Purification and partial characterization of an elastinolytic proteinase from Aspergillus flavus culture filtrates

 A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene (5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0. Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein, collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range of potential natural protein substrates. Received: 24 October 1995/Received last revision: 27 March 1996/Accepted: 30 March 1996     

Partial purification and characterization of an inhibitor from newborn-rat epidermis with activity against the proteinase of Schistosoma mansoni cercariae.

The penetration of cercariae through the skin initiates infection of the host with the human trematode parasite Schistosoma mansoni. Many larvae fail to migrate into the living epidermal cell layer. In order to determine if chemical as well as mechanical barriers to cercarial skin penetration exist, inhibitory activity of epidermal cell extracts against the proteinase obtained from cercarial secretions was assayed. An inhibitor was purified 50-fold by gel filtration on Sephadex G 75 and cation exchange chromatography at pH 5.8 and 4.9. The inhibitor has a relative molecular mass (Mr) of approx. 40 000-53 000. Oxidation of the inhibitor with N-chlorosuccinimide eliminated its inhibitory activity and thus indicated a critical methionine residue. The inhibitor was active against a wide spectrum of serine proteinases: porcine pancreatic elastase, human granulocyte elastase, bovine trypsin, and bovine alpha-chymotrypsin. However, no inhibition was detected against papain or clostridial collagenase. The inhibitor did not cross react with antiserum to human or rat serum alpha 1-proteinase inhibitor.     

Schistosoma mansoni: eicosanoid production by cercariae

Cercariae of Schistosoma mansoni are stimulated to penetrate skin by certain free fatty acids. The cercariae have an active arachidonate cascade, presumably using host skin essential fatty acids as cascade precursors. Exposing cercariae to 3.3 mM linoleate for 1, 10, and 60 min resulted in production of a wide variety of eicosanoids. Using high-performance liquid chromatography, eicosanoids coeluting with prostaglandin E2, D2, and A2, leukotriene B4, and 5-hydroxyeicosatetraenoic acid standards were identified, as well as unidentified peak positions. Radioimmunoassay confirmed the presence of immunoreactive prostaglandin E1, and E2, and 5- and 15-hydroxyeicosatetraenoic acids in cercarial extracts. No eicosanoid production occurred when cercariae were exposed to 3.3 mM oleate and 1 or 330 microM linoleate. Both high-performance liquid chromatography and radioimmunoassay data indicated that cercariae regulate the production of eicosanoids through time. It is postulated that arachidonate metabolism and subsequent eicosanoid production are required for successful cercarial penetration.     

Schistosoma mansoni: partial characterization of enzyme(s) secreted from the preacetabular glands of cercariae.

Secretions from the preacetabular glands of cercariae of Schistosoma mansoni were collected over skin surface lipid on a warmed glass surface in a system providing a temperature gradient. Proteolytic activity of these secretions was linear with respect to the numbers of cercariae secreting. The enzyme was relatively storage stable in water or glycine-NaOH buffer. It was highly sensitive to the buffer system, glycine-NaOH buffer providing the highest proteolytic activity against Azocoll and gelatin substrates. In the glycine buffer, the pH optimum for the enzyme was 8.5–8.8; the temperature optimum was 51 C with the Q₁₀ for Azocoll hydrolysis in the range of 30–50 C approximately 2.2. Of the activity 0.4% was lost at 5 C, 1.7% at 35 C, and 100% at 60 C within 10–15 min. Two Sephadex column chromatography fractions showed proteolytic activity against Azocoll. The fraction with greatest activity was not associated with either of the two peaks of high absorbancy at 280 nm, but the lower proteolytic peak coincided with the lower absorbancy peak in the area of peptides below 10,000 MW. Calibration of the Sephadex column with proteins of known molecular weight showed the MW of the main proteolytic fraction of the secretion to be approximately 25,000–27,000.     

