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Q&A: University of Cincinnati Researchers ID Proteins in Mycobacterium Afflicting Machinists

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JYadav.JPGName: Jagjit Yadav
Position: Associate professor, department of environmental health, University of Cincinnati College of Medicine, 2005 to present; assistant professor, department of environmental health, University of Cincinnati College of Medicine, 1998 to 2005
Background: Postdoc, University of Cincinnati, molecular genetics, biochemistry, and microbiology, 1995 to 1998; postdoc, Michigan State University, microbiology and public health, 1991 to 1995

In 2000, a bacterium called Mycobaterium immunogenum was identified as the cause of an ailment called hypersensitivity pneumonitis, an inflammation of the lungs that seemed to affect mostly machinists in metal-cutting factories.

The bacterium exists in contaminated metalworking fluids used to cool cutting tools that grind metals. The fluids and the infected particles become airborne during machining of metal parts and are inhaled by workers. Symptoms of hypersensitivity pneumonitis include fever, cough, chills, and difficulty breathing.

In a study appearing in the May issue of the Journal of Proteome Research, researchers set out to do immunoproteomic identifications of protein antigens in M. immunogenum. In the study, the authors said that "practically nothing is known about the antigens and other virulence factors in M. immunogenum.

"In the postgenomic era, [an] immunoproteomic approach based on 2-DE and mass spectrometry has proved useful in identification of the immunogenic proteins in several pathogenic organisms," they added. For their work, they set out to identify the immunogenic proteins in the mycobacterium with the long-term goal of gaining insight into its "virulence factors, drug targets, and/or targets for immunodiagnosis of the disease and/or exposure assessment."

They found 33 immunoreactive proteins, including four secretory, six cell wall-associated, 11 membranous, and 12 cytosolic proteins. Eight of the proteins were homologues of known mycobacterial antigens. The remaining 25 novel mycobacterium antigens showed homology with hypothetical proteins and other bacterial proteins with roles in virulence, survival and metabolic functions, the researchers said in the study.

The study, they said, is the first to identify antigens in M. immunogenum and to report on "the potential of its secretome proteins to induce host response."

ProteoMonitor spoke with Jagjit Yadav, the corresponding author of the study, this week. Below is an edited version of the conversation.

Describe the approach that you took, particularly the proteomics work.

This is a bacterium called Mycobacterium immunogenum and this is a bacterium that has been linked to hypersensitivity pneumonitis in machinists. We, for the first time, identified antigens in [the bacterium], antigenic proteins, using a proteomic approach.

Hypersensitivity pneumonitis is an immune-mediated disorder, so it is expected that the causative agents will have causative antigens in them, but nobody has attempted to identify them.

We [studied] secreted antigens, which are secreted outside the cell and then the antigens that are associated with the cell. The ones that are associated with the cell were fractionated into different factions — the ones with the cells of the bacterium, and the ones with the cell membrane, which is below the cell wall, and then the ones in the cytosol part of the cell.

So, we fractionated those fractions by different … speeds, and we isolated the ones that came out … by trichloroacetic acid precipitation. With all these fractions in hand, we did 2D gel electrophoresis, and then we did immunoblotting of the gel using polyclonal antibodies against the same organism that we are studying.

The proteins that were immunoreactive were identified by blotting and we went back and picked out those spots from the 2D gel and we identified [them] by MALDI-TOF analysis.

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Then we [used] the Mascot [search engine] for identification and compared those with known mycobacterium antigens. We had around 50 immunoreactive spots, but we conclusively identified 33 proteins from that.

Out of these 33, only eight were known in other mycobacterium species. The remaining 25 were new mycobacterium [proteins], so out of these 25, we started looking at the ones that …match no known protein — they are hypothetical proteins. There were 11 of them.

The remaining 14 are there in the bacterial database, but they were not identified as antigens before, so they are proteins with other functions … for example, survival of growth of the bacterium.

We were interested particularly in the secretory proteins because in these [metalworking fluids that machinists are exposed to] this mycobacterium grows … and people only look at the bacteria to detect [whether] there is mycobacterium or not. Nobody is looking at the secretome or secretory proteins, which are there in the fluids.

So they kill these bacteria, get rid of them, but they do not realize that they have already secreted enough antigens in these recirculating fluids. These fluids circulate for months and even years in some companies.

