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The research Laboratory of Roberta O'Connor, PHD in the Division of Geographic Medicine and Infectious Disease at Tufts Medical Center studies host-pathogen interactions of the zoonotic apicomplexan parasites, Cryptosporidium and Toxoplasma. Our work on these parasites encompasses immunobiology, molecular mechanisms of attachment and invasion and drug discovery. The drug discovery project is a serendipitous offshoot of a project conducted with collaborators at the Ocean Genome Legacy Center at Northeastern University, investigating the novel digestive strategy of shipworms, marine mollusks that burrow into and eat wood.
Significance: Cryptosporidium is a gastrointestinal apicomplexan parasite that is the etiological agent of water borne diarrheal disease outbreaks world-wide. In developing countries where it is endemic, it is the second most common cause of diarrhea in infancy, causing growth and developmental delays even when the infection is asymptomatic. This parasite is also an opportunistic infection of AIDS patients, in whom it can cause chronic, debilitating and sometimes fatal diarrheal disease. There is no vaccine and no universally effective treatment for Cryptosporidium. Furthermore, Cryptosporidium cannot be continuously cultured or genetically manipulated, leading to a paucity of information on this pathogen that might inform drug discovery or vaccine development. These difficulties have been compounded by the lack of investment in the research infrastructure needed to develop interventions for this disease. However, this area of research is now ripe for expansion of extramural funding. With the publication of the Gate’s funded Global Enteric Multicenter Study, which found that Cryptosporidium was second only to rotavirus as a cause of infant diarrhea, attention is again focused on this neglected pathogen.
For many years we have studied surface antigens that are involved in attachment and invasion of Cryptosporidium into host cells for the purposes of vaccine development. Because many Cryptosporidium surface antigens are mucin-like glycoproteins that are extensively O-glycosylated, my laboratory pioneered an expression system in Toxoplasma gondii to study these antigens. We showed that one of these glycoproteins, CpMuc4, was present on the surface of invasive sporozoite stages, appeared to interact with host cell receptors, and was a target of a neutralizing anti-CpMuc4 peptide antibody. All these data pointed to this antigen as an excellent vaccine candidate but further characterization of the post-translational modifications has proven difficult. Recently, our collaborator, Dr Weiss at Albert Einstein College of Medicine, has developed Toxoplasma strains knocked out for each of the O-glycosyltransferases and we are now starting to use these to express CpMuc4 glycosylation mutants to determine the specific role CpMuc4 plays in sporozoite invasion into host cells. Identification of the mechanisms of the receptor-ligand interaction will provide us with data to develop CpMuc4 as a vaccine candidate.
Bioactive compounds produced by environmental bacteria have been investigated for potential medical applications from pain medications to anti-microbials with great success. The search for new bioactive compounds has recently turned to mining the capacity of symbiotic bacterial communities, and many investigations suggest that this approach will be an extremely fruitful source of previously undiscovered secondary metabolites. Since marine mollusks are susceptible to infection with gregarine parasites, which are ancestral to all apicomplexan parasites, we hypothesized that mollusk symbiotic bacteria may have evolved the ability to produce compounds that would protect the host against parasitic infection. In support of this hypothesis, and with limited screening, we discovered a symbiotic bacterium that produces a compound inhibitory to intracellular growth of both T. gondii and Cryptosporidium parvum. Interestingly, secretion of this compound is co-incident with the formation of biofilms, suggesting a potential role for this compound in bacterial communication. Research to identify the compound and it’s mechanism of action are ongoing.
We have an ongoing drug discovery project to identify other potential anti-parasitic drugsThis project was started with funds from the Philippine Mollusk Symbiont-International Cooperative Biodiversity Group. The focus of the PMS-ICBG is to identify bioactive compounds produced by marine mollusks that can be developed into pharmaceuticals targeted against diseases of importance to the Philippines. The Philippines is a hot-spot for mollusk diversity and already many promising drug leads have been identified. The relative ease with which this anti-parasitic compound was identified suggests that this project will be remarkably fruitful.
Shipworms are marine bivalve mollusks that survive by burrowing into and eating wood; in the days of wooden ships they were the bane of sailors everywhere, and descriptions of them can be found in even in early Greek writings. Unlike other xylophagous animals, such as termites or ruminants, shipworms lack a bacterial community in the gut. They do, however, harbor abundant intracellular bacteria within the cells of their gills-an organ remote from the site of wood digestion in the gut. These bacteria are known to be cellulolytic, but it was unclear what role they could play in digestion given their location.
