The Trimeric Autotransporter Adhesin EmaA and Infective Endocarditis.

Infective endocarditis (IE), a disease of the endocardial surface of the heart, is usually of bacterial origin and disproportionally affects individuals with underlying structural heart disease. Although IE is typically associated with Gram-positive bacteria, a minority of cases are caused by a group of Gram-negative species referred to as the HACEK group. These species, classically associated with the oral cavity, consist of bacteria from the genera Haemophilus (excluding Haemophilus influenzae), Aggregatibacter, Cardiobacterium, Eikenella, and Kingella. Aggregatibacter actinomycetemcomitans, a bacterium of the Pasteurellaceae family, is classically associated with Aggressive Periodontitis and is also concomitant with the chronic form of the disease. Bacterial colonization of the oral cavity serves as a reservoir for infection at distal body sites via hematological spreading. A. actinomycetemcomitans adheres to and causes disease at multiple physiologic niches using a diverse array of bacterial cell surface structures, which include both fimbrial and nonfimbrial adhesins. The nonfimbrial adhesin EmaA (extracellular matrix binding protein adhesin A), which displays sequence heterogeneity dependent on the serotype of the bacterium, has been identified as a virulence determinant in the initiation of IE. In this chapter, we will discuss the known biochemical, molecular, and structural aspects of this protein, including its interactions with extracellular matrix components and how this multifunctional adhesin may contribute to the pathogenicity of A. actinomycetemcomitans.

Dual function of the O-antigen WaaL ligase of Aggregatibacter actinomycetemcomitans.

Protein glycosylation is critical to the quaternary structure and collagen-binding activity of the extracellular matrix protein adhesin A (EmaA) associated with Aggregatibacter actinomycetemcomitans. The glycosylation of this large, trimeric autotransporter adhesin is postulated to be mediated by WaaL, an enzyme with the canonical function to ligate the O-polysaccharide (O-PS) antigen with a terminal sugar of the lipid A-core oligosaccharide of lipopolysaccharide (LPS). In this study, we have determined that the Escherichia coli waaL ortholog (rflA) does not restore collagen binding of a waaL mutant strain of A. actinomycetemcomitans but does restore O-PS ligase activity following transformation of a plasmid expressing waaL. Therefore, a heterologous E. coli expression system was developed constituted of two independently replicating plasmids expressing either waaL or emaA of A. actinomycetemcomitans to directly demonstrate the necessity of ligase activity for EmaA collagen binding. Proper expression of the protein encoded by each plasmid was characterized, and the individually transformed strains did not promote collagen binding. However, coexpression of the two plasmids resulted in a strain with a significant increase in collagen binding activity and a change in the biochemical properties of the protein. These results provide additional data supporting the novel hypothesis that the WaaL ligase of A. actinomycetemcomitans shares a dual role as a ligase in LPS biosynthesis and is required for collagen binding activity of EmaA.

Regulation of adhesin synthesis in Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative bacterium associated with periodontal disease and a variety of disseminated extra-oral infections. Tissue colonization is mediated by fimbriae and non-fimbriae adhesins resulting in the formation of a sessile bacterial community or biofilm, which confers enhanced resistance to antibiotics and mechanical removal. The environmental changes experienced by A. actinomycetemcomitans during infection are detected and processed by undefined signaling pathways that alter gene expression. In this study, we have characterized the promoter region of the extracellular matrix protein adhesin A (EmaA), which is an important surface adhesin in biofilm biogenesis and disease initiation using a series of deletion constructs consisting of the emaA intergenic region and a promotor-less lacZ sequence. Two regions of the promoter sequence were found to regulate gene transcription and in silico analysis indicated the presence of multiple transcriptional regulatory binding sequences. Analysis of four regulatory elements, CpxR, ArcA, OxyR, and DeoR, was undertaken in this study. Inactivation of arcA, the regulator moiety of the ArcAB two-component signaling pathway involved in redox homeostasis, resulted in a decrease in EmaA synthesis and biofilm formation. Analysis of the promoter sequences of other adhesins identified binding sequences for the same regulatory proteins, which suggests that these proteins are involved in the coordinate regulation of adhesins required for colonization and pathogenesis.

The serotype a-EmaA adhesin of Aggregatibacter actinomycetemcomitans does not require O-PS synthesis for collagen binding activity.

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.

Contribution of adhesion proteins to Aggregatibacter actinomycetemcomitans biofilm formation.

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.

A Nonfimbrial Adhesin of Aggregatibacter actinomycetemcomitans Mediates Biofilm Biogenesis.

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.