This story originally ran on May 27.
Name: Jose M. C. Ribeiro
Position: Chief, Vector Biology Section, National Institute of Allergy and Infectious Diseases
Background: Assistant and Associate Professor, Harvard School of Public Health; Professor, Department of Entomology, University of Arizona
Using nano HPLC mass spec/mass spec, a team of researchers led by Jose Ribeiro has completed a preliminary characterization of the salivary proteins, or sialome, of Cimex lectularius — the common bed bug.
Bed bugs are among the roughly 20,000 insects and ticks that feed on blood. One of the key problems any blood-feeding organism must solve is disarming the hemostasis response of its host. For close to thirty years, Ribeiro has studied the saliva of blood-sucking arthropods, analyzing the proteins involved in combating hemostasis response in order to study the evolution of the process across many genera of blood-feeders.
In the last decade, Ribeiro has begun using mass spectroscopy to analyze the full sialomes — typically consisting of 100 to 150 different proteins each — of various blood-feeding insects. In a study published earlier this month in the Journal of Proteome Research, Ribeiro and his colleagues presented their characterization of the C. lectularius sialome, which was notable for its large quantity of a secreted acetylcholinesterase-type enzyme and a heme-group-carrying protein that C. lectularius uses as part of a novel approach to countering vasoconstriction on the part of its hosts.
This week ProteoMonitor spoke with Ribeiro, chief of the Vector Biology Section at the National Institute of Allergy and Infectious Diseases and first author on the JPR study.
Below is an edited version of the interview.
What interested you about the bed bug sialome?
I’ve been studying the saliva of blood-sucking arthropods for nearly 30 years now. There are about 20,000 insects and ticks that suck blood, and about 400 to 500 different genera of insects and ticks. Out of this there are about six to eight times that they evolved independently to blood-feeding mode. So there’s a very large diversity. Why that's interesting is that the saliva of these animals contains a large number of pharmacological reagents that prevent blood from clotting, that induce vasodilation, that prevent platelet aggregation, that modulate the immune response. In the past ten years with the new technologies of doing transcriptomes — which is, you know, making a cDNA library of the organism and then mass sequencing 1,000 to 2,000 clones and analyzing the information — we've started to learn that [blood-sucking arthropods] have hundreds of components in their saliva — up to 100 or 150 different proteins that are secreted in their saliva. Cimex belongs to a group of insects that we’d never looked into their salivary gland components. We’ve looked into mosquitoes, we’ve looked into fleas — and each of these evolved independently to the blood-sucking mode, so [their saliva] had completely different compositions. And the bed bug also revealed several very different solutions to anti-clotting, vasodilation, and so forth.
So your primary interest is in the evolutionary development of these organisms?
My interest is studying the evolution to blood feeding in general. When you try to remove blood you have all these issues. Most people think about blood clotting, but blood clotting is only one of the legs of a tripod, which is blood clotting, vasoconstriction, and platelet aggregation. Then you have the immune reactions that also occur, like inflammation. So it's very difficult to remove blood when you have these mechanisms around. That's what I'm interested in [is] how they solve this complex problem of disarming our hemostasis system.
What stood out about the Cimex sialome? Were there any surprises?
One thing is a big band that is a secreted acetylcholinesterase type of enzyme. We don't know what it is doing. It's very similar to enzymes of the acetylcholinesterase family, but normally acetylcholinesterase exists bound to muscle or nerve cells, and this doesn’t have the region that’s the anchor that binds it to the membrane — it's clearly a secreted enzyme. I have never found that amount of protein — sometimes we find lipases there — but not that much. It's a very thick band in the gel. The other thing, which we actually knew before from when we studied the vasodilator of bed bugs years ago — it uses nitrous oxide as a vasodilator. That's a very unstable gas, but [bed bug] salivary glands have a protein that has a heme group that binds to the nitric oxide and stabilizes it. That's very novel to Cimex. No other insect has developed this protein to carry nitrous oxide.
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Like you said, you've been doing this for thirty years or so. How have capabilities improved and research changed in terms of the ability to take something like a sialome and look at it — to be able to analyze these complex samples?
It's changed completely. Basically it was Baconian science to start with — hypothesis-driven research. The hypothesis was that there is anti-clotting, that there is an anti-platelet, that there is a vasodilator in the saliva, so let's grind and find what they are and characterize the protein, express the recombinant protein, and so on. But about ten years ago we started doing transcriptomes, so the process became not a hypothesis-driven approach but a discovery-driven approach. So I’m very much involved with the analysis of the libraries. I write a lot of the computer code. I wrote my own pipeline to assemble and analyze the data. I have a lab and I have very good senior people in the lab that are doing structural biology and hematology. Basically now what the lab does is study the function of the recombinant proteins that we discover in these transcriptomes. So instead of starting with the insect and going to do the biochemical work, we start with the transcriptome. So everything that is being done in the lab now follows the leads of interesting proteins that we find in the transcriptome. What we're doing in terms of equipment — we do mainly smart libraries done by PCR amplification. We're sequencing on regular ABI 370 machines which we have at a core facility in a Montana NIH lab. And we do regular gel electrophoresis, tryptic digest, and mass spec/mass spec using nano HPLC mass spec/mass spec.
Are you putting together any sort of databases with the information from this work?
I actually have a site at NIH, and at that site there’s some software that I've developed together with some people in bioinformatics at NIAID. There’s a page with all my transcriptomes, so all the transcriptomes that I’ve done are there and people can access the Excel spreadsheets, which are hyperlinked with all the proteins and all the mass spec data.
What other projects are you working on? Are you focusing primarily on Cimex?
No, I have several other transcriptomes that are in the pipeline. There are 400 genera of blood-sucking arthropods. I am 60 years old. It won’t be enough time to see them all, but I want to see at least five or six different genera per year. So maybe I will know about 10 or 15 percent of the different genera if I'm lucky.
So you're interested in characterizing the sialomes of all 400 genera? What will that allow you to do?
There are two reasons. One is to understand the evolutionary process and the speed of it and what it concentrated on and all of that. The other thing is that because of the extremely fast evolution [of proteins in the sialomes of blood-sucking arthropods], with every new gene you get about four or five completely new protein families. And these protein families that are very new are things they are making to affect our hemostasis or inflammation. We’ve been successful in decoding the functions of some of these proteins that we found in mosquitoes and other organisms. They’re usually very interesting. Some of them we’ve patented. So, basically, if you take the numbers — it takes about a year and a half or two years to work with one of these proteins — a full-time job for one person. So it's a lot of work. So if you think we have 400 different genera, and each one is going to generate five new proteins, you have 2,000 new proteins. So you have 2,000 years of a full-time job ahead of you, which is 10 people for 200 years or 100 people for 20 years. So I feel that I’m opening up the doors for a lot of people to do research after me. At 60 years old you start to think about who's coming next.
What other organisms are you working on right now?
There were many different genera of mosquitoes that were never investigated. I've just published a paper with a black fly from South America that was very different — it had many different protein families from the North American one. I'm going to get some black flies from the Amazon region in Brazil and hopefully some in Africa. I'm getting different genera of ticks right now to investigate. I'm getting horse flies. I’ve just completed an analysis of a tsetse fly, and so on. There's enough to keep me busy for the next two years, and then I'll start thinking about what I can get next. The problem is that [there aren't many] insects that exist in labs as cultures, so I'll have to start going and finding out how to collect these insects from the field. I already have a network of people that I'm working with to collect them.