RxPG News : Plasmodium

AgDscam gene Holds the Key to Broad-Based Pathogen Recognition

Fri, 23 Jun 2006 00:23:37 PST

( from http://www.rxpgnews.com ) Anything that's alive runs the risk of infection. How you respond to infection, however, depends on where you sit on the evolutionary tree. Humans and other vertebrates can fend off billions of pathogens by routinely recombining bits of genes for surface molecules on the cells charged with pathogen recognition. Insects and other invertebrates rely to a large degree on the pathogen recognition molecules (called pattern recognition receptors) they were born with. When a pattern recognition receptor detects a pathogenbased on what's known as its pathogen-associated molecular patternthe receptor can launch a direct attack that either engulfs the invader, through encapsulation or phagocytosis, or triggers signaling pathways that regulate immune system genes involved in killing the pathogen.

In a new study, Yuemei Dong, Harry Taylor, and George Dimopoulos found a mosquito gene that vastly boosts the ability of insect pattern recognition receptors to detect pathogens. Originally implicated in neuron development, the gene can create a plethora of receptors for the malaria vector Anopheles gambiae. The AgDscam geneshort for Anopheles gambiae Down syndrome cell adhesion molecule genehas 101 protein-coding regions (called exons) that can be mixed and matched after transcription to produce over 31,000 possible alternative splice forms with different properties. Thus, while B cell and T cell receptor diversity is generated largely at the gene sequence level before transcription, AgDscam diversity is produced by reshuffling sections of gene transcripts before translation into protein.

Dscam was first characterized in the fruitfly (Drosophila melanogaster), where it can generate about 38,000 splice forms with different recognition and binding specificities from 95 variable exons. It's been suggested that a diverse inventory of adhesion molecules may help olfactory nerves establish the proper connections during development. But the presence of high levels of Dscam in cells that function in the fly's innate immune system and evidence of involvement in phagocytosis raised the possibility that the gene also plays a diverse recognition role in immunity.

Dong et al. found that AgDscam, like the fly version, has three variable regions within a portion of the immunoglobulin (Ig) gene. Each region contains different numbers of alternative splicing exons: Ig4 has 14, Ig6 has 30, and Ig10 has 38, leading to a possible 31,920 alternative splice forms. The researchers worked with a mosquito immune cell line to investigate AgDscam's response to infection. Exposure to bacteria, fungi, and parasite surface molecules caused the cells to produce different AgDscam splice-form repertoires with different interaction properties. As with the cell lines, bacterial infection of adult mosquitoes also caused alternative splicing of AgDscam. Infecting mosquitoes with two different Plasmodium malaria parasites produced completely different AgDscam splice-form repertoires.

When the researchers cut AgDscam protein levels in half with a technique that silences a gene by degrading its transcript, they could link its function with phagocytosis of pathogens. Mosquitoes with a silenced AgDscam gene succumbed to bacterial infections (caused by two types of bacteria that produce different surface proteins) at much higher rates than did mosquitoes with a functioning AgDscam gene. Silencing AgDscam also resulted in a profound proliferation of opportunistic microbes, suggesting its essential role in defending the mosquito against bacterial infections. When gene-silenced mosquitoes fed on blood infected with malaria parasites, the researchers found a 65% increase of parasites on the insects' guts. The researchers confirmed the specificity of these associations between splice forms and particular pathogens by selectively silencing the exon transcripts induced by different bacteria. Disabling bacteria-specific exons reduced binding for the target bacteria but had no effect on other bacteria.

Altogether, these results show that infection-induced AgDscam splicing creates receptors better equipped to recognizeand defend againstthe invading pathogen. Cells generated different splice-form repertoires depending on the source of infection. Alternative splicing allows the insect to vastly increase its repertoire of pattern recognition receptors from one single gene and thereby fight infection more efficiently. This work suggests that a better understanding of how A. gambiae's hypervariable receptor AgDscam recognizes the Plasmodium parasite might suggest novel ways to control malaria by targeting the parasite inside its mosquito host.

Genes responsible for malaria parasite's survival pin pointed

Tue, 20 Jun 2006 19:11:37 PST

( from http://www.rxpgnews.com ) "While millions of dollars have been spent to develop a malaria vaccine, we still don't have a licensed product," says Associate Professor Elizabeth Winzeler of Scripps Research, who led the study. "Our findings may help in the vaccine-development effort, because they point to novel immunogens that could be targeted."

Winzeler adds the study also identified novel genes involved in the parasite's development of drug resistance-another critical issue in the fight against malaria.

Malaria is a nasty and often fatal disease, which may lead to kidney failure, seizures, permanent neurological damage, coma, and death. There are four types of Plasmodium parasites that cause the disease, of which falciparum, the subject of the recent study, is the most deadly.

