About
John Samuelson is a researcher with a substantial body of work focusing on parasitology and molecular biology, particularly involving the study of Schistosoma mansoni and Entamoeba histolytica. His research includes investigations into the surface antigens and immune evasion mechanisms of schistosomula, the larval form of Schistosoma mansoni, as well as the molecular and cellular changes during their transformation and interaction with the host immune system. Samuelson has contributed to understanding the biochemical and cellular processes such as the loss of surface glycoproteins and glycolipids, activation of human complement by the cercarial glycocalyx, and the role of septate junctions in parasite development. Additionally, his work extends to the molecular cloning and characterization of genes related to drug resistance, protein kinases, and membrane transporters in Entamoeba histolytica, a protozoan parasite responsible for amoebiasis. This includes studies on multidrug resistance mechanisms, gene expression related to P-glycoproteins, and the molecular biology of various enzymes and proteins critical to parasite physiology. Samuelson's research has also involved the development of diagnostic tools such as DNA hybridization probes and polymerase chain reaction techniques for clinical diagnosis and epidemiological studies of parasitic infections. His extensive publication record reflects a career dedicated to advancing the understanding of parasite biology, host-parasite interactions, and the molecular basis of drug resistance in parasitic protozoa.
Research topics
- Biology
- Cell biology
- Chemistry
- Biochemistry
- Microbiology
- Genetics
- Pathology
Selected publications
mSphere · 2025-03-04
articleOpen accessSenior authorABSTRACT Acanthamoebae , which are free-living amoebae, cause corneal inflammation (keratitis) and blindness, if not quickly diagnosed and effectively treated. The walls of Acanthamoeba cysts contain cellulose and have two layers connected by conical ostioles. Cysts are identified by in vivo confocal microscopy of the eye or calcofluor-white- or Giemsa-labeling of corneal scrapings, both of which demand great expertise. Trophozoites, which use a mannose-binding protein to adhere to keratinocytes, are identified in eye cultures that delay diagnosis and treatment. We recently used structural and experimental methods to characterize cellulose-binding domains of Luke and Leo lectins, which are abundant in the inner layer and ostioles. However, no antibodies have been made to these lectins or to a Jonah lectin and a laccase, which are abundant in the outer layer. Here, confocal microscopy of rabbit antibodies (rAbs) to recombinant Luke, Leo, Jonah, and laccase supported localizations of GFP-tagged proteins in walls of transfected Acanthamoebae . rAbs efficiently detected calcofluor white-labeled cysts of 10 of the 11 Acanthamoeba isolates tested, including six T4 genotypes that cause most cases of keratitis. Further, laccase shed into the medium during encystation was detected by an enzyme-linked immunoassay. Structural and experimental methods identified the mannose-binding domain (ManBD) of the Acanthamoeba mannose-binding protein, while rAbs to the ManBD efficiently detected DAPI-labeled trophozoites from all 11 Acanthamoeba isolates tested. We conclude that antibodies to four cyst wall proteins and the ManBD efficiently identify Acanthamoeba cysts and trophozoites, respectively. IMPORTANCE Free-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of unlabeled cysts by in vivo confocal microscopy of the eye or calcofluor-white (CFW) labeled cysts by fluorescence microscopy of corneal scrapings. Alternatively, Acanthamoeba infections are diagnosed by the identification of trophozoites in eye cultures. Here, we showed that rabbit antibodies (rAbs) to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently identify CFW-labeled cysts of 10 of the 11 Acanthamoeba isolates tested. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rAbs to the mannose-binding domain (ManBD) of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identify DAPI-labeled trophozoites of all 11 Acanthamoeba isolates tested. In summary, four wall proteins and the ManBD appear to be excellent targets for the diagnosis of Acanthamoeba cysts and trophozoites, respectively.
