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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]