The purpose of the present investigation was to study the impact of an internal perturbation, periodontitis, on bacterial gene expression profiles in three different compartments of the oral microbiota –plaque, tongue and saliva. We used contemporary metagenomics and metatranscriptomics analyses to test the hypothesis that periodontitis exerts an effect on bacterial gene expression not only locally in the plaque microbiota, but also in saliva and at distant sites such as the tongue. To the best of our knowledge, this is the first study to perform paired metagenomic and metatranscriptomic guided characterization of multiple compartments of the oral microbiota in oral health and disease.
As expected, major differences in taxonomic composition of predominant bacterial species identified in plaque, tongue biofilm and saliva were observed (Fig. 1C). In general, correlation was seen between tongue and saliva. Thus, data supports the present view of the tongue being the prime contributor shaping the salivary microbiota . On the other hand, while predominant bacterial species identified in plaque were almost completely absent in tongue, small amounts were identified in concomitant saliva samples. These findings suggest that at least a smaller proportion of the salivary microbiota is expelled from plaque, as previously suggested by concordance of specific bacterial species in subgingival plaque and saliva in patients with periodontitis [10, 30]. Notably saliva is sterile, when entering the oral cavity , and in the present study traces of the predominant plaque microbiota were observed in saliva not only from patients with periodontitis but also from healthy controls. Thus, our species-resolution data analysis confirms the salivary microbiota as a conglomerate of bacteria shed from various oral surfaces not only in periodontitis but also in healthy conditions.
In health, substantial site-specific variations in taxonomic composition were observed within some genera, as exampled by major differences in relative abundance of specific Rothia and Streptococcus species identified in plaque versus tongue. Specifically, Rothia dentocariosa and Streptococcus oralis were identified with high abundances in plaque, but almost absent in tongue, whereas Rothia mucilanginosa and Streptococcus salivarius were predominant in tongue (Fig. 1C). While species within the same genus are taxonomically closely related, site-specific preferences support the idea of co-evolution of the resident microbiota together with the host, which results in gradual adaptation of specific bacterial species to the environment offered at the various oral sites .
Bacterial gene expression is shaped by oral site in health, as seen by significant differences in β-diversity at pathway (Fig. 3A) as well as gene level (Fig. 4B). Moreover, bacterial pathway expression as measured by their RNA/DNA ratios identified at each site in oral health, demonstrated site-specific variations in term of general bacterial activity. This is clearly visualized in Fig. 2D with species such as L. hofstedii being placed in the upper left corner in plaque, in contrast to saliva and tongue. Likewise, species contribution of pathways being expressed significantly different in periodontitis and oral health were completely different at each site (Fig. 3C). Collectively, these findings confirm that taxonomic composition and bacterial gene expression of the resident microbiota in oral health is shaped by the prevailing ecological properties, which are present at each niche in the oral cavity .
As perturbation, periodontitis had a clear impact on the composition of the plaque microbiota, as seen by significant different β-diversity (Fig. 1B), together with significantly higher relative abundance of predominant species such as T. forsythia (Fig. 1C). Also, 25 bacterial species, including periodontal pathogens such F. alocis, P. micra, P. intermedia and T. denticola were observed with higher abundances in plaque from patients with periodontitis (Fig.S1A), which is in accordance with previous studies [30, 34]. Likewise, periodontitis had a significant effect on β-diversity of pathway and gene expression, as judged from their RNA/DNA ratios (Figs.S3A and 4A). Furthermore, pronounced differences were observed in specific bacterial gene expression in plaque at both pathway (Figs. 3A and S3B) and gene levels (Figs. 5A-B). Specifically, bacterial activity (RNA/DNA ratios) of pathways involved in carbohydrate metabolism, such as lactose and galatose degredation I and glucose and glucose-1 phosphate degradation were significantly lower in plaque from patients with periodontitis. Also, significant differences were observed with genes involved in carbohydrate metabolism, where glyceraldehyde-3-phosphate dehydrogenase was significantly higher expressed in periodontitis, as compared to fructose biphosphate aldolase, which was significantly less expressed. Collectively, these findings probably reflect that the microbiota compositionally and functionally adapts to the perturbation, i.e. ecological characteristics such as conditions of anaerobiosis and chronic inflammation being present in the local periodontal environment [34, 35]. Taken together with a recent theory suggesting that frequent carbohydrate consumption may induce inflammation in the periodontal tissues , our finding that periodontitis as perturbation impairs carbohydrate metabolism of the plaque microbiota, provides a possible explanation as to why excessive carbohydrate intake may contribute to the pathogenesis of periodontitis. Interestingly, periodontitis is linked with medical disorders such as type 2 diabetes, with conditions of systemic low-grade inflammation as the immediate communality . Thus, it is an interesting hypothesis that impaired bacterial carbohydrate metabolism could be a factor aggravating systemic low-grade inflammation, in general.
Periodontitis also had an effect on bacterial activity in saliva and the tongue, as visualized by specific pathways and their gene expression profiles (Figs. S3C and 5C). In addition, higher activity of specific bacterial species such as L. hofstadii was evident in saliva from healthy individuals (Fig. 2B). Specifically, significantly lower bacterial activity of the sucrose degradation III pathway was observed in saliva from patients with periodontitis. Furthermore, bacterial activity of genes related to lipid metabolism (glycerol-3-phosphate-cytidylyltransferase) and carbohydrate metabolism (beta fructofuranosidase) was significantly lower in tongue biofilm from patients with periodontitis. We have previously showed an impact of periodontitis on salivary bacterial activity . However, this is the first study to perform simultaneous characterization of bacterial pathways and their gene expression profiles in plaque, tongue and saliva. It is therefore interesting that pathways and genes identified with significantly different RNA/DNA ratios in saliva and tongue were not the same as those identified in plaque. Therefore, data suggest that periodontitis as perturbation acts differently on bacterial gene expression in different oral compartments as visualized in the present study by plaque, tongue and saliva.
Another interesting finding which was evident from analysis of our high-resolution dataset was that expression of Gingipain_K was solely identified in samples from patients with periodontitis (Fig. 5A). Gingipain_K is an endopeptidase with strict specificity for lysyl bonds, which is only produced by the periodontal pathogen P. gingivalis . Interestingly, previous in-vitro studies have shown that presence of P.gingivalis dramatically alters the transcriptomic profiles of oral commensals in an artificially grown biofilm , which is one of the reasons that P.gingivalis is the main act in the recently presented key stone hypothesis . Our finding of bacterial expression of Gingipain_K exclusively in samples from patients with periodontitis is therefore intriguing. However, future studies are warranted to reveal if P.gingivalis in vivo also alters the transcriptome of the resident microbiota or alternatively is counteracted by reliance mechanism of the commensals.
Some limitations apply to the present investigation, including the relatively small sample size, which however is comparable to other metatranscriptomic-based studies on the oral microbiota [41–43]. Furthermore, even though very deep sequencing of DNA and RNA was performed, it was still not possible to portray total gene expression of specific species with an overall low abundance. Accordingly, we could not determine if the transcriptomic profile of for example P.gingivalis differentiated between sites and in health versus disease.