Serotyping of Cryptococcus neoformans by dot enzyme assay

Belay, Tesfaye; Cherniak, Robert; O’Neill, Eric B.; Kozel, Thomas R.

doi:10.1128/jcm.34.2.466-470.1996

Cited by 30 publications

(9 citation statements)

References 27 publications

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“…); J9, J22, J11 (16), and 69 are clinical isolates from the Bronx, N.Y. NIH 34 was obtained from Robert Cherniak NIH 371 was obtained from J. E. Bennett, and SB4A and SB6A were obtained from E. Spitzer. Serotype A is represented by strains NIH 371, J11, SB4A, and SB6A; serotype B is represented by strain 24065; serotype C is represented by strains NIH 34 and ATCC 32608; and serotype D is represented by strains 24067, J9, and J22 (4). Strain 52817 is an acapsular strain.…”

Section: Methodsmentioning

confidence: 99%

Extracellular proteins of Cryptococcus neoformans and host antibody response

1997

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Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain-and growth conditiondependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and ␤-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection. Cryptococcus neoformans is an opportunistic fungal pathogen which causes an incurable, life-threatening meningoencephalitis in 6 to 8% of patients with AIDS. In New York City alone, there are over 1,000 cases of cryptococcosis per year (15). The immune response to and biological effects of the C. neoformans polysaccharide antigen have been extensively studied (13, 23, 36, 43, 45). The polysaccharide antigen glucuronoxylomannan (GXM) is the major component of the capsule and has an apparent molecular mass of ϳ1,000 kDa (6). Antigenic differences in GXM provide the basis for separating C. neoformans strains into four serotypes (13). C. neoformans strains lacking the polysaccharide capsule are avirulent (10, 20, 31). Antibodies to GXM have been shown to protect mice against cryptococcal infection (19, 41, 42). In contrast, little is known about the proteins secreted or released by C. neoformans, despite evidence that they elicit important immune responses. Immunization with a concentrated filtrate of cell culture supernatant known as CneF produced a delayed-type hypersensitivity response (44), although the active protein antigens were not identified. To date, systematic studies of C. neoformans supernatant proteins have not been undertaken, but several proteins have been described (1, 11, 22, 25, 26, 50, 54, 55). Identification and characterization of C. neoformans extracellular proteins are important because proteinases, esterases, and lipases have been associated with virulence in other pathogens (14, 30, 34, 37, 52, 53) and c...

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Section: Methodsmentioning

confidence: 99%

Extracellular proteins of Cryptococcus neoformans and host antibody response

1997

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“…DEA and ELISA. The preparation of antigen-coated nitrocellulose strips and their application in DEA were described previously (2,3). Peroxidase-labeled goat anti-mouse immunoglobulin G (whole molecule) and metal-enhanced DAB substrate (Pierce, Rockford, Ill.) were used for antibody detection.…”

Section: Methodsmentioning

confidence: 99%

“…The optimal conditions for preparing the microtiter plates and performing the ELISA analyses were described previously (1)(2)(3)11). The GXMs of different serotypes exhibit different efficiencies of binding to polystyrene microtiter plates.…”

Section: Methodsmentioning

confidence: 99%

“…Serotyping of C. neoformans isolates has been based on the reactivities of whole cells to polyclonal sera that have been selectively absorbed with whole yeast cells (2,3,29,31). The presence of at least eight antigenic factors, distributed among the serotypes of C. neoformans, has been proposed based on the reactivities of the factor sera in yeast cell agglutination reactions (29,31).…”

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confidence: 99%

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Reactivity patterns and epitope specificities of anti-Cryptococcus neoformans monoclonal antibodies by enzyme-linked immunosorbent assay and dot enzyme assay

et al. 1997

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Cryptococcus neoformans glucuronoxylomannans (GXM) are capsular polysaccharides important for virulence in cryptococcosis. This study used dot enzyme assays (DEA) and enzyme-linked immunosorbent assays (ELISA) to determine the reactivity patterns of 21 murine monoclonal antibodies (MAbs) with structurally defined GXMs from five serotypes. The MAbs were categorized into eight groups on the basis of DEA and five groups on the basis of ELISA. MAbs 302, 339, and 439 were studied extensively for their binding to various native and chemically modified GXMs. Quantitative variation in the inhibitory effects of GXMs on the binding of MAbs 302, 339, and 439 were observed by competitive ELISA. O-Deacetylation of serotype A, B, and D GXM resulted in the complete loss of their inhibitory properties. Carboxyl group reduction of GXMs from serotypes A and D resulted in a significant decrease of inhibitory activity for MAb. Xylomannans and methyl glycosides exhibited no detectable inhibitory activity on MAb binding to GXM. The results indicate (i) the existence of five to eight MAb-defined distinct epitopes in C. neoformans GXM that can elicit antibody responses, (ii) MAb detection of antigenic variation within GXMs assigned to a particular serotype, (iii) good correspondence between the patterns of MAb reactivities and polyclonal rabbit factor sera, (iv) good agreement between MAb molecular structure and serotype reactivity, and (v) a dependence of the serotype reactivity profile for a given MAb on the technique used to measure binding.

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“…grubii (serotype A); a former third variety (C. neoformans var. gattii, serotype B) was reclassified as a separate species C. gattii [10][11][12][13]. Recently, based on the analysis of genealogical concordance, coalescence-based, and species tree approaches, some researchers proposed that the current C. neoformans var.…”

Section: Introductionmentioning

confidence: 99%

Zinc Finger Proteins in the Human Fungal Pathogen Cryptococcus neoformans

Liu

2020

IJMS

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Zinc is one of the essential trace elements in eukaryotes and it is a critical structural component of a large number of proteins. Zinc finger proteins (ZNFs) are zinc-finger domain-containing proteins stabilized by bound zinc ions and they form the most abundant proteins, serving extraordinarily diverse biological functions. In recent years, many ZNFs have been identified and characterized in the human fungal pathogen Cryptococcus neoformans, a fungal pathogen causing fatal meningitis mainly in immunocompromised individuals. It has been shown that ZNFs play important roles in the morphological development, differentiation, and virulence of C. neoformans. In this review, we, first, briefly introduce the ZNFs and their classification. Then, we explain the identification and classification of the ZNFs in C. neoformans. Next, we focus on the biological role of the ZNFs functionally characterized so far in the sexual reproduction, virulence factor production, ion homeostasis, pathogenesis, and stress resistance in C. neoformans. We also discuss the perspectives on future function studies of ZNFs in C. neoformans.

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Serotyping of Cryptococcus neoformans by dot enzyme assay

Cited by 30 publications

References 27 publications

Extracellular proteins of Cryptococcus neoformans and host antibody response

Extracellular proteins of Cryptococcus neoformans and host antibody response

Reactivity patterns and epitope specificities of anti-Cryptococcus neoformans monoclonal antibodies by enzyme-linked immunosorbent assay and dot enzyme assay

Zinc Finger Proteins in the Human Fungal Pathogen Cryptococcus neoformans

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