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Gregory A. Buck , Ph.D.
Phone: (804) 828-2318 Address:
Center for the Study of Biological Complexity
Nucleic Acids Research Facilities Professional Experience
Research Interests: This laboratory is applying the strategies of discovery science and systems biology to studies of microbial pathogens. We are studying two primary pathogenic microbes: Trypanosoma cruzi and Cryptosporidium parvum . Both are responsible for extensive morbidity and mortality, T. cruzi in Latin America and C. parvum worldwide. In brief, we are attempting to dissect the pathogenesis of these organisms using high thoughput and system-wide technologies of genome sequence and analysis, DNA microarrays, proteomics, and bioinformatics, gene networks, and cellular modeling. Projects in the lab are briefly outlined below. I . Cryptosporidium parvum: C. parvum is an important causative agent of diarrhea in humans and animals worldwide. It is particularly associated with morbidity and mortality in immunocompromised individuals including AIDS patients. It is classified by the CDC as a Class B agent of biological terrorism. It has a global distribution and is one of few parasitic protozoa to affect developed countries, contaminating water supplies and resisting water treatment. In 1993, over 400,000 individuals were infected and over 100 patients died in a single epidemic in Milwaukee. There is currently no effective therapy for cryptosporidiosis in large part due to a poor understanding of its basic biology and pathogenicity. Elimination from water supplies is extremely difficult as the oocysts are resistant to most chemicals, including chlorine, and elimination requires physical measures (e.g., filtration). C. parvum is an apicomplexan protozoan related to the parasites that cause malaria and toxoplasmosis. 1. Genome sequencing of C. parvum . The goal of our two-component IRPG is the complete nucleotide sequence analysis of the ~9.5 Mbp Cryptosporidium parvum genome. Project 1 at VCU will focus on a human isolate TU502 (genotype 1or human isolate), and Project 2 at the University of Minnesota will focus on the calf isolate IOWA (genotype 2 or calf isolate). We propose to perform this analysis using a "modified total genome shotgun" approach (see below). We propose to sequence the C. parvum genome to a theoretical coverage of >>99% under the scope of this project. We propose directed "finishing sequencing" of ORFs and predicted genes. Final complete finished sequence will be provided for >99% of the genomes of both organisms. BAC contigs of each chromosome in the C. parvum TU502 isolate genome will assist in the modified shotgun approach (see below), to span sequence contig gaps, and so that all sequences will be readily available for future studies. We have elected to focus on a genotype 1 isolate because: 1) genotype 1 isolates are most relevant for public health; 2) genotype 1 isolates have been the most difficult to study because of their restricted host range (primates); and 3) because we have recently developed for the first time an experimental animal model for propagation of these isolates. Of genotype 1 isolates, we will study the TU502 isolate because this isolate has been propagated the most successfully and for the longest period of time. Progress . We have completed a ~10X shotgun sequence of the C. parvum genome and generated a ~5X coverage with large insert clones (BACs). Our current assembly of a data freeze on Oct. 10, 2003 contains less than 300 supercontigs. We have entered the finishing phase of the project and are beginning to analyze the data. Comparative genomics and other data mining projects have begun. Projects ongoing or available . 1) Finishing sequencing; 2) data mining; characterizing the C. parvum genome; 3) comparative genomics; studying the relationships between the genomes of C. parvum and other apicomplexan parasites (malaria, Toxoplasma , Theileria , etc.); 4) establishing DNA microarrays for C. parvum ; 5) examining expression profiles of differentiating C. parvum ; 6) examining the host response of mammalian cells exposed to infectious C. parvum . 