Aggregatibacter, a Low Abundance Pathobiont That Influences Biogeography, Microbial Dysbiosis, and Host Defense Capabilities in Periodontitis: The History of a Bug, and Localization of Disease

Aggregatibacter actinomycetemcomitans, the focus of this review, was initially proposed as a microbe directly related to a phenotypically distinct form of periodontitis called localized juvenile periodontitis. At the time, it seemed as if specific microbes were implicated as the cause of distinct forms of disease. Over the years, much has changed. The sense that specific microbes relate to distinct forms of disease has been challenged, as has the sense that distinct forms of periodontitis exist. This review consists of two components. The first part is presented as a detective story where we attempt to determine what role, if any, Aggregatibacter plays as a participant in disease. The second part describes landscape ecology in the context of how the host environment shapes the framework of local microbial dysbiosis. We then conjecture as to how the local host response may limit the damage caused by pathobionts. We propose that the host may overcome the constant barrage of a dysbiotic microbiota by confining it to a local tooth site. We conclude speculating that the host response can confine local damage by restricting bacteremic translocation of members of the oral microbiota to distant organs thus constraining morbidity and mortality of the host

Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments.

Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2) some caution should be used when interpreting the effect attributed to targeted gene mutations when seen in a competitive in vivo environment

Plasmids used in this study.

<p>Plasmids used in this study.</p

Biofilm congo red staining and expression of <i>pga</i>C gene.

<p>Staining of the biofilm using congo red image (A) show that there was a decreased EPS production in RhAa-VS2. Data of bound congo red on to the biofilm (B) show that RhAa-VS2 strain produced significantly lower (<i>P</i><0.0001) level of EPS production compared to RhAa3. Further, the EPS production was restored in RhAa-VS3 strain (<i>P</i> = 0.0035). The result of qRT-PCR (C) show that the genes responsible for EPS production, <i>pga</i>C was significantly decreased in RhAa-VS2 (<i>P</i> = 0.0076) compared to RhAa3, which was restored in RhAa-VS3 (<i>P</i> = 0.0031) using 5srRNA gene as the internal control.</p

Confocal microscopic measurement of biofilm depth.

<p>Biofilm depth was determined by performing Z- axis plane scans and determined by integrating fluorescence intensity across the Z-stack image (A). The biofilm depth was significantly reduced (<i>P</i> = 0.008) in RhAa-VS2 and RhAa-VS3 strains compared to RhAa3. The complemented strain RhAa-VS3 showed no significant increase in biofilm depth (B). The 16 biofilm cells were serially diluted and plated. Data shows CFU from different strains (C). Significant differences in biofilm depths and CFU were calculated by one-way ANOVA with Tukey’s post-hoc multiple comparison test. *<i>P</i><0.05 was considered as significance.</p

Hydroxyapatite binding assay.

<p>The hard tissue binding comparing the ratios of bound to unbound <i>A</i>. <i>actinomycetemcomitans</i> strains to HA showed a significant difference in their binding efficiencies. The RhAa-VS2 strain showed a significantly decreased binding ability compared to RhAa3 (Fig, <i>P</i><0.006). The complemented strain RhAa-VS3 strain showed a significant increase (<i>P</i><0.002) in binding compared to RhAa-VS2. Significant differences in binding abilities of the strains were calculated by one-way ANOVA with Tukey’s post-hoc multiple comparison test. *<i>P</i><0.05 was considered as significance.</p