UC Scientist Searching for Clues to Deadly Ancient Organism

You don’t need to be a biologist to know about the dangerous bugs out there.

One pathogen or another, the news media direly report, is working its way around the world, from Central Africa, to Thailand, to China and to the slums of every major city in Europe.

Whether it’s bird flu, Ebola fever, SARS, West Nile virus or HIV/AIDS, it seems deadly infection is only a casual contact or an incoming international flight away.

But while these microscopic threats to world health are the “stars” for epidemiologists and doomsayers alike, a prevalent, less exotic organism that’s more likely to carry us off is still playing cat-and-mouse with researchers.

It’s an ancient parasitic fungus called Pneumocystis carinii, and it’s as common as the often deadly pneumonia it causes in the sick, the aged, infants and, above all, people whose immune defense system is suppressed by chronic illness like AIDS, or by the medications with which they are being treated.

Pneumocystis left the closeted world of bioscience and appeared increasingly in everyday language when the AIDS epidemic exploded in the 1980s. As the No. 1 AIDS-associated infection, it became a major diagnostic feature of the disease.

“P. carinii,” in scientific shorthand, is what’s called an archaeascomycete, meaning it’s as old as dirt—and equally prevalent. A “silent infection” often hiding behind other chronic diseases, it’s been living in our lungs—and killing us—since humankind split off from the reptiles in the so-called mammalian diversion.

So, if it’s been around so long, why hasn’t medical science been able to take it out?

The reason is that for nearly a century since researchers first realized pneumocystis existed, and the widespread threat it posed, it has dodged identification and definition, let alone any really effective attempts to target it with drugs.

But now, thanks to work under way by University of Cincinnati scientists and other researchers involved in the international Pneumocystis Genome Project, the game might finally be up for this elusive killer.

The difficulty in nailing pneumocystis, explains UC research microbiologist Melanie Cushion, PhD, project leader of the Pneumocystis Genome Project, has been in finding a way to culture it, or grow it, outside the human lung. Only then can researchers see what they’re really dealing with and determine how to control it.

“We all basically have it,” Dr. Cushion says, “and one of the areas of research my laboratory is getting into is to understand this relationship better, in terms of its colonization. Is it always there? Do we get rid of it for a time? And are we reinfected?

“We don’t know whether pneumocystis is a transient or long-term colonizer. It’s very elusive.”

Dr. Cushion, a professor in UC’s infectious diseases division and a world-renowned authority on pneumocystis, works at the Cincinnati VA Medical Center and recently received the VA’s Research Career Scientist (RCS) Award, which pays her salary, to continue the study of pneumocystis for five more years. She is only the third Cincinnati scientist to receive the RCS, the highest award for nonclinician VA scientists, in the last 20 years.

Behaving like the true host-dependent parasite it is, says Dr. Cushion, pneumocystis has long existed unseen, unsuspected and for the most part harmlessly in human lungs, and also as a “species specific” guest in certain other mammals.

“Pneumocystis doesn’t want to kill us,” says Dr. Cushion, “unlike a nasty organism like Ebola, which jumps species—something that pneumocystis doesn’t do—and hasn’t been able to establish this balance with its host.”

In contrast to some of the current big name bugs, pneumocystis is a relatively well-behaved

live-in. That is until its host is weakened—by sickness, age, chemotherapy, radiation therapy or some other treatment that suppresses the body’s defensive immune system.

And then it too becomes a killer.

Although pneumocystis was first identified in 1912, no one was able to “grow it out” to provide material for study, and it sank into obscurity.

“It’s a mysterious organism,” Dr. Cushion says. “We don’t know how it’s transmitted, although we think it’s through the air. With most fungi you can find a spore … but with pneumocystis we don’t even know what the agent of transmission looks like. Nor do we understand what it is that causes the infection, or how it gets out of the host.

“It’s able to camouflage itself … it’s very clever that way,” she says. “That’s probably how it’s been able to live in a normal host without causing infection, and to avoid immune surveillance and destruction.”

It was a deadly pneumonia epidemic among undernourished children in European orphanages after World War II that finally blew the silent killer’s cover.

Back then, no one knew what had caused the outbreak, Dr. Cushion explains. But when scientists studied left-over tissue in the 1950s, with the advantage of improved tissue-staining technology, they realized the culprit was pneumocystis.