2. Reverse vaccinology of C. parvum . We (VCU) have nearly completed the genome sequence of the genotype 1 (human) C. parvum , and our collaborators at the University of Minnesota are completing the genotype 2 (calf or Iowa) strain. We have begun analysis of gene expression profiles during C. parvum differentiation, and host expression profiles during infection, using custom and commercial arrays. In this project, we propose to use the sequence information generated to apply 'reverse vaccinology' approaches to identification of potential vaccinogens for C. parvum infection, and to examine the efficacy of these potential vaccinogens to block infection by C. parvum in vitro and in vivo. The specific aims of the project are to: 1) identify potential vaccinogens using available and custom bioinformatics tools; 2) over express these vaccinogens (domains or subdomains) in bacterial or yeast expression systems, and to purify these synthetic polypeptide products; 3) examine these products for potential immunologic recognition during human infections using Western analysis; 4) explore protection in tissue culture by examining blockade of attachment and/or invasion in vitro; 5) examine genetic determinants of human susceptibility to C. parvum infection. This work is being performed in collaboration with colleagues at UVA, Va Tech and UVT. Progress . We have completed a ~10X shotgun sequence of the C. parvum genome and generated a ~5X coverage with large insert clones (BACs) and are finishing the assembly. Preliminary analysis shows that there are ~5000 genes, approximately 60% of which have identifiable homology to genes with known functions. Approximately 10% of these genes have motifs suggesting they may be secreted or membrane protein genes. Projects ongoing or available . 1) Searching C. parvum genes for known or likely antigenic motifs and domains using 'in silico' approaches; 2) develop new strategies for 'in silico' characterization of putative vaccinogens; 3) over express selected genes in appropriate bacterial/plant vectors; 4) work with collaborators to characterize possible vaccine candidates. We expect to be able to identify and test several dozen potential vaccinogens in the course of this project. II. Trypanosoma cruzi . Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi , remains one of the most serious public health concerns in Latin America. Despite recent advances in control of the disease, nearly 20 million people are infected with T. cruzi and up to 100 million people are at risk. In terms of disability adjusted life years, Chagas' disease is globally ranked behind only malaria and schistosomiasis as the third most serious parasitic disease. Despite decades of research on T. cruzi , no vaccines have been proven effective and only toxic drugs such as nifurtimox and benznidazole have shown efficacy in the acute phase of the disease. No treatments are available for chronic Chagas Disease. Thus, there is a significant need for new approaches to treatment of T. cruzi infections. T. cruzi exhibits a complex and interesting life cycle involving the alternate infection of a mammalian host and transmission by an insect vector, a large family of blood sucking reduviid bugs. Humans are infected by mechanical introduction of T. cruzi from the excreta of the insect into the bite wound or through mucus membranes. The infecting parasites are non-dividing metacyclic trypomastigotes that have differentiated from non-infective complement sensitive epimastigotes that propagate in the GI tract of the insect. Much interest in recent years has focused on the differentiation of the noninfectious "insect form" epimastigotes into the infectious metacyclic trypomastigotes. It is generally believed that a better understanding of the transition from the relatively innocuous epimastigote into the infectious non-replicative metacyclic trypomastigote, termed metacyclogenesis, will lead to new treatment or disease control strategies. Metacyclic trypomastigotes invade mammalian cells and subsequently differentiate into amastigotes. Amastigotes replicate intracellularly, lyse the host cell after 2-5 days, and differentiate into non-replicative infectious trypomastigotes, which invade other host cells. Trypomastigotes are ingested by the insect vector and differentiate into extracellular replicative epimastigotes in the gut of the vector. 1. Transcriptional profiling of differentiation in T. cruzi . We have created a T. cruzi microarray containing probes for ~4000 T. cruzi genes. We are currently using available T. cruzi DNA sequence and cDNA to generate a comprehensive UNIGENE library of T. cruzi sequences to complete our T . cruzi microarray. This array is being used to study gene expression profiles of T. cruzi during metacyclogenesis, as well as during differentiation into amastigotes, trypomastigotes and epimastigotes. Progress. We have established a 4000 component array of T. cruzi genes. We have used this array to begin to profile metacyclogenesis. Projects ongoing or available . 