Structural characterization of a “Signature” phosphatidylethanolamine as the major 10‐hydroxy stearic acid‐containing lipid of <i>Cryptosporidium parvum</i> oocysts

Schrum, David; Alugupalli, Srinivas; Kelly, Sean T.; White, David C.; Fayer, Ronald

doi:10.1007/s11745-997-0102-4

Cited by 4 publications

(9 citation statements)

References 13 publications

(9 reference statements)

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“…Our analysis of oocyst wall proteins yielded 5 major bands, while wall residues obtained after lipid extraction yielded 12 major bands. Our results can be compared to those of Harris and Petry (14), who reported finding seven protein bands under 66 kDa at 54, 48,46,43,38,21, and 14.5 kDa in samples of similarly purified oocyst walls. While it is difficult to reconcile the differences between our results and theirs because of the different methods we employed, it is significant that our results for post-lipid extraction wall residues showed 12 major proteins, with nine bands under 66 kDa, similar to the results of Harris and Petry (14), who for starting material used oocysts purified after treatment with ethyl ether to extract lipids.…”

Section: Discussionsupporting

confidence: 73%

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Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts

Jenkins¹,

Eaglesham²,

Anthony³

et al. 2010

Appl Environ Microbiol

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The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium-and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.Previous studies of the survival of Cryptosporidium parvum under natural and laboratory conditions have shown that the oocyst phase is a durable stage in the life cycle of this apicomplexan parasite and is crucial for parasite transmission. A major public health problem is the resistance of oocysts to chlorine at normal concentrations used in water treatment systems. Oocysts have the reputation of being tough, durable structures; however, they can be inactivated by many physical and chemical disinfectants, including UV radiation, ozone, ammonia, high temperature, desiccation, freezing, and exposure to extreme alkaline or acidic conditions (10,11,12,17,18,22,35). Low temperatures above freezing extend oocyst viability and infectivity for very long times (12,18,19,20,35). Environmental temperature is a major factor controlling oocyst survival (23,24,32). While there have been many studies documenting the significance of temperature for oocyst survival and the influence of temperature on stored energy reserve utilization has been recognized (see references 10 and 32 for reviews), the effects of temperature on the key oocyst wall structures a...

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Section: Discussionsupporting

confidence: 73%

“…Plates were developed in a 30:8:1 (vol/vol/vol) chloroform-methanol-water mixture as outlined by Schrum et al (38). Glycolipids were identified by using alpha-naphthol and heating at 105°C.…”

Section: Thin-layer Chromatography (Tlc)mentioning

confidence: 99%

Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts

Jenkins¹,

Eaglesham²,

Anthony³

et al. 2010

Appl Environ Microbiol

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“…The standards (3-hydroxy eicosanoic acid, 12-hydroxy stearic acid, and 6-hydroxy stearic acid) were purchased from Matreya (Pleasant Gap, PA). Chromatography-grade solvents of chloroform, methanol, acetone, and water were also used (4,5).…”

Section: Methodsmentioning

confidence: 99%

“…Parasites. Oocysts of C. parvum used in this study were purified from calf feces as previously described (4,5).…”

Section: Methodsmentioning

confidence: 99%

“…For this reason, the focus of the assay was shifted to the highly unusual fatty acid, 10-OH 18:0. Previous investigators had shown that the traces of 10-OH 18:0 were localized at the sn-2 position of phosphatidylethanolamine (PE) (5). However, the combined efforts of parent/derived fragment ion mass spectrometry and high-performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS) by R.S.…”

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The signature 10‐hydroxy stearic acid thought to correlate with infectivity in oocysts of Cryptosporidium species is an artifact

Burkhalter¹,

Smith²,

White

et al. 1998

Lipids

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Heating or freezing leads to loss in infectivity of oocysts of Cryptosporidium parvum toward neonatal BALB/c mice and is reflected in the profile of the polar lipid fatty acids. Upon loss of infectivity, the ratio of polar lipid to neutral lipid fatty acid decreased and the relative proportions of 18:1n-9 also decreased; proportions of 18:2n-6 and 20:5n-6 increased, whereas the proportions of 16:0 remained constant with freezing. During these investigations, a novel fatty acid, 10-OH 18:0, was discovered in the glycolipid fraction. The identification of a fatty acid unique to species of Cryptosporidium was thought to provide a specific biomarker for this organism. Cryptosporidium also demonstrated fluctuations in absolute quantities of 10-OH 18:0 with events that lead to loss of infectivity. This led to the presumed correlation of this biomarker with infectious Cryptosporidium. The 10-OH 18:0 was putatively localized at the sn-2 position of phosphatidylethanolamine. High-performance liquid chromatography/electrospray ionization mass spectrometry revealed that the 10-OH 18:0 existed principally in the free fatty acid form. Herein, we establish that the free fatty acid 10-OH 18:0 was, in actuality, an artifact of the procedures for sample preparation.

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Cryptosporidium: The analytical challenge

Smith¹,

Thompson²,

Smith³

et al. 2007

Cryptosporidium

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Structural characterization of a “Signature” phosphatidylethanolamine as the major 10‐hydroxy stearic acid‐containing lipid of Cryptosporidium parvum oocysts

Cited by 4 publications

References 13 publications

Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts

Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts

The signature 10‐hydroxy stearic acid thought to correlate with infectivity in oocysts of Cryptosporidium species is an artifact

Cryptosporidium: The analytical challenge

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