July 2007
If researchers were to describe the oral bacterium Porphyromonas gingivalis, three adjectives likely would come to mind: well adapted, opportunistic, and destructive. And with good reason. This anaerobic, or oxygen-eschewing, microbe has adapted well to living with other bacterial species in the low-oxygen environment of the gingival crevice, the shallow space between a tooth and gum, or gingiva, and is most strongly associated with human periodontal disease. But if conditions in the crevice permit, P. gingivalis turns into a Jekyll-like pathogen that infects nearby gingival cells. As the infection spreads, P. gingivalis sooner or later will encounter white blood cells called monocytes that are part of the body’s innate, or natural, immune system. What’s never been clear is how monocytes sense live P. gingivalis or its constituent parts, a key interaction in understanding the onset of periodontitis and its tissue-damaging inflammation. These parts include:
- Lipopolysaccharide, or LPS, a distinctively shaped protein affixed like scales to the outer membrane of the bacterium. They can shed from the bacterium and induce an immune response on its own.
- Fimbriae, or FimA, the thin, foot-like appendage that the bacterium uses to probe and attach to various surfaces, including gingival cells. It, too, can be removed from P. gingivalis and trigger an immune response.
In the June issue of the Journal of Proteome Research, a team of NIDCR supported scientists and colleagues take a closer look at how a monocyte senses live bacteria, LPS, or FimA. The Inside Scoop spoke to the paper’s senior author, Dr. Salomon Amar, a scientist in the Department of Periodontology and Oral Biology at the Boston University School of Dental Medicine. As Amar noted, his data mark a starting point in using comprehensive protein-profiling, or proteomic, approaches to map out signaling pathways in the monocyte and, hopefully, to identify new ways to control the destructive inflammation of chronic periodontitis.
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In this paper your group looks at how human monocytes respond to LPS, FimA, and live whole P. gingivalis. What did you discover?
Our primary finding is monocytes do seem to respond a little differently to each. Based on previous work, we had strong suspicions that this might be the case. But these data really begin to nail it down.
Why is this important to know?
Because it leads to a larger question that we have right now on the table with periodontitis. I believe acute and chronic periodontal infections are triggered differently. In an acute infection, the monocyte generally senses the whole bacteria. There is no time to focus specifically on the LPS, fimbriae, or any other byproducts. It’s a short response, quick and acute.
What about a chronic infection?
Well, once the immune system is not capable of completely eliminating the bacteria - because of immune dysfunction or the virulence of the micro-organism - the process moves into a form of pseudo healing. In other words, the infection never heals. That means the monocyte senses the whole bacteria, but because the immune response is perpetual, it also senses residual LPS or fimbriae that is shed from dead or damaged bacteria. The monocytes recognize both as belonging to P. gingivalis and mistakenly mount an immune response as though they have detected live bacteria.
How did you determine that monocytes sensed each differently?
Through an increasingly popular approach in biological research called expression proteomics. In relation to our work, the term essentially means that we exposed monocytes one at a time to whole bacteria, LPS, and FimA, and generated a comprehensive protein read out, or profile, for each exposure. Importantly, we also had a profile of unexposed monocytes as our control. With these four distinct profiles in hand, we could compare them and look for increases or decreases in the presence of myriad proteins throughout the cell.
What exactly do you mean by "profile?"
When a monocyte encounters LPS, for example, it generates a signal on the cell surface that is relayed internally to the nucleus. In response to the signal, the monocyte modifies to some degree the genes that it expresses and thus the proteins that it makes to deal with whatever potential danger that it’s sensing. Think of the process as an input leading to an output. We profiled the whole protein output.
Why not just profile the genes?
Well, both approaches are complementary, and combining them helps to minimize false positives. By that, I mean we certainly need to know which genes are expressed. Our laboratory and others already have compiled genetic profiles, and they have provided invaluable information. But you can’t be sure that a gene transcript is translated into a protein. You take it on faith, and that’s why you must take the additional step of broadly profiling the actual protein “outputs” in the monocyte. The two levels of evidence - gene and protein - are extremely powerful in telling you what’s happening inside the cell. Our ultimate determination of a differentially expressed gene remains at the protein level.
In this case, what did these protein profiles show?