Despite a century of effort to globally control malaria, the disease remains endemic in many parts of the world. With some 40 percent of the world's population living in these areas, the need for more effective vaccines and treatments is profound. The spread of drug-resistance adds to the urgency.

In the study, the scientists used gene-chip technology to compare the genomes of 14 different field and laboratory strains of Plasmodium falciparum collected from four continents. Of the parasite's roughly 5,000 genes, about 500 were found to be highly variable across the different strains, indicating that these genes are evolving at a faster-than-neutral rate.

"These genes exhibit variability far above and beyond basic housekeeping genes," notes Winzeler. "Most genes in the malaria parasite are highly conserved, but these appear to be evolving rapidly."

Why? According to the study, "guilt by association" would indicate that the genes that are rapidly evolving are the very genes responding to our best attempts to eradicate the parasite. "The two largest forces exerting selection pressures on the parasite are our immune system and anti-malarial drugs, particularly chloroquine," says Winzeler.

Previous to this study, no systematic overview of these potential targets in the parasite's genome existed. The study's results include known drug and vaccine targets and intriguingly, areas of the genome not currently under investigation.

One example of a promising potential target highlighted by the research is the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Downstream members of this pathway are targeted by several widely used antimalarials, and authors speculate that an amplification of the GTP cyclohydrolase enzyme facilitates parasite resistance to antifolate drugs.

"I'm super excited about the paper," says Winzeler. "It's going to have an impact on the research community."

Malaria parasite plasmodium impairs key immune system cells

Wed, 12 Apr 2006 13:35:37 PST

( from http://www.rxpgnews.com ) Plasmodium, the parasite responsible for malaria, impairs the ability of key cells of the immune system to trigger an efficient immune response. This might explain why patients with malaria are susceptible to a wide range of other infections and fail to respond to several vaccines. In a study published today in the open access journal Journal of Biology, researchers show that if dendritic cells, the key cells involved in initiating immunity, are exposed to red blood cells infected with Plasmodium chabaudi, they initiate a sequence of events that result in compromised antibody responses. The researchers show that this is due to the presence of hemozoin, a by-product of the digestion of hemoglobin by Plasmodium, in infected red blood cells. These observations also explain why vaccines for many diseases are so ineffective during malaria infection, and suggest that the use of preventive anti-malarial drugs before vaccination may improve vaccine-induced protection.

In a study funded by the Wellcome Trust, Owain Millington and colleagues from the University of Strathclyde, UK, studied the effects of Plasmodium chabaudi, the mouse Plasmodium, on mice antigen-presenting dendritic cells in culture and confirmed their findings in live mice.

Millington et al.'s results show that dendritic cells exposed to P. chabaudiinfected red blood cells do not activate normally. They express lower levels of membrane molecules that stimulate other cells of the immune system, and their cytokine production is lower than that of normal dendritic cells. Millington et al. demonstrate that this is caused by exposure to hemozoin present in infected red blood cells.

Millington et al. then show that P.chabaudi-infected dendritic cells fail to activate helper T cells properly T cells are activated but show reduced proliferation and cytokine production in culture. Importantly, during malaria infection, T cells fail to migrate to B-cell areas of lymph nodes or spleen, and this results in the failure of B-cell activation and antibody production.

How Plasmodium falciparum sneaks past the human immune system

Thu, 29 Dec 2005 16:22:38 PST

( from http://www.rxpgnews.com ) The world's deadliest malaria parasite, Plasmodium falciparum, sneaks past the human immune system with the help of a wardrobe of invisibility cloaks. If a person's immune cells learn to recognize one of the parasite's many camouflage proteins, the surviving invaders can swap disguises and slip away again to cause more damage. Malaria kills an estimated 2.7 million people annually worldwide, 75 percent of them children in Africa.

Howard Hughes Medical Institute (HHMI) international research scholars in Australia have determined how P. falciparum can turn on one cloaking gene and keep dozens of others silent until each is needed in turn. Their findings, published in the December 28, 2005, issue of Nature, reveal the mechanism of action of the genetic machinery thought to be the key to the parasite's survival.

A DNA sequence near the start of a cloaking gene, known as the gene's promoter, not only turns up production of its protein, but also keeps all other cloaking genes under wraps, according to Alan Cowman and Brendan Crabb, HHMI international research scholars at the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia, and their co-authors. "The promoter is all you need for activation and silencing," Cowman said. "It's the main site of action where everything is happening."

Malaria parasites enter human blood from infected mosquitoes. The organisms invade and promptly remodel red blood cells. They decorate the surface of the cells they occupy with a protein called PfEMP1, made by the var gene family.

