The study, scheduled for publication on-line in the preview edition of Public Library of Science Biology (PLoS Biology) on August 20, reveals that an unusually high percentage of genes – at least 60 percent -- were "turned on," or expressed, at the third and critical stage in the parasite's life cycle – when it invades red blood cells and wreaks the havoc that causes the disease's symptoms.
Moreover, the analysis showed that the pattern, or timing, of gene expression was unique, with most genes expressed only once during the stage, and in a certain order, for a simple, continuous cascade of gene expression, beginning with genes that correspond to normal cellular processes and ending with highly specialized functions for invading red blood cells. The order and timing of expression resembled a molecular "just in time" factory, whereby cellular components are created only as part of a precisely timed process, rather than on demand.
The discovery of the pronounced level of activity, in which a gene is transcribed into messenger RNA on the path toward producing a protein, points to numerous new potential targets for drug and vaccine therapy. And the timing of expression of individual genes offers insights into the functions of the proteins they encode.
In addition, the revelation that the genes were expressed in a relatively rigid pattern suggests a possible vulnerability in the organism's manufacturing process. Normally, unicellular organisms that cause disease, such as bacteria and parasites, are dynamic entities capable of shifting their patterns of gene expression in response to environmental conditions. But the expression pattern seen in Plasmodium falciparum suggests that a relatively small number of transcription factors could regulate the entire cascade.
"If the gene expression program of malaria is merely a simple linear progression of activation, rather than some baroque network of factors that respond to the environment, then this could be the Achilles heal of the organism. In such a system, disruption of a key regulatory protein would lead to disruption of all the downstream processes as well. The orderly and precisely timed cascade of expression would be fouled, ultimately leading to the death of the organism," says the senior author of the paper, Joseph DeRisi, PhD, assistant professor of biochemistry & biophysics and Gordon M. Tomkins Chair at the University of California, San Francisco.
"This work lays the foundation for dissecting the mechanisms – the transcription factors -- that control this gene expression program." Plasmodium falciparum infects 200 to 300 million people world wide each year, mostly in tropical climates, such as sub Saharan Africa, and kills 700,000 to 2.7 million people annually, mostly children under the age of four.
The disease causes fever and flu-like symptoms, and if not treated quickly can cause kidney failure, seizures, mental confusion, coma, and death. The disease can be cured if treated promptly, but few drugs work to prevent the disease, and thus far no successful vaccine has been developed. In addition, in recent years there has been an increase in drug-resistant strains of the disease.
The researchers submitted their paper to Public Library of Science Biology, rather than a traditional scientific publication, because they wanted to ensure that it would be freely accessible to scientists around the world, many of whom cannot afford to subscribe to scientific journals, says DeRisi. "Malaria is a major killer in the Third World. But many scientists in malaria-endemic areas, such as South America, South East Asia and Africa, who are carrying out very good science on malaria, don't have first world budgets," he says.
In addition to the scheduled publication in the preview edition on-line this month, the paper is planned for publication in the inaugural issue of PLoS Biology, which is scheduled to appear both online and in print in October. The paper already is accessible for free at http://malaria.ucsf.edu and http://plasmodb.org.
Recently, researchers elsewhere sequenced the Plasmodium falciparum's genome, and determined that it contained 5,409 genes, more than 60 percent of which coded for proteins of unknown function. As most of these genes of unknown function do not appear to resemble those found in other organisms, scientists face a particular challenge in determining their role over the course of the parasite's three-stage life cycle.
In the current study, led by Zbynek Bozdech, PhD, and Manuel Llinas, PhD, postdoctoral fellows in the DeRisi lab, the researchers sought to identify the genes involved in carrying out the third, and harmful stage of the parasite's life cycle -- after it has passed from an infected mosquito to the liver of an infected person – when it invades an individual's red blood cells.
The researchers first grew a strain of the parasite in a suspension of human red blood cells, then collected samples of the blood that had been infected at one-hour time intervals over the course of the 48-hour developmental cycle.
Then, to determine the levels and patterns of gene expression over the course of the cycle, the scientists applied samples from each time point to a custom-built DNA microarray, developed in the DeRisi lab, representing thousands of malaria gene sequences.
The researchers tracked the genetic activity of the parasite's three DNA packages – the 14 linear chromosomes, the circular "plastid" genome and the linear mitochondrial genome – within the red blood cells, through the ring, trophozoite and schizoint stages of parasite development.
The pattern of expression revealed sets the organism apart from all other known one-celled organisms, says DeRisi. "Most of the genes were expressed just once in a cycle and in a particular order."
Genes expressed in the early phases appeared to code for proteins involved in metabolic functions. Many of those expressed in the later stages appeared to code for antigens -- proteins expressed on the surface of a host cell, such as that of a human, after a foreign organism, such as a parasite, has taken over its cellular machinery. While antigens are produced by the cell to alert the immune system to a foreign invader – and are the prime target for vaccines against Plasmodium falciparum -- the parasite has evolved ways to change the amino acid sequence of its antigens, to avoid being recognized by the immune system.
Notably, while the nucleotide sequences of the genes expressed in the parasite were relatively fixed, as seen in a comparison with the control strain, a comparison of the genes expressed in the subtelomeric region at the end of the parasite's chromosomes revealed vast differences. Nucleotides had been deleted or changed, indicating that they were zones of genomic instability – which would lead to the production of new antigens that would not be recognized by the human immune system.
"In Plasmodium falciparum, we see a genome whose regulation appears to be uncomplicated and streamlined, yet there is a hidden complexity in the arrays of genes the parasite uses for antigen diversity," says DeRisi. "Identifying these zones of instability lays the foundation for dissecting the molecular mechanism by which malaria evades host immune processes, and may lead to better selection of antigens for vaccine development."
The study also reveals an unusual product of nature. "In its simple design," says DeRisi, "it looks more like a glorified virus than what we think of as a unicellular organism. It's like a different form of life." Other co-authors of the study were Brian Lee Pulliam, Edith D. Wong and Jingchun Zhu.
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