In collaboration with Department of Energy’s Joint Genome Institute, we sequenced the Bankia setacea shipworm gill bacterial metagenome and the genomes of four isolates cultured from B. setacea gill then used these databases to inform proteomic studies of the gill and the caecum contents. As would be expected for a bacteria-containing tissue, the gill proteome consisted of primarily housekeeping and core metabolism proteins, membrane proteins and chaperonins. In stark contrast to the gill proteome, the proteome of the caecum contents consisted primarily of PCWP-active proteins. Moreover, we showed that these PCWP-active proteins, encoded in the gill bacterial genomes, are the most abundant proteins found in the caecum and are active against a variety of PCWP components. These studies uncovered a completely new mechanism of digestion, in which intracellular bacteria produce digestive enzymes that are transported out of the bacterial cell, out of the host cell and are selectively transported from the gill to the lumen of the digestive tract, there to breakdown food products for the host animal. Not only does this work describe a novel mode of digestion but these observations also extend the range of known functions of intracellular bacteria.
The unusual digestive system of the shipworm relies on sterility of the caecum that is maintained in the face of abundant substrate for bacterial growth and in the marine environment which is rife with a wide diversity and abundance of bacteria. We are very interested in the source and character of the compounds that maintain the sterility of the shipworm caecum, especially for what this system can reveal about avoiding drug resistance. Towards this end we are characterizing the genomes of the gill bacteria and identifying their secondary metabolite loci. The bacterial strain found to produce the anti-parasitic compound described above is a shipworm gill endosymbiont. We believe that continued investigation of these bacteria will reveal novel and efficacious antimicrobials.
Our previous work examined immune responses to Cryptosporidium polymorphic antigens in Bangladeshi and South Indian children and characteristics of HIV-Cryptosporidium co-infections in Kenyan adults. Much to our surprise, there is virtually no data on cryptosporidiosis in Philippine children. As an adjunct to the work funded by the PMS-ICBG, we are trying to identify Filipino collaborators to design and conduct clinical studies of cryptosporidiosis in Filipino children. Many people residing in rural areas in the Philippines lack access to clean water, and with the new and highly sensitive tools we now have to diagnose infection, we expect to document a significant burden of infection as has been described in other sites. Interestingly, many Filipinos in coastal areas consume shipworms and it is possible that this addition to the diet is protective against gastrointestinal infections; baseline epidemiological studies would allow us to design studies to address this question.
1. R.M. O’Connor, J.M. Fung, K.H. Sharp, J. Benner, C. McClung, S. Cushing, E. Lamkin, A. Fomenkov, B. Henrissat, Y. Londer, M. B. Scholz, J. Posfai, S. Malfatti, S.G. Tringe, T. Woyke, R.R. Malmstrom, D. Coleman-Derr, M.A. Altamia, S. Dedrick, S. T. Kaluziak, M.G. Haygood and D.L. Distel. Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk. Proc Natl Acad Sci U S A. 2014 Nov 10. pii: 201413110. [Epub ahead of print]**
**This work was featured in Science magazine’s Editor’s Choice (vol346, issue 6214, p1196) and has been highlighted in several press releases.
2. Paluszynski, J., Monahan, Z., Williams, M., Lai, O. Morris, C. Burns, P and O’Connor, RM*. Biochemical and functional characterization of CpMuc4, a Cryptosporidium surface antigen that binds to host epithelial cells. Mol. Biochem Parasitol. 193(2):114-21, 2014
3. Wanyiri, J.W., Kanyi, H., Maina, S., Wang, D.E., Steen, A., Mungai, A., Gachuhi, K., O’Connor, RM, Mwamburi, M., Wamae, N. and Ward, H.D. Cryptosporidiosis in HIV/AIDS patients in Kenya. AJTMH, 91(2):319-28, 2014.
4. Wanyiri, J.W., Kanyi, H., Maina, S., Wang, D.E., Steen, A., Mungai, A. , Gachuhi, K., O’Connor, RM, Mwamburi, M., Wamae, N. and Ward, H.D. Infectious Diarrhea in HIV/AIDS patients in Nairobi, Kenya. Trans R Soc Trop Med Hyg. 107:631-8, 2013
5. Betcher, M.A., Fung, J.M., Han, A.W., O'Connor, R.M., Seronay, R., Concepcion, G.P., Distel, D.L., Haygood, M.G. Microbial distribution and abundance in the digestive system of five shipworm species (bivalvia: teredinidae). PLoS One; 7(9):e45309, 2012.
6. Lai, O., Morris, C., Ahmed, S., Karim, M.M., Khan, W., Ward, H. and O’Connor, RM*. Serum antibody responses to a polymorphic Cryptosporidium mucin antigen in Bangladeshi children with cryptosporidiosis. AJTMH, 85:464-70, 2011
7. Hira, K.G., Mackay, M.R., Hempstead, A.D., Ahmed, S., Karim, M.M., O'Connor, RM, Hibberd, P.L., Calderwood, S.B., Ryan, E.T., Khan, W.A., Ward, H.D.. Genetic diversity of Cryptosporidium spp. from Bangladeshi children. J Clin Microbiol. 49:2307-10, 2011.