They showed some unique modifications for each exposure. The laboratory term for this concept is “differential expression.” We found 12 differentially expressed proteins following exposure to live whole bacteria, 11 differentially expressed proteins after LPS exposure, and nine differentially expressed proteins for FimA. What’s really nice here is these data correspond to the matching genetic profiles that we generated in previous studies.
Can you take the next step and plug these differences into the internal circuitry, or signaling pathways, of the cell?
Absolutely. There are enough online tools out there now to allow us to lay out a skeleton map of known signaling pathways. We have data to show where some of the enzymes are activated. Once you know that, you know the identity of their ligand. That allows you to go back and work out the molecular biology, looking at receptor, phosphorylation of proteins, kinases that help to regulate the process. You just start building your model. Matter of fact, we have an upcoming paper that lays out a novel signal transduction pathway in the monocyte that is preferentially activated upon sensing LPS as opposed to live bacteria. We track the signal from the cell surface to the nucleus. But you’ll have to wait a little while for that to be published.
We started our conversation talking about the basic differences between acute and chronic infections. How might defining the pathways influence care for acute and chronic periodontitis?
Well, in theory, a differentially activated pathway might tell you what it is that the monocytes are sensing at the moment. Is it live P. gingivalis or LPS? Or is it both?
And that would give you greater specificity in treating the infection?
Exactly. I say “in theory” because we still need to do the work and demonstrate that this is indeed the case. But that’s the current thinking, and that’s where the work is headed.
Couldn't you also detect the developing disease before it's visible?
Again, in theory. Let’s say we have several defined, well characterized molecular profiles of monocytes reacting against various pathogens. It might be possible to collect a blood sample, isolate the monocytes, and query their molecular behavior. If the monocytes exhibit a known genetic or proteomic profile, that tells you that the patient might have an early infection that isn’t yet visible that can be preemptively treated. With the profile in hand, we’ll know how precisely to think about targeting the infection before it progresses to a full-blown infection.
These research tools that measure gene transcripts and protein production, in essence, are like snapshots. They give you one freeze frame in time. But the interaction between a monocyte and P. gingivalis is a dynamic, ongoing process. Is it possible to take myriad snapshots and develop a composite of the process over time?
Certainly. The research tools are available. It’s just a matter of rolling up our sleeves, framing the research questions correctly, and doing the work.
P. gingivalis obviously doesn't operate alone. What about widening the investigative lens and capturing the interactions between P. gingivalis and the other oral pathogens that contribute to periodontitis?
I think that will happen, too. Once a skeleton map of the primary signaling pathways within the monocyte has been completed, it will be possible to complicate our research questions by layers.
What do you mean?
Dr. Sigmund Socransky and his group at The Forsyth Institute in Boston have reported that bacterial species exist in complexes in subgingival plaque. The most pathogenic complex, otherwise named “Red Complex,” consists of P. gingivalis, Tannerella forsythia, and Treponema denticola. This complex relates strikingly to the clinical measures of periodontal disease, particularly pocket depth of the gingival crevice and bleeding on probing. So, you’re right. We want to know about the dynamics of their interactions, too. Once we have defined a pathway in the monocyte over which inputs travel from the cell surface to the nucleus, it’s possible to evaluate the traffic along that pathway when the monocyte senses P. gingivalis alone or in tandem with other bacteria.
So, the issue here is the pathway, not necessarily the number of bacterial species.
Exactly. This work boils down to the question: How is the immune response dysfunctional in periodontitis? That’s what we want to figure out, and the answers will provide the fundamental understanding that will greatly improve treatment for people with chronic periodontitis. So, from our perspective, oral bacteria are triggers of an immune response. We don’t necessarily need to understand how they aggregate to form a biofilm. That said, however, it certainly will be important to test whether multiple bacteria activate one pathway as opposed to another.
Or whether certain combinations of bacteria induce a more rapid or intense signal to the nucleus.
That’s right. The key point here is not that our current paper is noteworthy because it solves a longstanding treatment issue. It doesn’t. What’s significant is it shows that the research tools are now available to develop these skeleton maps of the immune response and develop genetic and protein profiles that are highly specific, highly testable clinically, and, most importantly, of great potential benefit to the millions of people with chronic periodontitis.
In other words, major discoveries could be around the corner?
Absolutely.