Everyone has dreams of their teeth falling out, but while waking up is a relief from that nightmare, the reality is that teeth can fall out, and not only when you’re under the age of ten.
Periodontitis, also known as gum disease, is a disease that can cause teeth to fall out in adulthood. It affects 42% of people over the age of 30 and causes inflammation, destruction of soft tissue in the mouth, gum (or gingival) recession, bone resorption, and, yes, loss of teeth. Simply put, this disease can decay your teeth and gums, potentially causing tooth loss.
Therefore, periodontitis is of great concern to dentists and toothbrushers everywhere because no one wants their teeth to fall out. Thankfully, there is active research on the causes of this disease.
Research shows that the primary culprit of periodontitis is the bacteria Porphyromonas gingivalis (P. gingivalis). Researchers have found that P. gingivalis causes periodontitis by colonizing below the gum line (subgingival pocket). There, it can produce virulence factors called gingipains and fimbrillin. These factors damage gum tissue and disrupt the microbes that live in the gum line. This disrupts the homeostasis that the resident microbes maintain and leads to the progression of periodontitis. The disruption of microbes that usually live below the gum line triggers the host inflammatory response, releasing pro-inflammatory factors and causing inflammation.
Figure 1. Comparison of a healthy tooth to one that is suffering from periodontitis.
P. gingivalis releases virulence factors below the gum line through outer membrane vesicles (OMVs). P. gingivalis is a gram-negative bacterium, meaning that it has an outer membrane. To release virulence factors, a part of the outer membrane can form a vesicle containing these factors and separate from the cell. P. gingivalis is the most prolific producer of OMVs, which suggests to researchers that these play an important role in how P. gingivalis causes periodontitis.
Gram-negative bacteria like P. gingivalis produce outer membrane vesicles (OMVs) that contain virulence factors released into the host cell. An OMV acts like a stealth agent, containing instructions such as sRNAs that sabotage the host cell.
In P. gingivalis, OMVs contain virulence factors like fimbriae, gingipains, and lipopolysaccharide (LPS). These factors are inflammatory and can regulate immune cells. In addition to containing virulence factors, OMVs can contain macromolecules like proteins, lipids, and nucleic acids. OMVs can contain small RNAs (sRNAs), which have demonstrated gene regulatory functions.
Figure 2. The formation of outer membrane vesicles (OMVs) from gram-negative bacteria. IM means inner membrane. PG stands for peptidoglycan. OM is the outer membrane. LPS refers to lipopolysaccharide. OMP means outer membrane proteins.
The paper titled "Porphyromonas gingivalis Outer Membrane Vesicles Promote Apoptosis via msRNA-Regulated DNA Methylation in Periodontitis" by Fan et al. focuses on the overlap of these components of P. gingivalis. Researchers hypothesized a role for sRNAs in periodontitis after observing that sRNAs interact with a region on human cells that silences RNA, thereby inhibiting host immunity and allowing bacteria to survive intracellularly.
Figure 3. Many mechanisms of sRNA gene regulation. RBS in purple stands for the ribosome-binding site, and CDS stands for the coding sequence.
Researchers have found that P. gingivalis produces a class of sRNAs that are microRNA-sized, called msRNA. This small size allows OMVs to be contained and enables host cells to take them up. Researchers also found that there is an overlap in human gene regulatory regions and the P. gingivalis genome. Researchers found that they align with regions marked by histones.
Histones are DNA organization molecules in the human genome, which can regulate gene expression through a closed or open histone complex. When histones coil densely, they are known as heterochromatin, which represses gene transcription. When histones spread apart, they become available for gene transcription and are known as euchromatin.
Figure 4. The transition from euchromatin to heterochromatin.
The overlap in genes suggested to the researchers that the OMVs from P. gingivalis transported sRNAs into host cells, where they played a regulatory role in gene expression and the immune response.
To examine the role of sRNAs in causing periodontitis by P. gingivalis, Fan et al. conducted a study that investigated the role of OMVs and their contents in promoting apoptosis in human periodontal ligament cells (hPDLCs). hPDLCs are cells that promote tooth stability and anchor them to the jawbone. Examining the role of the sRNAs in apoptosis would determine the role that P. gingivalis plays in decaying the gum line and bone resorption of teeth in periodontitis.
The researchers first showed that OMVs from P. gingivalis did cause apoptosis in hPDLCs, but they wanted to explain how. To do this, they examined how the treatment of cells with P. gingivalis OMVs changed the expression of proteins associated with apoptosis. They examined the expression level of the protein p53, which is known to play a role in promoting apoptosis, and saw that the presence of P. gingivalis OMVs increased the expression. They further observed that the presence of OMVs from P. gingivalis downregulated the anti-apoptotic protein Bcl-2 relative to control groups.
They further examined the effect P. gingivalis OMVs had on immune cells to examine how they affected inflammation in hPDLCs. They found that the presence of P. gingivalis OMVs increased immune system cells that promote inflammation: IL-1β, IL-6, and TNF-α. From this, the researchers concluded that P. gingivalis OMVs promote both apoptosis and inflammation in the hPDLCs.