8. Chatterjee, A., Banerjee, S., Steffen, M., Moore, L. L., O’Connor, R. M., Ward, H. D., Robbins, P. W., Samuelson, J. Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall. Eukaryotic Cell 2010; 9:84–96.
9. Ajjampur, SS, Liakath FB, Kannan A, Rajendran P, Sarkar R, Moses PD, Simon A, Agarwal I, Mathew A, O'Connor RM, Ward H, Kang G. Multi-site study of cryptosporidiosis in Indian children with diarrhea. J Clin Microbiol. 48:2075-81, 2010. (PMID: 20392919).
10. J.W. Wanyiri, P. Techasintana, R.M. O’Connor, M. Blackman, K. Kim and H. Ward. Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvum infection in vitro. Eukaryotic Cell, 8:470-477, 2009. (PMID: 19168760)
11. O’Connor R. M*, P.B. Burns, T. Ha-Ngoc, K. Scarpato, W. Khan, G. Kang, and H. Ward. The polymorphic mucin antigens CpMuc4 and CpMuc5 are integral to Cryptosporidium parvum infection in vitro. Eukaryotic Cell, 8:461-469, 2009. (PMID: 19168754)
12. O’Connor R. M*, J. W. Wanyiri, A.M. Cevallos, J. W. Priest, and H. D. Ward Cryptosporidium parvum glycoprotein gp40 localizes to the sporozoite surface by association with gp15. Mol Biochem Parasitol 156: 80-83, 2007
13. O’Connor R. M*., Wanyiri, J., Wojczyk, B., Kim, K., and Ward, H. D. Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii. Mol Biochem Parasitol 152: 149-58, 2007
14. Wanyiri, J. W., O'Connor, R.M.., Allison, G., Kim, K., Kane, A., Qiu, J., Plaut, A. G., and Ward, H. D. Proteolytic processing of the Cryptosporidium spp. glycoprotein gp40/15 by human furin and by a parasite-derived furin-like protease activity. Infect Immun 75:184-192. 2007
15. O’Connor, R.M*., Kim, K., Khan, F., and Ward, H. Expression of Cpgp40/15 in Toxoplasma gondii: a surrogate system for the study of Cryptosporidium glycoprotein antigens. Infect Immun, 71:6027-34, 2003.
16. Leav, B.A., Mackay, M.R., Anyanwu, A., O’Connor, R.M., Cevallos, A.M., Kindra, G., Rollins, N.C., Bennish, M.L., Nelson, R.G., and Ward, H.D. Analysis of sequence diversity at the highly polymorphic Cpgp40/15 locus among Cryptosporidium isolates from HIV-infected children in South Africa. Infect Immun, 70:3881-90, 2002.
17. O’Connor, R.M*, Thorpe, C.M., Cevallos, A.M., and Ward, H. Expression of the highly polymorphic Cryptosporidium parvum Cpgp40/15 gene in genotype I and II isolates. Mol Biochem Parasitol, 119:203-15, 2002.
18. Allred, D.R., Carlton, J.R., Satcher, R.L., Long, J.A., Brown, W.C., Patterson, P.E., O'Connor, R.M., and Stroup, S.E. The ves multigene family of B. bovis encodes components of rapid antigenic variation at the infected erythrocyte surface. Molecular Cell, 5:153-62, 2000.
19. O’Connor, R.M. and Allred, D.R. Selection of Babesia bovis-infected erythrocytes for adhesion to endothelial cells coselects for altered variant erythrocyte surface antigen isoforms. J Immunol, 164:2037-45, 2000.
20. O’Connor, R.M., Long, J., and Allred, D.R. Cytoadherence of Babesia bovis-infected erythrocytes to bovine brain capillary endothelial cells provides an in vitro model for sequestration. Infect Immun, 67:3921-8, 1999.
21. O’Connor, R.M., Long, J., and Allred, D.R. Selection and recovery of minor parasite populations expressing unique surface antigen phenotypes. Mol Biochem Parasitol, 100:125-9, 1999.
22. Riggs, M.W., McNeil, M., Perryman, L.E., Stone, A.L., Scherman, M.S., and O'Connor, R.M. Cryptosporidium parvum sporozoite pellicle antigen recognized by a neutralizing monoclonal antibody is a β-mannosylated glycolipid. Infect Immun, 67:1317-22, 1999.
23. O'Connor, R.M., Lane, T.J., Stroup, S.E., and Allred, D.R. Characterization of a variant erythrocyte surface antigen (VESA1) expressed by Babesia bovis during antigenic variation. Mol Biochem Parasitol, 89:259-70, 1997.
Roberta M. O'Connor, PhD