What did you expect to see when you were looking at the secretory proteins?

We were trying to identify the antigens there and then we did identify some antigens from secretory fractionation. …Out of the 33, there were four that were identifiable, but there were other spots that we could not identify but they were immunoreactive, they were showing the signals on Western blots.

There was a major spot on the 2D gel that was immunoreactive, so we cut out that band and we reacted it with … lung macrophages and we did see an immune response … so we [correlated] these proteins to the immune response that we can expect in the host.

So, for the first time we showed the antigens in this organism and for the first time we are showing the secretory proteins, the secretory antigens, are immunoreactive — they do trigger an immune response.

In the study, you said that in subsequent work done during the writing of the article, more was revealed about the secretome. Can you share with me what you found during this work?

What happened was when we originally ran the gel, there was a big spot on the gel from the secretory fraction. It was not cell-associated. The secretome was not resolving properly, but when we were reacting with antibodies, it was reacting. It was the major spot, so we studied that.

When we were writing the manuscript, we did separate this in a more resolved fashion, so we did have multiple spots.

What we [were] trying to [say was that in] this first initial gel, which had poor resolution, there was one big spot with multiple antigens there. In the second phase, we could isolate them to more separated spots.

Why did you choose the MALDI platform? Was there something particular about that platform that was more suitable than an electrospray instrument?

Well, we had this [instrument] available, and it's cheaper than going with a more expensive [platform], but we have planned studies now with electrospray to go after each spot and identify the ones which we could not identify [before], and to go after even the ones we did identify and know their sequence, so that we can then design probes, and then track them in these fluids and do some kind of cloning and all those things to get more antigens by recombinant production.

That is the next phase, but [for this study] it was a proteomic profiling study, so MALDI was handy.

Do you have any plans to go beyond identifications of the proteins to look at interactions, both protein-protein interactions and interactions between proteins and other molecules?

We are planning a full study. We are pursuing funding right now. This is in the grant proposal stage. We have planned a full study on these antigens [to investigate] how we can eventually identify and segregate them into the ones [that] are diagnostically important, the ones that are important as vaccine candidates. … If they are important from a vaccine point of view, do they interact with other proteins? All those detailed studies have been proposed.

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So what do we know now about this bacterium based on this study? We know that these proteins seem to trigger some kind of an immune response, but we don't know much more about their biology.

Well, we have [the eight that have been identified in other bacterium species, and of the 25 novel mycobacterium antigens], we know the biology about [14 of them] … but we don't know if they're antigens. In this bug, they seem to be antigens, and even in the [proteins] that are known, the eight candidates, it's possible the epitopes are different in this species than in tuberculosis or another species.

So we'll look at those specific epitopes. That's one immediate goal — we want to sequence them and identify epitopes, species-specific epitopes, so that we can then track them either in the patient or outside in the [metalworking] fluids for immunodiagnosis or immunomonitoring.

Did you do any de novo protein sequencing for this study?

No, we haven't done any sequencing yet. That's planned. We are collaborating with somebody on that.

Sequencing, cloning … all that will be part of [future work]. We are also thinking of sequencing this species, the genome sequence, but that's kind of [further in the future]. We are making some good libraries of this … but right now we are depending on this protein-based probe to fish out the genes for their antigens so that we can express them in E. coli or some other host.

Is the main application of what you're doing for the detection of contamination in the workplace, or for diagnosis of the mycobacterium in a patient?

Both. We are targeting both. Diagnosis in the patient is very challenging. …If the person has been exposed, we should be able to identify the signals of these antigens.

And the power of a diagnostic tool that may come from this study is that it would be able to differentiate it from another mycobacterium, such as tuberculosis?

That part has already been done. We have a number of tests, molecular-based DNA assays, which speciate this particular species. …We recently published a method in which we developed specific DNA-based probes. Because DNA is hydrophobic, that goes in the cell pretty well. We have developed a series of other methods based on PCR, so all that work has been done … by my lab.

Detecting the bacterial DNA or the intact bacterium, that is already possible.

But this particular [study] has identified secretory antigens, which will allow us to detect their activities in the fluids even if the bacterium is gone. … We don't need the bug to be there. If the antigen is there, we still have the phenotype.

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