Creation of FLEXGene Repositories of Human Pathogens

Leonardo Brizuela, Ph.D.
Anu Anumanthan, Ph.D.
Dongmei Zuo, M.S.
Joy Qiu, B.S.
Wendi Mar, B.S.

For the biomedical sciences, the beginning of this century is marked by 1) the excitement and expectations created by the completion of the Human Genome Sequencing Project and related projects (covering a large number of experimental organisms and pathogens) and by 2) the daunting challenge of effectively managing and eliminating re-emerging infectious diseases.

Most public attention has centered on the Human Genome initiative and its milestone achievements and potential consequences. However, often overlooked are the efforts and similar initiatives that has been undertaken and completed for a large number of human pathogens, including clinically relevant viruses, bacteria and protozoa.

Diseases such as tuberculosis, malaria and dengue fever are characterized by an increase of disease incidence due to evolution of drug-resistance mechanisms and expansion of geographical distribution. They represent some of the major challenges facing public health in this new century, (see 2000 WHO report: http://www.who.int/infectious-disease-report/2000/index.html). The magnitude of the problem presented by these diseases is alarming and the potential impact of genomic research in this area is immense.

According to the WHO, tuberculosis kills 2 million people each year. The global epidemic is growing and becoming more dangerous. The breakdown in health services, the difficult treatment regimen and emergence of multidrug-resistant TB are contributing to worsening the impact of this disease in both industrialized and developing countries. It is estimated that between 2000 and 2020, nearly one billion people will be newly infected, 200 million people will get sick, and 35 million will die from TB - if control is not further strengthened. Malaria, on the other hand, affects over 300 million people and kills 5,000 children under the age of five every day. Evolution of drug resistance to field medications and modern patterns of human migration also contribute to widen malaria’s distribution.

The vast amount of information generated by the various genome-sequencing projects (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html) has given raise to a new paradigm in experimental biology. This new paradigm invokes experimentation and data analysis at genome-wide scales, as well as the generation of new technologies and resources that take full advantage of the available sequence information.

This new approach brings numerous opportunities for novel approaches for the diagnosis, target identification and treatment of infectious diseases. New analytical tools, such as DNA micro arrays and proteomics, exploit genomic information to generate new hypotheses about the various aspects of host-pathogen interactions as well as provide powerful means of validation through new molecular and bioinformatic approaches. These genomic tools make it possible for the first time to address scientific questions on a whole cell, or system-wide basis, in contrast to the classical reductionist approaches.

Consequently, obtaining and exploiting the genomic information of the etiological agents involved in the major infectious diseases will certainly aid in 1) the identification of new targets for therapeutic intervention and for dealing with known drug resistance issues and 2) the identification of specific antigens for use in vaccination and diagnostic approaches.

The Institute of Proteomics at Harvard Medical School (HIP) is building a comprehensive, characterized, arrayed and flexible gene repository that will allow full exploitation of the genomic information of pathogen organisms by enabling functional genomics as well as protein expression, purification and analysis on a genome wide scale. In other words, HIP is using information from the genome sequencing projects to create a physical resource -- the actual genes in an expression-ready format -- that will be required for genome–scale experimentation.

Pseudomonas Aeruginosa Project

We are now collaborating with Dr. Stephen Lory, also at Harvard Medical School, to generate a repository of genes from the bacterium Pseudomonas aeruginosa PAO1 (with funding from the Cystic Fibrosis Foundation). This organism is the leading cause of death in cystic fibrosis patients and it is a significant cause of morbidity and mortality in hospitalized patients. The goal of this work is to use “genome-based” approaches to drive research in several areas of Pseudomonas pathogenesis and host response including drug discovery, vaccine development, and the toxicology of various agents developed for the therapy of CF.

We have successfully completed the capture of 5290 genes (95% of the predicted ORF’s for this organism) using recombination-cloning techniques. We are now in the process of developing E. coli and Pseudomonas expression vectors to express and purify recombinant proteins and to use in complementation experiments respectively.

Plasmodium Falciparum Project

Presently the open reading frames for almost all of the approximately 5,600 genes in Plasmodium falciparum 3D7 have been completed. The final annotation is in progress with the expected published release of the genome due in mid 2002. Functional analysis of the P. falciparum genome will continue to be complex because of genotype and phenotypic diversity from life cycle stages to strain variation. Almost 50% of the genes have introns necessitating quality cDNA preparations from multiple stages. More than a third of the genes have no homologies to known genes based on their predicted protein primary structure.

In collaboration with David Sullivan (JHU), Joao Aguiar and Dan Carucci (Naval Medical Research Center) we are conducting a pilot experiment to clone and sequence 200 genes from this organism and to set up the protocols for cloning the totality of the predicted ORF’s. Particular interest will be paid to the development of cDNA template for the PCR amplification of that large proportion of intron-containing genes.

Arboviruses Project

In collaboration with Dr.Irene Bosch, at the University of Massachusetts Medical School (Center for Infectious Disease and Vaccine Research), we are cloning and expressing the genes encoding the proteins expressed by Arboviruses of clinical relevance. Dengue (strains 2 and 3) and West Nile viruses have been originally targeted for the initial cloning and expression efforts on members of the Flaviviridae family of viruses.