Contribution of adhesion proteins to <i>Aggregatibacter actinomycetemcomitans</i> biofilm formation

Danforth, David R; Melloni, Marcella; Tristano, Jake; Mintz, Keith P.

doi:10.1111/omi.12346

Cited by 6 publications

(11 citation statements)

References 38 publications

(66 reference statements)

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“…The functionality of EmaA expressed in E. coli was determined by the ability of these cells to increase the mass of biofilm formed compared to the control cells, since the EmaA adhesin contributes to biofilm formation in A. actinomycetemcomitans independent of Waal ligase activity (26, 48). E. coli strain MG1655 formed very little biofilm, whereas transformation of this strain with the plasmid expressing emaA , under the regulation of the endogenous promoter, resulted in a greater than a 40-fold increase of the mass of the formed biofilm compared with the control strain (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Dual function of the O-antigen WaaL ligase ofAggregatibacter actinomycetemcomitans

Danforth

Melloni

Thorpe

et al. 2022

Preprint

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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, co-expression 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.

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Section: Resultsmentioning

confidence: 99%

“…5A). Expression of emaA in another E. coli strain has been shown to enhance the biofilm potential of the cell (48). In both of the E. coli strains used in this study (MG1655 and a hemolytic strain synthesizing a complete O-PS) we also observed an increase in biofilm generated when EmaA was expressed (Fig.…”

Section: Discussionmentioning

confidence: 99%

Dual function of the O-antigen WaaL ligase ofAggregatibacter actinomycetemcomitans

Danforth

Melloni

Thorpe

et al. 2022

Preprint

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“…The promoter regions of each of these adhesins contain predicted transcriptional regulator binding sites similar to those present in the emaA regulatory region (Figure 8). The promoters of aae and apiA have not been characterized, but our laboratory has used the intergenic regions as promoters in complementation studies (Danforth et al., 2021). The fimbrial ( flp ) promoter has been better characterized (Figurski et al., 2013; Kram et al., 2008), but extensive studies of the regulatory elements of the promoter have not been performed.…”

Section: Discussionmentioning

confidence: 99%

Regulation of adhesin synthesis in Aggregatibacter actinomycetemcomitans

Tristano

Danforth

Wargo

et al. 2023

Molecular Oral Microbiology

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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.

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“…The canonical function of A. actinomycetemcomitans WaaL to ligate O‐PS to the core polysaccharide of LPS was confirmed by increased immunoreactivity of the transformed strain when compared with the E. coli waaL mutant, to a level similar to the parent strain (Figure 4a), even if this was not accompanied by an observable change in the O‐PS profile, must likely due to the lack of resolution of this gel system. Expression of emaA in another E. coli strain has been shown to enhance the biofilm potential of the cell (Danforth et al., 2021). In both of the E. coli strains used in this study (MG1655 and a hemolytic strain synthesizing a complete O‐PS), we also observed an increase in biofilm generated when EmaA was expressed (Figure 4b), which suggested that the adhesin was properly expressed and localized to the outer leaflet of the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…The functionality of EmaA expressed in E. coli was determined by the ability of these cells to increase the mass of biofilm formed compared to the control cells, because the EmaA adhesin contributes to biofilm formation in A. actinomycetemcomitans independent of Waal ligase activity (Danforth et al, 2019(Danforth et al, , 2021. E. coli strain MG1655 formed very little biofilm, whereas transformation of this strain with the plasmid expressing emaA, under the regulation of the endogenous promoter, resulted in a greater than a 40-fold increase of the mass of the formed biofilm compared with the control strain (Figure 4b).…”

Section: Functional Expression Of Waal and Emaa In E Colimentioning

confidence: 99%

Dual function of the O‐antigen WaaL ligase of Aggregatibacter actinomycetemcomitans

Danforth,

Melloni,

Thorpe

et al. 2023

Molecular Oral Microbiology

Self Cite

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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.

show abstract

Contribution of adhesion proteins to Aggregatibacter actinomycetemcomitans biofilm formation

Cited by 6 publications

References 38 publications

Dual function of the O-antigen WaaL ligase ofAggregatibacter actinomycetemcomitans

Dual function of the O-antigen WaaL ligase ofAggregatibacter actinomycetemcomitans

Regulation of adhesin synthesis in Aggregatibacter actinomycetemcomitans

Dual function of the O‐antigen WaaL ligase of Aggregatibacter actinomycetemcomitans

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