1) Development of the T. cruzi UNIGENE library; 2) Expression profiling of early events during metacyclogenesis; 3) Profiling of middle and late events during metacyclogenesis; 3) Profiling of events during formation of amastigotes, trypomastigotes or epimastigotes. 4) Proteome profiling of events during metacyclogenesis using mass spectrometry. 2. Transcription profiling of host response to invasion by T. cruzi . T. cruzi metacyclic trypomastigotes exhibit a tropism for cardiomyocytes. In collaboration with investigators at FIOCRUZ, Rio de Janeiro, we have developed a model to study the transcriptional response of primary murine cardiomyocytes to infection by metacyclic or tissue culture trypomastigotes. Progress. We have used Affymetrix GeneChip® arrays to study the profile of host cell transcription responses to infection with metacyclic and cell culture trypomastigotes. The preliminary results show a fascinating cellular response, some of which is clearly host defense related, but much of which is clearly parasite-directed. Projects ongoing or available . 1) Mining of the data from the study of primary cardiomyocytes infection with T. cruzi ; 2) Use of other T. cruzi isolates that use other mechanisms of infection in the cardiomyocytes model; 3) Use of other cell types, some sensitive to and others resistant to, T. cruzi infection, to compare the host cell response to T. cruzi infection. 3. Proteomic analysis of differentiation in T. cruzi . Using the models established in the previous section, we will apply proteomics analysis to validate and extend our conclusions from transcription profiling during differentiation of T. cruzi . We have shown that many T. cruzi genes are regulated post-transcriptionally, and these events cannot be measured using transcriptional profiling. Progress . We have established a proteomics pipeline including 2D gel electrophoresis, automated selection and extraction of proteins from the gel, preparation for mass spectrometry and analysis by mass spectrometry. Projects ongoing or available . 1) Characterization of proteins localized in macromolecular complexes precipitated with antibodies against splicing proteins; 2) Characterization of proteins localized in macromolecular complexes precipitated with antibodies against transcription factors; 3) Characterization of proteins expressed during metacyclogenesis of T. cruzi ; 4) Characterization of proteins expressed during different differentiation stages of T. cruzi ; 5) Examination of different T. cruzi strains for expression of similar proteomic profiles at various life cycle stages. 4. Global interactomics of T. cruzi . We have applied yeast 1-, 2-, and 3-hybrid analyses to identify transcription and splicing factors in T. cruzi . We are now applying yeast and bacterial 2- and 3- hybrid systems to identify global macromolecular interactions in this parasite. We hope to develop a complete interactomics profile. Progress. Yeast 2-hybrid and 3-hybrid libraries of T. cruzi proteins and RNAs have been developed. We have used these libraries to identify several dozen potential interacting partners and are in the process of verifying these interactions. Projects ongoing or available . 1) Establishing a high throughput pipeline for identification of interacting proteins in T. cruzi ; 2) Identification of proteins that interact with other proteins in T. cruzi ; 3) Identification of proteins that interact with RNAs in T. cruzi ; 4) development of a comprehensive interactome map for T. cruzi . 5. Virtual Parasite Project . We are working with mathematical modelers and complexity theorists to develop an 'in silico' model of T. cruzi . The objective is to create models of critical stages of the T. cruzi life and infectious cycle that will permit prediction and interpretation of the pathogenesis of the parasite. Progress . A mathematical model of T. cruzi motility in solution has been generated. This model consists of parallelized differential equations describing the motion of T. cruzi in a three dimensional volume. The algorithm describing this process runs on high performance parallel LINUX/UNIX OS systems. This model has been translated graphically using OpenGL. Host cells have been incorporated into the model to permit the modeling of the invasion process. Projects ongoing or available
. 1) Establish the equations that define motility of T.
