking EmaA structures, but still expressing fimbriae, were observed to have reduced biofilm potential. In strains lacking both EmaA and fimbriae, biofilm mass was reduced by 80%. EmaA enhanced biofilm formation in different strains, independent of the fimbriation state or serotype. Confocal microscopy revealed differences in cell density within microcolonies between the EmaA positive and mutant strains. EmaA-mediated biofilm formation was found to be independent of the glycosylation state and the precise three-dimensional conformation of the protein, and thus this function is uncorrelated with collagen binding activity. The data suggest that EmaA is a multifunctional adhesin that utilizes different mechanisms to enhance bacterial binding to collagen and to enhance biofilm formation, both of which are important for A. actinomycetemcomitans colonization and subsequent infection.

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontal disease, as well as other systemic diseases. The ability of the bacterium to bind collagen, the principal component of the extracellular matrix, is mediated by the extracellular matrix protein adhesin A (EmaA). EmaA forms antennae-like appendages on the bacterial cell surface that are comprised of three monomers. The functional domain, of approximately 30 nm in length, is located at the distal end of the adhesin and is subdivided into three subdomains (SI-SIII) [1]. Glycosylation of EmaA adhesins is critical for collagen binding, and it has been demonstrated that this post-translational modification exploits the same pathway as the O-polysaccharide (O-PS) of the lipopolysaccharide [2]. However, it is still unclear how glycosylation facilitates collagen binding. In this study we have analyzed the 3D structure of the functional domain of the EmaA adhesin from mutant strains with a disrupted glycosylation mechanism (rmlC and waaL mutants). The rmlC mutant strain lacks the rhamnose epimerase and the waaL mutant strain does not express the O-antigen ligase, WaaL, an essential component of the O-PS glycosylation pathway. Structural comparison of the glycosylated and non-glycosylated adhesins will help to determine if this modification promotes a structural conformation that is required for collagen binding.EmaA adhesins from both the rmlC and the waaL mutant strains were analyzed by electron tomography of whole-mount negatively-stained preparations of the bacteria as previously described for other strains [3]. Bacteria were adsorbed on carbon-coated grids pretreated with a colloidal gold solution and negatively stained with Nano W (2% methylamine tungstate). Tomographic single-axis tilt series were acquired over a ±64° angular range in 2° intervals with a calibrated 3.08 Å pixelsize at the specimen scale. Single-axis tilt series were processed using the IMOD processing software to generate tomograms. EmaA adhesins were selected from the tomograms by marking two points on their axis. For each selected adhesin, a tilt series of subprojections was extracted and subvolumes were calculated with the adhesin's axis approximately parallel to the Y-axis using algorithms in both Spider and EMIRA [4,5]. These subvolumes were visualized in Chimera [6] and further aligned to a reference subvolume of the wild-type EmaA [1]. Lower quality EmaA subvolumes were removed from further processing (<10% subvolumes removed). Probabilistic Principal Component Analysis (PPCAEM) implemented in EMIRA was used to assess differences between all the EmaA subvolumes and estimate the missing data [5,7]. The 3D reconstructions were divided into groups of similar structures. The subvolumes were further aligned to a reference selected from the volumes present in each group. Chimera [6] was used to generate an average subvolume for each group.The 3D electron microscopy analysis of the EmaA adhesins from the mutant strain suggests that glyco...

Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans, a Gram-negative oral pathobiont causing aggressive periodontitis and systemic infections, demonstrates serum resistance. We have identified a dsDNA-tailed bacteriophage, S1249, which was found to convert from this microorganism inducible by human serum into a lytic state to kill the bacterium. This phage demonstrated active transcripts when exposed to human serum: 20% of genes were upregulated more than 10-fold, and 45% of them were upregulated 5-10-fold when the bacterium was grown in the presence of human serum compared to without the presence of human serum. Transcriptional activation when grown in equine serum was less pronounced. This phage demonstrated a tail with inner rigid tubes and an outer contractile sheath, features of Myoviridae spp. Further characterization revealed that the lysogenized integration of the phage in the chromosome of A. actinomycetemcomitans occurred between the genes encoding cold-shock DNAbinding domain-containing protein (csp) and glutamyl-tRNA synthetase (gltX). Both phage DNA integrated lysogeny and nonintegrated pseudolysogeny were identified in the infected bacterium. A newly generated, lysogenized strain using this phage displayed similar attributes, including 63% growth inhibition compared to its isogenic phage-free strain when in the presence of human serum. Our data suggest that bacteriophage S1249 can be induced in the presence of human serum and enters the lytic cycle, which reduces the viability of infected bacteria in vivo.

Aggregatibacter actinomycetemcomitans , a causative agent of periodontitis and non-oral diseases, synthesizes a trimeric extracellular matrix protein adhesin A (EmaA) that mediates collagen binding and biofilm formation. EmaA is found as two molecular forms, which correlate with the serotype of the bacterium. The canonical protein (b-EmaA), associated with serotypes b and c, has a monomeric molecular mass of 202 kDa. The collagen binding activity of b-EmaA is dependent on the presence of O-polysaccharide (O-PS), whereas biofilm activity is independent of O-PS synthesis. The EmaA associated with serotype a strains (a-EmaA) has a monomeric molecular mass of 173 kDa and differs in the amino acid sequence of the functional domain of the protein. In this study, a-emaA was confirmed to encode a protein that forms antenna-like appendages on the surface of the bacterium, which were found to be important for both collagen binding and biofilm formation. In an O-PS-deficient talose biosynthetic (tld) mutant strain, the electrophoretic mobility of the a-EmaA monomers was altered and the amount of membrane-associated EmaA was decreased when compared to the parent strain. The mass of biofilm formed remained unchanged. Interestingly, the collagen binding activity of the mutant strain was similar to the activity associated with the parent strain, which differs from that observed with the canonical b-EmaA isoform. These data suggest that the properties of the a-EmaA isoform are like those of b-EmaA, with the exception that collagen binding activity is independent of the presence or absence of the O-PS.