Glycobiology · 2025-09-01 · 1 citations
articleOpen accessToxoplasma gondii is a highly successful intracellular mammalian and avian pathogen that must adapt to a wide range of intracellular and extracellular environments. A mechanism that may support this is the modification of hydroxyamino acid rich sequences of nucleocytoplasmic proteins with O-fucose. O-fucosylation of possibly hundreds of proteins is mediated by a single highly conserved nucleocytoplasmic enzyme. Deletion of the SPY O-fucosyltransferase gene is tolerated but inhibits parasite proliferation in fibroblasts and their accumulation in mouse brains. A prior ectopic expression study suggested that O-fucose is required to detect proteins considered essential. To distinguish whether the SPY requirement was specific to the method or for protein expression per se, GPN1, an RNA polymerase chaperone, was epitope-tagged at its endogenous locus in both normal and SPYΔ strains. GPN1 was shown to be substantially and quantitatively O-fucosylated and exhibited a modest 24% reduction in level in SPYΔ cells. Proteomic analysis of its interactome indicated that fucosylation did not affect its association with RNA polymerase subunits. GPN1 was mostly cytoplasmic based on super-resolution immunofluorescence microscopy, and this localization was not affected by O-Fuc. A fusion of its O-fucosylated serine-rich domain to yellow fluorescent protein behaved similarly. In comparison, the abundance of a Zn-finger containing protein also depended on SPY, whereas the abundance and localization of ERK7 were not affected nor were levels of two other proteins. Thus O-fucose directly but modestly promotes the accumulation of select targets, but it does not enforce their localization in nuclear assemblies that are highlighted by immunofluorescence studies.
mSphere · 2025-02-06 · 4 citations
articleOpen accessABSTRACT Cellular adaptations to change often involve post-translational modifications of nuclear and cytoplasmic proteins. An example found in protists and plants is the modification of serine and threonine residues of dozens to hundreds of nucleocytoplasmic proteins with a single fucose (O-fucose). A nucleocytoplasmic O-fucosyltransferase occurs in the pathogen Toxoplasma gondii , the social amoeba Dictyostelium , and higher plants, where it is called Spy because mutants have a spindly appearance. O-fucosylation, which is required for optimal proliferation of Toxoplasma and Dictyostelium , is paralogous to the O-GlcNAcylation of nucleocytoplasmic proteins of plants and animals that are involved in stress and nutritional responses. O-fucose was first discovered in Toxoplasma using Aleuria aurantia lectin, but its broad specificity for terminal fucose residues on N- and O-linked glycans in the secretory pathway limits its use. Here we present affinity-purified rabbit antisera that are selective for the detection and enrichment of proteins bearing fucose-O-Ser or fucose-O-Thr. These antibodies detect numerous nucleocytoplasmic proteins in Toxoplasma, Dictyostelium , and Arabidopsis , as well as O-fucose occurring on secretory proteins of Dictyostelium and mammalian cells except when blocked by further glycosylation. The antibodies label Toxoplasma , Acanthamoeba , and Dictyostelium in a pattern reminiscent of O-GlcNAc in animal cells including nuclear pores. The O-fucome of Dictyostelium is partially conserved with that of Toxoplasma and is highly induced during starvation-induced development. These antisera demonstrate the unique antigenicity of O-fucose, document the conservation of the O-fucome among unrelated protists, and enable the study of the O-fucomes of other organisms possessing O-fucosyltransferase-like genes. IMPORTANCE O-fucose (O-Fuc), a form of mono-glycosylation on serine and threonine residues of nuclear and cytoplasmic proteins of some parasites, other unicellular eukaryotes, and plants, is understudied because it is difficult to detect owing to its neutral charge and lability during mass spectrometry. Yet, the O-fucosyltransferase enzyme (OFT) is required for optimal growth of the agent for toxoplasmosis, Toxoplasma gondii , and an unrelated protist, the social amoeba Dictyostelium discoideum . Furthermore, O-fucosylation is closely related to the analogous process of O-GlcNAcylation of thousands of proteins of animal cells, where it plays a central role in stress and nutritional responses. O-Fuc is currently best detected using Aleuria aurantia lectin (AAL), but in most organisms, AAL also recognizes a multitude of proteins in the secretory pathway that are modified with fucose in different ways. By establishing the potential to induce highly specific rabbit antisera that discriminate O-Fuc from all other forms of protein fucosylation, this study expands knowledge about the protist O-fucome and opens a gateway to explore the potential occurrence and roles of this intriguing posttranslational modification in bacteria and other protist pathogens such as Acanthamoeba castellanii .