cruzi in a three dimensional liquid environment; 2) Establish the
equations that define the interaction of the T. cruzi epimastigotes
with susceptible mammalian host cells in a three dimensional liquid
environment; 3) Establish algorithms that describe the interaction and
invasion of susceptible host cells with metacyclic cell culture
trypomastigotes (e.g., van der Waal's equations, necessary quantum
calculations, metabolic network dynamics, etc.). 4) Work with biologists to
validate and modify the models to represent real systems. Selected Publications: Xu, P., Widmer, G., Wang, Y., Ozaki, L.S., Alves, J., Serrano, M., Puiu, D., Manque, P., Akiyoshi, D., Mackey, A., Pearson, W., Dear, P., Bankier, A., Peterson, D., Abrahamsen, MS., Kapur, V., Tzipori, S., and Buck , GA . The Genome of Cryptosporidium hominis . Nature 431: 1107 – 1112 (2004). Kier, L.B., Bonchev, D., and Buck, G. A.. Modeling biochemical networks: a cellular automata approach. Chemistry & Biodiversity 2: 233 – 243 (2005). Bonchev, D., and Buck , G. A. Quantitative Measures of Network Complexity. In: Complexity in Chemistry, Biology and Topology, D. Bonchev and D. H. Rouvray, Eds., KLUWER Academic, New York. (in press). Abrahamsen, M S, Templeton, T J Enomoto, S, Abrahante, J E, Zhu, G, Lancto, C A, Deng, M, Liu, C, Widmer, G, Tzipori, S, Buck, G A, Xu, P, Bankier, A T, Dear, P H, Konfortov, B A, Spriggs, H F, Iyer, L, Anatharaman, V, Aravind, L, Kapur, V.The complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science. 304: 441-5 (2004). Krieger, M., Fragoso, S., Ozaki, L.S., Xu, P, Carvalho, M.R., Buck, G A, and Goldenberg, S. Messenger rna mobilization to polysomes is a major mechanism of gene expression regulation in Trypanosoma cruzi. ( submitted ). Lee, J. K., Laudeman, T., Kanter, J., James, T., Siadaty, Mir S., Knaus, W. A., Prorok, A., Bao, Y., Freeman, B., Puiu, D., Wen, L.M., Buck , G.A., Schlauch, K., Weller, J., Mangalam, H., and Fox, J. W. GeneX Va : VBC Open Source Microarray Database and Analysis Software for Multiple Users in Biomedical Research. Biotechniques 36: 634-638 (2004). Collins, A.M., Ikutani, M., Puiu, D., Buck, G.A ., Nadkarni, A., Gaeta, B., and Sewell, W. Rearranged immunoglobulin genes partitioned by analysis of the 5' to 3' distribution of somatic point mutations show D-D fusions and D inversions to be rare events. J. Immunol. 172: 340-348 (2004). Puiu, D., Enomoto, S., Buck, G.A. , Abrahamsen, M.S., and Kissinger, J.C. CryptoDB: the Cryptosporidium genome resource. Nucl. Acids Res. 32: 1-3 (2004). Brisse, S. J. Henriksson, C. Barnabé, E.J.P. Douzery, D. Berkvens, M. Serrano, M. R.C. Carvalho, G.A. Buck, J.C. Dujardin, and M. Tibayrenc. Evidence for genetic exchange and hybridization in Trypanosoma cruzi based on nucleotide sequences and molecular karyotype. Infect. Genet. & Evol. 2 173183 (2003). Stern, A.G., Carvalho, M.R.C., Buck, G.A., Adler, R.A. Rao, T.P. Disler, D., and Moxley, G. Association of Erosive Hand Osteoarthritis with a Single Nucleotide Polymorphism on the Gene Encoding Interleukin-1 Beta. Osteoart Cartil. 11: 394-402 (2003). Ventura, R.M., Takeda, G.F., Silva, R.A.M., Nunes, V.L.B., Buck, G.A., and Teixeira, M.M.G. Genetic relatedness among Brazilian stocks of Trypanosoma evansi from domestic and wild mammals by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis. Int J Parasitol. 