Using this versatile surface protein, the parasite evades the host's immune system using two basic strategies. First, the protein sticks infected red blood cells to the blood vessel lining, removing the infected cells from circulation, where they would probably be destroyed.

But the protein cannot protect the parasite from patrolling immune cells, which eventually detect the invader and recruit troops to fight it. So, during a malaria infection, a small percentage of each generation of parasites switches to a different version of PfEMP1 that the body has never seen before. In its new disguise, P. falciparum can invade more red blood cells and cause another wave of fever, headaches, nausea, and chills.

"It's like a leopard being able to change its spots," Cowman said. "New forms come up, and the immune system beats them down again. Because of this a lot of people think you need five years of constant exposure to malaria in its different disguises to gain immunity."

Many children do not survive malaria long enough to develop immunity. And without continuous exposure, hard-won immunity may disappear. For example, adults in Papua New Guinea who move to work in the mining industry, which is in mountainous regions that are mosquito-free, lose their immunity within a short time, he said.

The diverse genetic sequences of the 60 var cloaking genes all code for remarkably similar protein structures, the malaria researcher added. The genes are generally found at the ends of P. falciparum's 14 chromosomes, although some of them cluster in internal regions.

In April 2005, Cowman, Crabb, and colleagues showed that var genes are regulated by the chromosome packaging, which unwraps one gene to be expressed at a time and literally packs away the inactive genes. In chromosomes, DNA can be encased so securely by some proteins that other proteins cannot access the nucleic acid for transcription, a process known as epigenetic silencing.

In their new paper, the researchers show that the activation of a var gene promoter is all it takes to trigger both the production that gene's protein and the epigenetic silencing of the 59 other var genes. As in a previous study, they found that the physical location of the promoter within the nucleus seems to make a difference. The genetic activity occurred at the edge of the nucleus, with the activated promoter surrounded by chromosome ends containing silenced var genes.

To do this research, the scientists had to master the difficult technique of cloning large DNA sequences with a var promoter attached to various genes, inserting them into plasmid vectors, and introducing them into red blood cells infected by malaria parasites.

In one experiment, they set up a system where var gene expression could be studied using drug selection rather than the immune pressure that is normally needed to select variants in the field. Using this system they found that the information required for switching var genes on and off was contained within a promoter and that when active this could silence all of the var genes in the parasite.

"This is the first time anyone has actually been able to infiltrate an antigenic variation program," Cowman said. "We forced the cell to switch our gene on and others off." Their system can be used to study blood samples from people in the field to determine how they gain immunity over time.

Kirk Deitsch's lab at Cornell University found that a piece of shared DNA--discarded in the process of translating the protein from its genetic instructions--was a key regulator of var gene silencing and activation. The HHMI researchers confirmed that this gene segment caused tighter packaging for the silenced genes, but they also showed that it wasn't vital.

The researchers are continuing to disassemble the var gene machinery, piece by piece. They want to identify the proteins that unpack and activate the promoter region and learn more about the other proteins in the nuclear compartment that make it the prime spot for var gene activation. Eventually, they think their work may lead to new types of therapies that interfere with the parasite's immune evasion strategies.

How Plasmodium breaks in to blood cells

Tue, 30 Aug 2005 01:16:38 PST

( from http://www.rxpgnews.com ) Plasmodium falciparum, the most lethal malaria parasite, is a housebreaking villain of the red blood cell world. Like a burglar searching for a way in to his targeted premises, the parasite explores a variety of potential entry points to invade the red blood cells of its human victims. When a weak point is found, the intrusion proceeds.

Scientists have known about the parasite's housebreaking habit for a decade, but just how it breaks in to blood cells has been unknown.

Now, an international team of scientists, led by WEHI's Professor Alan Cowman, has discovered the gene - known as PfRh4 - that the parasite uses as a tool to switch between potential invasion points. More specifically, the gene provides the parasite with the ability to switch from receptors on red blood cells that contain sialic acid to those that do not.

In effect, if the gene finds all the doors locked, then it will try all the windows until it finds one it can force open.

The team who performed the research work consisted of Janine Stubbs, Ken Simpson, Tony Triglia, David Plouffe, Christopher J. Tonkin, Manoj T. Duraisingh, Alexander G. Maier and Elizabeth Winzeler. Professor Cowman and his team at WEHI worked with researchers from the Scripps Research Institute (TSRI) in La Jolla, California and the Genomics Institute of the Novartis Research Foundation in San Diego, California.

This discovery made by the group will have a profound impact upon the design of new anti-malarial vaccines, since the inactivation of this single protein could block multiple entry points currently open to the parasite.