bioRxiv (Cold Spring Harbor Laboratory) · 2024-10-17
preprintOpen accessABSTRACT Cellular adaptations to change often involve post-translational modifications of nuclear and cytoplasmic proteins. An example found in protists and plants is the modification of serine and threonine residues of dozens to hundreds of nucleocytoplasmic proteins with a single fucose (O-Fuc). A nucleocytoplasmic O-fucosyltransferase (OFT) occurs in the pathogen Toxoplasma gondii , the social amoeba Dictyostelium , and higher plants, where it is called Spy because mutants have a spindly appearance. O-fucosylation, which is required for optimal proliferation of Toxoplasma and Dictyostelium , is paralogous to the O-GlcNAcylation of nucleocytoplasmic proteins of plants and animals that is involved in stress and nutritional responses. O-Fuc was first discovered in Toxoplasma using Aleuria aurantia lectin, but its broad specificity for terminal fucose residues on N- and O-linked glycans in the secretory pathway limits its use. Here we present affinity purified rabbit antisera that are selective for the detection and enrichment of proteins bearing fucose-O-Ser or fucose-O-Thr. These antibodies detect numerous nucleocytoplasmic proteins in Toxoplasma, Dictyostelium , and Arabidopsis , as well as O-Fuc occurring on secretory proteins of Dictyostelium and mammalian cells, although the latter are frequently blocked by further glycosylation. The antibodies label Toxoplasma , Acanthamoeba , and Dictyostelium in a pattern reminiscent of O-GlcNAc in animal cells including nuclear pores. The O-fucome of Dictyostelium is partially conserved with that of Toxoplasma and is highly induced during starvation-induced development. These antisera demonstrate the unique antigenicity of O-Fuc, document conservation of the O-fucome among unrelated protists, and will enable the study of the O-fucomes of other organisms possessing OFT-like genes. IMPORTANCE O-fucose, a form of mono-glycosylation on serine and threonine residues of nuclear and cytoplasmic proteins of some parasites, other unicellular eukaryotes, and plants, is understudied because it is difficult to detect owing to its neutral charge and lability during mass spectrometry. Yet the O-fucosyltransferase enzyme (OFT) is required for optimal growth of the agent for toxoplasmosis, Toxoplasma gondii , and an unrelated protist, the social amoeba Dictyostelium discoideum . Furthermore, O-fucosylation is closely related to the analogous process of O-GlcNAcylation of thousands of proteins of animal cells, where it plays a central role in stress and nutritional responses. O-Fuc is currently best detected using Aleuria aurantia lectin (AAL), but in most organisms AAL also recognizes a multitude of proteins in the secretory pathway that are modified with fucose in different ways. By establishing the potential to induce highly specific rabbit antisera that discriminate O-Fuc from all other forms of protein fucosylation, this study expands knowledge about the protist O-fucome and opens a gateway to explore the potential occurrence and roles of this intriguing posttranslational modification in bacteria and other protist pathogens such as Acanthamoeba castellanii .