32: 53-63 (2002). Ventura, R. M., Paiva, F., Silva, R.A.M., Takeda, G.F., Buck, G.A., and Teixeira, M.M.G. Trypanosoma vivax: characterization of the spliced-leader gene of a Brazilian stock and species-specific detection by PCR amplification of an intergenic spacer sequence. Exp Parasitol. 99: 37-48 (2001). Wen, L., Xu, P., Benegal, G., Carvalho, M.R.C., Butler, D.R. and G.A. Buck. Trypanosoma cruzi: exogenously regulated gene expression. Exp. Parasitol. 97: 196-204 (2001). Xu, P., Wen, L, Benegal, G., Wang, X., and G.A.Buck. Identification of a spliced leader RNA binding protein from Trypanosoma cruzi . Mol. Bioch. Parasitol. 112: 39-49 (2001). Wen, L., Xu, P., Benegal, G, Carvalho, M.R.C., and G. A. Buck. PPB1, a putative spliced leader RNA gene transcription factor in Trypanosoma cruzi. Mol. Bioch. Parasitol. 110: 207-221 (2000). Santos, W., and Buck, G.A. Simultaneous stable expression of neomycin phosphotransferase and green fluorescence protein genes in Trypanosoma cruzi . J. Parasitol. 86:1281-1288 (2000). Widmer, G., Akiyoshi, D., Buckholt, M.A., Feng, X., Rich, S.M., Deary, K.M., Bowman, C., Xu, P., Wang, Y., Wang, X., Buck, G.A., Tzipori, S. Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum . Mol. Biochem. Parasitol. 108: 187-197 (2000). Santos, W., Metcheva, I., and Buck, G.A. Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium. Mem. Inst. Os. Cruz. 95:111-114 (2000). Buck, G.A., Fox, J.W., Guthorpe, M., Hager, K.M., Naeve, C.W., Pon, R.T., Adams, P.S., and Rush, J. Design strategies and performance of custom DNA sequencing primers. BioTechniques 27: 528-536 (1999). Serrano, M.G., M. Campaner, G. A. Buck, M.M.G. Teixeira and E. P. Camargo. PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears. FEMS Microb. Let. 176: 241-249 (1999). Santos, W. and G.A. Buck. Polymorphisms at the topisomerase II gene locus provide more evidence for the partition of Trypanosoma cruzi into two major groups. J. Euk. Microbiol. 46: 17-23 (1999). Pinho, J.R., Zanotto, P.M., Ferreira, J.L., Sumita, L.M., Carrilho, F.J., da Silva, L.C., Capacci, M.L., Silva, A.O., Guz, B., Goncales, F.L. Jr., Goncales, N.S., Buck, G.A., Meyers, G.A., Bernardini, A.P. High prevalence of GB virus C in Brazil and molecular evidence for intrafamilial transmission. J Clin Microbiol. 37: 1634-1637 (1999). Serrano, M.G., Nunes, L.R., Campaner, M., Buck, G.A., Camargo, E.P., and Teixeira, M.M.G. Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene. Exp. Parasitol. 91: 268-279 (1999). Knight, H., Reynolds, T.R., Meyers, G.A. Pomponio, R.J., Buck, G.A., Wolf, B. Structure of the human biotinidase gene. Mammalian Genome 9: 327-330 (1998). Pomponio, R.J., J. Hymes, A. Pandya, B. Landa, P. Melone, R. Javaheri, R. Mardach, S. W. Morton, G. A. Meyers, T. Reynolds, G. A. Buck, and B. Wolf. Prenatal diagnosis of heterozygosity for biotinidase deficiency by enzymatic and molecular analyses. Prenat. Diagn. 18: 117-122 (1998). Stedman, T. D. R. Butler, and G. A. Buck. The HSP70 gene family and in Pneumocystis carinii : molecular and phyogenetic characterization of cytoplasmic members. J. Euk. Microbiol. 45: 589-599 (1998). Pinho, J.R., D. Takahashi, A. Fava, N. Goncales, F. Carrilho, R. Stucchi, F. Boncales, L. da Silva, M. Soares, G. Bensabath, G.A. Buck, G. Meyers, and P. Bernardini. Transfusion-transmitted virus (TTV) in Brazil. Priliminary Report. Rev. Inst. Med. Trop. S. Paulo 40: 335-336 (1998). Norrgard, K.J., R.J. Pomponio, K.L. Swango, J. Hymes, T.R.Reynolds, G.A.Buck, and B. Wolf. Double mutation (A171T toD444H) is a common cause of projound biotinidase deficiency in children ascertained by newborn screening in the United States. Human Mutation, Mutation in Brief #128 (1997) on-line. Floeter-Winter, L.M., Souto, R., Stolf, B., Nunes, L.R., Carvalho, M.R.C., Zingales, B. and G. A. Buck. Is the rRNA promoter a marker for speciation in Trypanosoma cruzi ? Exp. Parasitol. 