In the 1960s and 1970s, when cancer chemotherapy became widely available, pneumocystis attracted attention again.

“By then we could study it using electronmicroscopy, and that’s when we really got a handle on it and what it looked like,” Dr. Cushion says.

Then, in the 1980s, the worldwide AIDS epidemic began.

“And boom, there it was again!” says Dr. Cushion.

Pneumocystis was back, big time, says Dr. Cushion. But this time, thanks to genetic analysis, “we were able to rip off the camouflage.”

“Since we didn’t have a culture system to grow it and study its metabolism,” she says, “we had to see what genes it has, what it can do.”

A large part of the painstaking work has involved collecting enough material to study. But working with rat tissue, Dr. Cushion and colleagues George Smulian, MD, Jim Stringer, PhD, Scott Keely, PhD, and Brad Slaven have been able to determine the chemical arrangement of some of the host genome (the genetic “map” of an organism), some bacteria from immunosuppressed lung tissue, and almost all of the pneumocystis genome.

“So we’ve been be able identify what’s host, what’s pneumocystis and what are contaminants, then assemble the pneumocystis genome,” she says.

Although these gene “sequencing” techniques are fairly well worked out in fungi, bacteria, human and other genomes, says Dr. Cushion, “When we finally release the complete pneumocystis genome it will be the first host-dependent, eukaryotic pathogen (it has a nucleus and is relatively genetically complex) to be sequenced.

“Before we started this project, only about 20 pneumocystis genes were known. We estimate now it’s got about 4,000,” says Dr. Cushion.

“That’s a pretty small genome, actually, but we’re finding that unlike in the human genome, there’s not a lot of extraneous ‘junk DNA.’

“Pneumocystis isn’t like many other parasitic genomes that have lost a lot of stuff because they’re in a very intimate relationship with their host. It’s retained much of its biosynthetic ability. So that’s promising. We can start to target some of these things.”

Problems remain, however. After overcoming the shortage of purified study material, the researchers had difficulty cloning some of the pneumocystis genome regions, due to the complex way they encode their “building block” amino acids.

In addition, pneumocystis has a lot of repetitive DNA “gene families,” especially at the end of the chromosome. These repetitive genes, which encode the surface of the organism, are the antigens that alert the body’s defense system that it has a target. With at least 100 encoding genes on its surface, pneumocystis seems able to baffle the immune system by reorganizing genetic variations of these genes.

But there is good news—“One of the breakthroughs we have right now is the PCR, or polymerase chain reaction, which can amplify segments of the genome, so long as we know what the sequence is. If you have a DNA sequence to a gene, the polymerase enzyme will make copies, and keep making copies so it becomes detectable.”

While pneumocystis is troublesome enough for patients with depressed immune systems, it now appears to be spreading into different subpopulations. It’s been recognized in underlying chronic diseases like chronic obstructive pulmonary disorder (COPD) and cancer, especially lung carcinoma, and there’s now evidence from Chile that it might also be involved in sudden infant death syndrome (SIDS).

“Now the questions is whether it’s a ‘comorbidity factor’ … is it contributing to those underlying chronic disease states? Case reports are coming out that they’re finding pneumocystis in combination with these other diseases, but the smoking gun isn’t there. It’s very evasive.”

One of her team’s major goals, says Dr. Cushion, is to use genetic analysis to find drug targets in pneumocystis, so new medications can be developed to treat it.

And the need for new drugs is getting urgent.

Although a commonly used antibiotic, Bactrim, works well against pneumocystis associated with AIDS, half of the patients can’t tolerate it, and evidence is emerging that the organism is mutating genetically in a way that helps it resist the standard treatment.

“We’re finding these drug-resistant mutations in pneumocystis and other organisms, like malaria, all over the globe,” Dr. Cushion says. “They’ve already spread through the human population within a decade.

“That’s pretty impressive, and it’s a major problem.”

“People thought we were crazy when we began the Pneumocystis Genome Project,” Dr. Cushion observes, “because all the other organisms that have been sequenced have a nice culture system that allows you to grow buckets of them, then isolate the DNA and RNA so you have as much as you need to work with.

“But genetic analysis is where the action is, and that’s the fun and interesting part of pneumocystis research.”