Plugging escape routes



Pathogens have a tendency to develop resistance. The usual pathway is through chance mutations that have inborn resistance. These mutants, though in small numbers, survive the attack by drugs and the body’s mechanisms against the original organisms, and they thrive, because the drugs reduce their competitors for resources. Mutants hence multiply and grow into a drug-resistant strain, in good numbers.

Malaria is a notable example of this method of keeping a step ahead of drugs and has been rampant in the tropics, where the temperature and humidity favour the growth of the malaria parasite. In a paper just published in the journal, Science (of the American Association for the Advancement of Science), Elizabeth A Winzeler and a team of scientists from California, Missouri, Massachusetts, New York, Geneva and Madrid describe an innovative strategy to track down the sensitive spot in the parasite’s genome, to block the facility with which it changes form.

The authors explain that a single human infection may result in more than a thousand billion blood-stage parasites. With even a mutation rate of one instance in a billion, at least one parasite that resists known drugs or body defences can turn up in a few reproductive cycles. And this mutant strain can grow unopposed, for months, till effective drugs are developed.


While this rapid proliferation and turnaround of generations leads to successive, new drugs becoming obsolete, the same features allow the process of evolution, under pressure of anti-malarial substances, to be initiated and observed in the laboratory. As drug-resistant forms arise in the laboratory cultures, minute genetic changes can be traced, by analyses of the genome of the succession of parent and mutant offspring, to uncover the changes that bring about drug resistance. The paper notes that watching for the changes that accompany the onset of resistance is also an effective method of isolating the target, or mechanism of action, of a known anti-malarial agent. These methods, however, have focused, so far, on isolated genetic effects of single compounds.

In the present study, the group systematically examined the changes in the DNA of the falciparum malaria parasite, over its life cycle, as it evolved and developed resistance against a range of anti-malarial substances. The bulk of therapeutic drugs consist of organic chemical molecules that are small in comparison with structures like proteins. These molecules act by binding at specific regions of large biological molecules or cells or act as initiators of immune or other responses. As small molecules can be absorbed through the intestinal wall, it is possible for such drugs to be administered orally. The study was hence conducted with a collection of plasmodium falciparum parasites of the same genealogy, which had developed resistance against an array of 37 different small molecules. Most of these were known anti-malarials, while the others had significant chemical structures.

To isolate the genetic bases of the drug-resistance developed, the DNA structures of 204 mutant varieties of the falciparum parasite were fully sequenced. The DNA structure is a pair of strings, each of which is a series made up of four kinds of chemical units known as A,T,G and C. The units in the pair of strings are interconnected, to form the shape of a spiralling ladder, with the rule that A must pair with T and C must pair with G. The sequence of bases in one string thus defines the sequence in the other string and this is what makes it possible for each string to build up the complementary string when the DNA splits into two during cell division.

Segments of DNA, each one called a gene, are the code on the basis of which the cells of an organism generate different proteins. The structure of the DNA thus administers the functional viability of a species and permitted variations in the DNA structure lead to differences in the individuals of the species. These are the differences among the descendents of pathogen organisms that may lead to some growing resistant to drugs or other agents that threaten the organism.

In 262 analyses of the sequences of millions of units (which are called base pairs) in the DNA, the study discovered 1,277 instances of a change in a single base pair and 668 small insertions or deletion of base pairs. Single base pair damage, called point mutations, can have far ranging effects and there is a list of serious genetics disorders arising from them. This study, like others that have gone before, thus finds single base pair damage implicated in malarial drug resistance.

In addition to single pair variations, the study found frequent instances of base pairs or series of base pairs being repeated, a feature known as copy number variants, which contribute to drug-resistance. While the human DNA, with three billion base pairs, codes for 30,000 genes, it was believed that the DNA usually had two copies of each gene. But it has been discovered that there are often many copies and great variety in the number of copies of even large sections of DNA, often in parts that do not code for genes.

Such variations lead to individual differences, due to which functional genes are differently presented for becoming active. That leads to differences in traits, including susceptibility to disease, or, in the case of the malaria parasite, in the sensitivity to drugs. True to being difficult to handle, the paper notes that even in this feature of copy number variants, the structure of p falciparum is “A-T rich”, which makes it problematic to detect copy number variation. The team hence developed a case specific algorithm for detecting copy number variants.

The team notes that the study, through “a controlled examination of anti-malarial drug resistance acquisition”, has identified a collection of genes that would aid clinical studies and drug development. “It is notable that we were able to identify a likely target or resistance gene for every compound for which resistant parasites were generated. Our characterisation of the chemo genetic landscape of p falciparum will guide the design of small-molecule inhibitors against this deadly eukaryotic pathogen,” the paper says.