86: 232-234 (1997). Norrgard, K.J., R.J. Pomponio, K.L.Swango, J. Hymes, T. Reynolds, G.A. Buck, and B. Wolf. Mutation Q456H is the most common cause of projound biotinidase deficiency in children ascertained by newborn screening in the United States. Biochem. Mol. Medicine 61: 22-27 (1997). Nunes, L.R., Carvalho, M.R.C., and G.A. Buck. Trypanosoma cruzi strains partition into two groups based on the structure and function of the SL RNA and rRNA gene promoters. Mol. Biochem. Parasitol. 86: 211-224 (1997). Pomponio, R.J., T.R. Reynolds, H. Mandel, O. Admoni, P. Melone, G.A. Buck, and B. Wolf. Profound biotinidase deficiency caused by a point mutation that creates a downstream cryptic 3' splice acceptor site within an exon of the human biotinidase gene. Hum. Mol. Genet. 6: 739-745 (1997). Pomponio, R.J., J. Hymes, T.R. Reynolds, G.A. Meyers, K. Fleischhauer, G.A. Buck and B. Wolf. Mutations in the human biotinidase gene that cause profound biotinidase deficiency in symptomatic children: molecular, biochemical and clinical analysis. Pediat. Res. 42: 840-848 (1997). Pomponio, R.J., K.J. Norrgard, J. Hymes, T.R. Reynolds, G.A. Buck, and B. Wolf. Arg538 to Cys mutation in a CpG dinucleotide of the human biotinidase gene is the second most common cause of profound biotinidase deficiency in symptomatic children. Hum. Gen. 99: 506-512 (1997). Nunes, L.R., M. R. Carvalho, A. Shakarian and G.A. Buck. Characterization of the spliced leader gene promoter in Trypanosoma cruzi. Gene 188: 157-168 (1997). Teixeira, M.M.G., M.G. Serrano, L. R. Nunes, M. Campaner, G. A. Buck and E. P. Camargo. Trypanosomatidae: a spliced-leader-derived probe specific for the genus Phytomonas . Exp. Parasitol. 84: 311-19 (1996). Buck, G.A., T.A. Stedman, and I. Metcheva. Molecular analysis of BiP and other HSP70 gene homologs in Pneumocystis carinii . Mem. Inst. Oswaldo Cruz 91: 7-9 (1996). Pomponio, R.J., V. Narashimhan, T.R. Reynolds, G.A. Buck, L.F. Povirk, and B. Wolf. Deletion/insertion mutation that causes biotinidase deficiency may result from the formation of a quasipalindromic structure. Hum. Molec. Gen. 5: 1657-1661 (1996). Stedman, T. and G. A. Buck. Identification, characterization and expression of the BiP ER resident chaperonins in Pneumocystis carinii . Inf. Im. 64: 4463-4471 (1996). G.A. Buck, Pon, R. T. (shared first author), K.M. Hager, C.W.Naeve, R.L. Niece, M. Robertson, and A.J. Smith. A multi-facility survery of oligonucleotide synthesis and an examination of the performance of unpurified primers in automated DNA sequencing. BioTechniques 21: 680-685 (1996). Metcheva, I., Stedman, T. and G. A. Buck. An arrayed bacteriophage P1 library of Pneumocystis carinii . J. Euk. Microbiol. 43: 171-176 (1996). Espinel-Ingroff, A., A. Quart, L. Steele-Moore, I. Metcheva, G.A. Buck, V.L. Bruzzese, and D. Reich. Molecular karyotyping of multiple yeast species isolated from nine patients with AIDS during prolonged fluconazole therapy. J. Med.& Vet. Mycol. 34: 111-116 (1996). |
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