Schistosoma mansoni: chemical stabilization of cercariae by aldehydes

Chemically stabilized cercariae of Schistosoma mansoni have been developed by inactivating surface glycoproteins which are essential for their survival. The inactivation was achieved by reaction with 0.01-0.1% glutaraldehyde, 0.1-1% formaldehyde, and 0.37-3.7 microliters citraconic anhydride. The cercariae lost their viability but retained the ability to exclude trypan blue for up to 2 years in a manner similar to live cercariae and in contrast to cercariae killed by other means, which took up the dye immediately. The chemically stabilized cercariae reacted with polyclonal and monoclonal antischistosome antibodies in an indirect immunofluorescence assay for up to 2 years, indicating the retention and preservation of surface antigens. Chemically stabilized cercariae revealed the presence of antischistosome antibodies as early as 1 week after infection when used for immunodiagnosis of mouse and rat infections. The presence of Fc receptors for human IgG on the stabilized cercariae interfered in their use as an immunodiagnostic reagent of human schistosomiasis. The stabilized cercariae were also used to screen cultures for monoclonal antischistosome antibodies. Preliminary results indicated that immunization of mice with glutaraldehyde stabilized cercariae imparted protective immunity to mice.     

Murine immunization by cesium-137 irradiation attenuated Schistosoma mansoni cercariae

Cesium-137, becoming a more readily available ionizing gamma radiation source for laboratory use, was shown to effectively attenuate Schistosoma mansoni cercariae for vaccine production. In parallel comparison studies with the murine model, cesium-137 attenuated cercariae consistently afforded better (P greater than 0.05) protection than did the cobalt-60 prepared vaccine. Dose-response data indicated that the optimal total irradiation with cesium-137 was between 45 and 50 Krad.     

Ultraviolet disinfection of Schistosoma mansoni cercariae in water

BackgroundSchistosomiasis is a parasitic disease that is transmitted by skin contact with waterborne schistosome cercariae. Mass drug administration with praziquantel is an effective control method, but it cannot prevent reinfection if contact with cercariae infested water continues. Providing safe water for contact activities such as laundry and bathing can help to reduce transmission. In this study we examine the direct effect of UV light on Schistosoma mansoni cercariae using ultraviolet light-emitting diodes (UV LEDs) and a low-pressure (LP) mercury arc discharge lamp.MethodologyS. mansoni cercariae were exposed to UV light at four peak wavelengths: 255 nm, 265 nm, 285 nm (UV LEDs), and 253.7 nm (LP lamp) using bench scale collimated beam apparatus. The UV fluence ranged from 0–300 mJ/cm² at each wavelength. Cercariae were studied under a stereo-microscope at 0, 60, and 180 minutes post-exposure and the viability of cercariae was determined by assessing their motility and morphology.ConclusionVery high UV fluences were required to kill S. mansoni cercariae, when compared to most other waterborne pathogens. At 265 nm a fluence of 247 mJ/cm² (95% confidence interval (CI): 234–261 mJ/cm²) was required to achieve a 1-log₁₀ reduction at 0 minutes post-exposure. Cercariae were visibly damaged at lower fluences, and the log reduction increased with time post-exposure at all wavelengths. Fluences of 127 mJ/cm² (95% CI: 111–146 mJ/cm²) and 99 mJ/cm² (95% CI: 85–113 mJ/cm²) were required to achieve a 1-log₁₀ reduction at 60 and 180 minutes post-exposure at 265 nm. At 0 minutes post-exposure 285 nm was slightly less effective, but there was no statistical difference between 265 nm and 285 nm after 60 minutes. The least effective wavelengths were 255 nm and 253.7 nm. Due to the high fluences required, UV disinfection is unlikely to be an energy- or cost-efficient water treatment method against schistosome cercariae when compared to other methods such as chlorination, unless it can be demonstrated that UV-damaged cercariae are non-infective using alternative assay methods or there are improvements in UV LED technology.