My laboratory uses a wide variety of approaches to study infectious diseases. These approaches include the use of genomic technologies and bioinformatics for the study of P. falciparum, small RNA viruses, and diseases of unknown etiology. These research targets are served by a core interest in developing new technologies for biomedical applications.
The majority of the world's infectious disease burden, especially in children under 5, can be attributed to just a handful of pathogens. Among these, Plasmodium falciparum ranks as the most vicious and arguably one of the most neglected afflictions known to humankind. This protozoan parasite is the causative agent of the most deadly form of human malaria: approximately 1 million people with this disease will perish every year, in addition to a far greater number who will suffer repeated debilitating infections. Beyond the immediate human toll, the burden of malaria has a profoundly negative impact on socioeconomic development in those regions where the disease is endemic. There is renewed interest in reviving global eradication campaigns, despite the failures of the past. A major component of this effort will rightfully focus on vector control. However, treating diseased individuals must always be a priority. To assist in these efforts, there is a dire need for new, cheap antimalarial therapeutics.
In addition to seeking to advance fundamental understanding of Plasmodium molecular physiology and genomics, my laboratory is pursuing the discovery and development of new malaria drugs. Although the causative agent of malaria has been known for more than a century, our knowledge of malaria molecular biology is shockingly sparse when compared to knowledge of a model organism such as Saccharomyces cerevisiae, which has a comparable genome size. We believe that pushing the frontiers of Plasmodium biology will yield insights that will ultimately aid both drug and vaccine development.
Drug discovery. In collaboration with Kip Guy (University of California, San Francisco/St. Jude Children's Research Hospital, Memphis), we have engaged in an intense hit-to-lead development program, whereby compounds with antimalarial activity are further developed to evaluate their realistic potential for becoming a therapeutic. The majority of antimalarial compounds discovered in high-throughput screening assays against whole parasites have no known target. Therefore, an important component of our program is identification of molecular targets.
A primary method for identifying a target is to implement large-scale culture systems in which parasites are kept under continuous drug pressure. Given a large starting population, rare genetic mutants that are resistant or less susceptible to the drug pressure will be selected and may be recovered. Individual clones can then be subjected to ultra-deep sequencing to identify all genetic changes that occurred with drug pressure. By comparing individual clones, we can separate passenger mutations from driver mutations and identify the genetic determinants. Knowledge of the drug target or mechanism of resistance then allows rational optimization of the lead series of compounds.
Plasmodium cell biology. For nearly 20 years since the discovery of the apicoplast, the function of this unique and essential plastid organelle in blood-stagePlasmodium parasites has eluded researchers. Recently, we showed that parasites that have lost their apicoplast can be “rescued” by the addition of isoprenoid precursor IPP (isopentenyl pyrophosphate) demonstrating that the sole function of the apicoplast in blood-stage parasites is the biosynthesis of isoprenoid precursors. This discovery resolves a long-standing mystery inPlasmodium biology and presents new opportunities for investigation into the apicoplast and its role in malaria pathogenesis. Given the importance of the apicoplast for drug development, this insight also provides the ability to identify essential and specific apicoplast targets for therapeutic intervention and opens new avenues for the study of this intriguing “weak spot” of the parasite.
Plasmodium genomics. Our laboratory seeks to discover and dissect fundamental regulatory mechanisms that govern Plasmodium development. To facilitate our expression experiments, we have pioneered the development of multiliter bioreactor techniques for growing large quantities of parasites, thus enabling us to conduct high-time-resolution experiments. Our first large-scale effort was to profile the intraerythrocytic developmental life cycle (IDC) of P. falciparum. Sampling from the bioreactor on the hour, every hour, for more than 50 hours produced a detailed portrait of the malarial transcriptome. This portrait revealed a continual cascade of gene expression, whereby the vast majority of genes are induced once, and only once, per life cycle. The biochemical functionalities represented in the transcriptome are well ordered and are reminiscent of a "just-in-time" assembly process. Such a tightly regulated and well-ordered cascade implies that small perturbations in gene regulation may cause catastrophic failure for the parasite.
Recently, my laboratory has pioneered the use of ultra-deep sequencing to characterize previously inaccessible aspects of malaria molecular biology, including alternative splicing. To facilitate the analysis of the large amounts of data generated by deep sequencing, we have also developed novel informatics approaches and software. As always, we make our software, protocols, and data freely available to all.
Virology and Viral Discovery
A second focus of our lab is the pursuit of viral agents associated with diseases of unknown etiology. In past years, we had developed a screening system capable of detecting essentially all known sequenced viruses and potentially new, previously undiscovered viruses. Among other projects, we have used the virus chip to identify the etiological agent of SARS (severe acute respiratory syndrome), as part of the global emergency effort initiated by the Centers for Disease Control and Prevention and the World Health Organization. This work was pursued in collaboration with former HHMI investigator Don Ganem (University of California, San Francisco), now vice president at Novartis, Inc.
Recently, we have pioneered the use of ultra-deep sequencing to complement the virus chip. Sequencing allows us to discover viruses that have little if any obvious similarity to known viruses. Over the past few years, we have identified numerous agents, including a novel avian bornavirus, which we proved was the cause of a mysterious disease in psittacines (parrots). We have also used this approach to investigate the health of honeybee colonies in the context of a commercial beekeeping operation. By using a novel de novo assembly algorithm created in my lab, we were able to identify a virus in honeybees that was the most abundant component of the honeybee microbiome, yet was unrecognized because of its extreme divergence from known viruses. We are now using ultra-deep sequencing and new approaches to de novo assembly to pursue an expanding range of applications in infectious disease.
In addition to discovery efforts, my laboratory is also engaged in further characterization of specific RNA viruses, especially picornaviruses. We are developing new deep-sequencing and proteomic approaches to uncover molecular determinants of picornavirus replication and recombination.
Grants from the National Institute of Allergy and Infectious Diseases provided partial support for the malaria projects.
As of May 30, 2012