bioRxiv (Cold Spring Harbor Laboratory) · 2024-10-17
preprintOpen accessSenior authorCorrespondingAbstract Acanthamoebae , which are free-living amoebae, cause corneal inflammation (keratitis) and blindness, if not diagnosed and effectively treated. While trophozoites adhere to and damage the cornea, Acanthamoeba cysts, the walls of which contain cellulose and have two layers connected by conical ostioles, are the diagnostic form by microscopy of the eye or of corneal scrapings. We recently used structural and experimental methods to characterize cellulose-binding domains of Luke and Leo lectins, which are abundant in the inner layer and ostioles. However, no antibodies have been made to these lectins or to a Jonah lectin and a laccase, which are abundant in the outer layer. Here we used confocal microscopy to show that rabbit antibodies to recombinant Luke, Leo, Jonah, and laccase generally support localizations of GFP-tagged proteins in walls of transfected Acanthamoebae. Rabbit antibodies to all four wall proteins efficiently detected calcofluor white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC, including five T4 genotypes that cause most cases of keratitis. Laccase shed into the medium during encystation was detected by an enzyme-linked immunoassay. We also used structural and experimental methods to characterize the mannose-binding domain of an Acanthamoeba mannose-binding protein and showed that rabbit antibodies to the mannose-binding domain efficiently detected trophozoites of all 11 Acanthamoeba isolates. We conclude that four wall proteins are all excellent targets for diagnosing Acanthamoeba cysts in the eye or corneal scrapings, while the mannose-binding domain is an excellent target for identifying trophozoites in cultures of corneal scrapings. Importance Free-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of cysts by microscopy of the eye or of corneal scrapings, using calcofluor-white that unfortunately cross-reacts with fungi and plants. Alternatively, Acanthamoeba infections are diagnosed by identification of trophozoites in cultures of scrapings. Here we showed that rabbit antibodies to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently detect calcofluor-white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rabbit antibodies to the mannose-binding domain of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identifies trophozoites of all 11 ATCC isolates. In summary, four wall proteins and the ManBD appear to be excellent targets for diagnosis of Acanthamoeba cysts and trophozoites, respectively.
mSphere · 2024 · 6 citations
Senior authorCorresponding- Biology
- Biochemistry
- Chemistry
resistant to surface disinfectants, hand sanitizers, contact lens sterilizers, and antibiotics applied to the eye. The goal here was to understand better how proteins are targeted to specific locations in the cyst wall. To this end, we identified three new proteins in the outer layer of the cyst wall, which may be targets for diagnostic antibodies in corneal scrapings. We used structural predictions and mutated proteins to show linear arrays of aromatic amino acids of two unrelated wall proteins are necessary for binding cellulose and proper wall localization. We showed early expression during encystation causes proteins to localize to the outer layer, while later expression causes proteins to localize to the inner layer and the ostioles.
bioRxiv (Cold Spring Harbor Laboratory) · 2024-02-02
preprintOpen accessSenior authorCorrespondingABSTRACT The cyst wall of the eye pathogen Acanthamoeba castellanii contains cellulose and chitin and has ectocyst and endocyst layers connected by conical ostioles. Previously, we used mass spectrometry of purified walls to identify an abundant laccase and three families of lectins (Jonah, Luke, and Leo). Here we show that frameshifts in the protein prediction in AmoebaDB, which incorrectly add 12 transmembrane helices, cause Jonah to mislocalize to a ring around ostioles rather than to the ectocyst layer. RT-PCR, double labels with GFP and RFP or mCherry, and promoter swaps show that ectocyst localization does not just correlate with but is caused by earlier expression, while localization in the endocyst layer and ostioles is caused by later expression. A chitin-binding domain from an Entamoeba chitinase shows chitin forms thick fibrils in the ectocyst layer and a honeycomb in the endocyst layer. AlphaFold shows Ac wall proteins originate from bacteria by horizontal gene transfer (β-helical folds of Jonah and three cupredoxin-like domains of the laccase), share common ancestry with wall proteins of slime molds (β-jelly-roll folds of Luke), or are unique to Acanthamoeba (four disulfide knots of Leo). Ala mutations show linear arrays of aromatic amino acids in β-jelly-roll folds of Luke and disulfide knots of Leo are necessary for binding cellulose and proper localization of proteins in the cyst wall. Finally, rabbit antibodies to recombinant Jonah, Luke, Leo, and laccase efficiently detect calcoflour white-labeled cysts of 10 of 11 Acanthamoeba isolates tested, suggesting all four proteins are excellent diagnostic targets. IMPORTANCE Acanthamoebae are free-living amoeba in the soil and water that cause Acanthamoeba keratitis in under-resourced countries, where water for washing hands may be scarce. Acanthamoeba is an emerging pathogen in the United States, because of its association with contact lens use. Here we show early expression during encystation causes a Jonah lectin and a laccase to localize to the outer layer of the cyst wall, while later expression cause Luke and Leo lectins to localize to the inner layer and the conical ostioles that connect the layers. We used structural predictions to identify the aromatic amino acids of Luke and Leo necessary for binding cellulose in the wall and to identify domains of Jonah and laccase useful for making recombinant proteins to immunize rabbits. Rabbit antibodies to Jonah, Luke, Leo, and laccase all efficiently detected cysts of ten Acanthamoeba isolates, including five T4 genotypes that cause most keratitis cases.