The researchers then aimed to figure out which component of OMVs was responsible for the apoptosis and inflammation they observed. They analyzed the transcripts produced by P. gingivalis OMVs and found transcripts linked to gene regulation, mRNA processing, and cell cycle processes. Due to the potential of OMVs to affect gene regulation, the researchers aimed to find sRNAs that P. gingivalis expresses at high levels in OMVs. They used bioinformatics to analyze expression levels and identify targets for certain RNAs. Researchers discovered one sRNA, sRNA45033, which likely plays a role in regulating apoptosis and is predicted to target the gene CBX5.
CBX5, known as chromobox 5 heterochromatin protein α-1, is a protein that binds DNA to form and maintain heterochromatin. CBX5 is important to heterochromatin inflammatory responses, apoptosis, and death receptor signaling.
Researchers examined expression levels of CBX5 in the presence of P. gingivalis OMVs compared to the control group. They found that the presence of OMVs decreased CBX5 expression.
Since the researchers observed decreased levels of CBX5 expression, they thus concluded that sRNA45033 was repressing the expression of CBX5. sRNAs often repress the translation of genes by binding to their 3’ untranslated region (UTR) of the mRNA transcript, a region at the end of the transcript that is not translated.
The researchers wanted to see how sRNA45033 affects CBX5 expression. To ask whether the RNA controls the sRNA in a common region called the 3'UTR, they compared the normal CBX5 gene with a mutant version that had changes in the 3'UTR. Researchers used a luciferase assay to determine the effect of the sRNA on the gene. A luciferase assay connects the expression of a gene to the amount of “glow” shown in the form of fluorescence, so the higher the “glow,” the higher the gene expression. In the presence of the sRNA, the wild-type CBX5 gene had decreased luciferase activity and thus reduced expression relative to the mutant version of the CBX5 gene. This indicated that the sRNA targets the 3’UTR of the CBX5 gene, reducing its expression (Fig. 5).
Figure 5. Relative luciferase activity between CBX5 wild-type and mutant genes. Mimics-NC stands for the sRNA45033 negative control, while mimics in grey stand for the sRNA45033 mimic.
The researchers wanted to see if sRNA45033 in hPDLCs also reduces cell viability or causes apoptosis, like the P. gingivalis OMVs did. Researchers found that in hPDLCs, the presence of sRNA45033 increased cell apoptosis, and when they inhibited the sRNA in these cells, they observed the rescue of normal cell growth. This indicated that sRNA45033 plays a direct role in cell viability and induces apoptosis (Fig. 6).
Figure 6. Propidium iodide (PI) staining which binds to dead cells, and calcein AM staining, which stains only live cells. The merged row shows the combination of calcein AM and PI staining, showing both the prevalence of alive and dead cells at each condition. Inhibitor+OMV conditions mean an inhibitor to the sRNA45033 mimic, and the inhibitor-NC+OMV is an inhibitor to the sRNA4503 mimic control, and the P. gingivalis OMVs are present.
They also saw a similar upregulation of the expression of apoptotic-inducing protein p53 and a decrease in the expression of antiapoptotic factor Bcl-2. This was further supported by the inhibition of sRNA45033 showing the reverse trend: upregulating Bcl-2 and downregulating p53. The results backed the researchers' idea that sRNA45033 causes cell death by blocking CBX5 activity (Fig. 7).
Fan et al.'s research found that sRNA45033 in P. gingivalis OMVs causes apoptosis and inflammation by inhibiting the CBX5 gene. CBX5 controls heterochromatin, which is silent DNA, and this inhibition promotes cell death. This leads to the death of gum line cells that P. gingivalis colonizes, and the decay of the support structure of teeth, which leads to tooth decay or loss (Fig. 8).
Figure 8. Adapted from Fan et al. Mechanism of apoptosis by the secretion of OMVs into hPDLCs carrying sRNA45033. Figure made with BioRender.com.
While this research elucidates a potential mechanism of sRNAs influencing periodontitis, the study by Fan et al. had some limitations. They only investigated one sRNA, while other sRNAs and mRNAs transported by OMVs may also impact the pathways they examined. Additionally, the apoptotic pathway is complex, and the researchers only examined one possible pathway. Investigation of additional pathways could reveal more mechanisms by which sRNAs and mRNAs carried in OMVs may regulate apoptosis.
This work identifies a central mechanism for the periodontitis caused by P. gingivalis, and other researchers could use it to develop treatments that target the specific sRNA or cells affected.
About the Author:
Erin Desmond ‘25 is an Anthropology and Biochemistry double major from Massachusetts. They enjoy listening to music, knitting, and reading. Erin currently works in the Berry lab, studying RNA-protein interactions. They plan to continue their career in research by earning a Ph.D in Biochemistry and Molecular Biophysics from UC San Diego.
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