Ben Cohn
BBSI – Summer 2005 Research Proposal
Analyzing Cryptosporidium hominis' genome for putative transcriptional motifs.

Introduction:

Cryptosporidium are parasitic protists and pathogens of humans and animals. Members of the phylum apicomplexa, they are characterized by an apical complex organelle that allows the organism to invade its host, as well as aiding in locomotion1,5. Unlike other members of apicomplexa, Cryptosporidium lacks an apicoplast, an organelle that aids in anabolic metabolism, instead employing glycolysis to produce energy. Additionally, evidence suggests that an RNA-less mitochondrion-like organelle is, in fact, a relict of an ancestral mitochondrion, lost along with accompanying oxidative phosphorylation machinery to reductive evolution2.

As a pathogen, Cryptosporidium is highly resilient to chlorine and other chemical treatment. Also, it does not need a vector to infect humans (like Plasmodium falciparum, which transmits the malaria virus through a mosquito vector) and can complete its life cycle within in a single host1. It is transmitted by the fecal-oral route through the ingestion of oocysts1 and is of major concern in water and food contamination5. The extent of infection may range from gastroenteritis (inflammation of the intestines) to severe diarrhea, which may turn life-threatening in immunocompromised individuals, such as AIDS patients1,5. Particularly pertinent to humans are the species Cryptosporidium parvum and Cryptosporidium hominis, which differ only subtly in their gene complements and were, in fact, once considered to be different genotypes of the same species. The most obvious differences between the two, however, lie in their host range, genotype and pathogenicity1. Whereas C. parvum is known to infect both humans and other mammals, C. hominis is restricted to humans. This host preference makes C. hominis very difficult to study in vitro, although isolates can be successfully cultivated in gnotobiotic animals.

Many water-borne outbreaks of Cryptosporidium have been recorded, in both developing and developed nations around the world1. They are usually caused by fecal contamination in either municipal drinking water supplies or recreational waters bodies. In Milwaukee in 1993, sewage overflow led to the infection of over 400,000 people and the deaths of at least 100. Unfortunately, cryptosporidiosis incidents are notoriously underreported and this particular incident was one of many that led to a renewed call for a solution. During the last 20 years, researchers have intensified efforts to find an effective treatment for cryptosporidiosis5.

Recently, the C. parvum and C. hominis genomes have been sequenced1,5, which may hopefully lead to innovative drugs and new treatments for Cryptosporidiosis. In particular, discovery of regulatory sequences along the Cryptosporidium genome may allow researchers to target and disable the core metabolic mechanisms5 that allow the organism to function. Transcriptional regulating elements such as transcription factors, coactivators, corepressors, enhancers, silencers and insulators probably play roles in the transcription of the Cryptosporidium genome. However, as the genome remains largely unmined, many of these elements remain to be located.

Methods:

I plan to search for transcriptional regulatory elements of Cryptosporidium hominis, being the genome of focus for the VCU team. I will use the programming language BioLingua to search for such elements of transcriptional start points, attempting to establish motifs common to C. hominis genes.

Many sequences of regulatory elements are already known. Particularly notable are so-called “TATA” boxes, which are observed about 25bp upstream of the transcriptional start point of many organisms. I am currently and will continue to research known regulatory sequences of similar organisms. Using loop routines within BioLingua, I will automate a search for these sequences along with any others I may observe. Specifically, it will probably be most helpful to search for these sequences within 200bp upstream of forward transcribing genes and vice versa for backward transcribing genes. Additionally, it may also be helpful to search for palindromic sequences in the same regions, as they have been known to indicate binding sites. By establishing common sequential motifs, especially in relation to accompanying transacting elements, I hope to help elucidate some of transcriptional processes of C. hominis.

Possible Results and their Implications:

The identification of regulatory elements in C. hominis will aid researchers in developing methods of treating cryptosporidiosis by focusing on vital transcriptional regions of the hominis genome. Drugs that target the genes producing vital proteins and enzymes would be especially useful in controlling C. hominis. Currently, there is no treatment for this disease, so insights on the transcriptional processes of hominis will undoubtedly prove valuable in the future.

References:

1. Ping Xu, Giovanni Widmer, Yingping Wang, Luiz S. Ozaki, Joao M. Alves, Myrna G. Serrano, Daniela Puiu, Patricio Manque, Donna Akiyoshi, Aaron J. Mackey, William R. Pearson, Paul H. Dear, Alan T. Bankier, Darrell L. Peterson, Mitchell S. Abrahamsen, Vivek Kapur, Saul Tzipori, Gregory A. Buck. 2004. The Genome of Cryptosporidium hominis. Nature. 431: 1107-1112.

2. Janet S. Keithly, Susan G. Langreth, Karolyn F. Buttle, Carmen A. Mannella. 2005. Electron Tomographic and Ultrasound Analysis of the Cryptosporidium Relict Mitochondrion, its Associated Membranes, and Organelles. Journal of Eukaryotic Microbiology. 52(2): 132-140.

3. Julie Nardone, Dong U Lee, K Mark Ansel, Anjana Rao. 2004. Bioinformatics for the “bench biologist”: how to find regulatory regions in genomic DNA. Nature Immunology. 5(8): 768-774.

4. James A. Shapiro. 2005. Retrotransposons and regulatory suites. Bioessays. 27.2: 122-125.

5. Mitchell S. Abrahamsen, Thomas J. Templeton, Shinichiro Enomoto, Juan E. Abrahante, Guan Zhu, Cheryl A. Lancto, Mingqi Deng, Chang Liu, Giovanni Widmer, Saul Tzipori, Gregory A. Buck, Ping Xu, Alan T. Bankier, Paul H. Dear, Bernard A. Konfortov, Helen F. Spriggs, Lakshminarayan Iyer, Vivek Anantharaman, L. Aravind, Vivek Kapur. 2004. Complete Genome Sequence of the Apicomplexan, Cryptosporidium. Science. 304: 441-445.