Fucopeptides-ETD raw data Supplemental Fig. 8
Figshare · 2022-01-01
datasetOpen accessMass Spectrometry raw data files supporting data on supplemental Fig. 8
Fucopeptides-HCD raw data Fig. 9
Figshare · 2022-01-01
datasetOpen accessMass Spectrometry HCD raw data files supporting information on Figure 9.
Glycobiology · 2022-10-17 · 5 citations
articleOpen accessO-GlcNAcylation is a prominent modification of nuclear and cytoplasmic proteins in animals and plants and is mediated by a single O-GlcNAc transferase (OGT). Spindly (Spy), a paralog of OGT first discovered in higher plants, has an ortholog in the apicomplexan parasite Toxoplasma gondii, and both enzymes are now recognized as O-fucosyltransferases (OFTs). Here we investigate the evolution of spy-like genes and experimentally confirm OFT activity in the social amoeba Dictyostelium-a protist that is more related to fungi and metazoa. Immunofluorescence probing with the fucose-specific Aleuria aurantia lectin (AAL) and biochemical cell fractionation combined with western blotting suggested the occurrence of nucleocytoplasmic fucosylation. The absence of reactivity in mutants deleted in spy or gmd (unable to synthesize GDP-Fuc) suggested monofucosylation mediated by Spy. Genetic ablation of the modE locus, previously predicted to encode a GDP-fucose transporter, confirmed its necessity for fucosylation in the secretory pathway but not for the nucleocytoplasmic proteins. Affinity capture of these proteins combined with mass spectrometry confirmed monofucosylation of Ser and Thr residues of several known nucleocytoplasmic proteins. As in Toxoplasma, the Spy OFT was required for optimal proliferation of Dictyostelium under laboratory conditions. These findings support a new phylogenetic analysis of OGT and OFT evolution that indicates their occurrence in the last eukaryotic common ancestor but mostly complementary presence in its eukaryotic descendants with the notable exception that both occur in red algae and plants. Their generally exclusive expression, high degree of conservation, and shared monoglycosylation targets suggest overlapping roles in physiological regulation.
Recent grants
NIH · $4.0M · 2017
Structure and Development of Oocyst and Sporocyst Walls
NIH · $1.8M · 2015–2020
NIH · $524k · 1994
Genetic modification of cultured Cryptosporidium to test the autoinfection model
NIH · $453k · 2017–2019
The Biochemistry and Cell Biology of the SpindlyO-fucosyltransferase of Toxoplasma
NIH · $2.1M · 2020–2024
Frequent coauthors
- 49 shared
Phillips W. Robbins
Boston University
- 28 shared
Julie Nixon
University of Edinburgh
- 28 shared
Catherine E. Costello
Boston University
- 28 shared
Andrew G. McArthur
Berkeley College
- 28 shared
Mitchell L. Sogin
Marine Biological Laboratory
- 27 shared
Hilary G. Morrison
Marine Biological Laboratory
- 26 shared
Jike Cui
- 25 shared
Michael J. Cipriano
University of Georgia
Labs
Education
- 1984
M.D.
Harvard University
- 1984
Ph.D.
Harvard University
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