title: Purification and identification of amylases released by the human pathogen Trichomonas vaginalis that are active towards glycogen.
abstract: The parasitic protist Trichomonas vaginalis is the causative agent of the sexually transmitted infection trichomoniasis. In the laboratory, T. vaginalis is typically cultured in a serum-containing medium with maltose or glucose as the carbon source. The nature of the carbohydrates used by the organism in the environment of its host is unclear. However, the vagina contains substantial amounts of glycogen, which is believed to provide a growth substrate for the vaginal microbiota. We have shown previously that T. vaginalis releases glucosidases that are active towards glycogen into its environment. Here we purify and identifying these glucosidases. Using ammonium sulfate precipitation and precipitation with ethanol/glycogen, we purified glucosidase activity from conditioned growth medium, achieving over 300-fold enrichment. Maltose was released when glycogen was incubated with the glucosidase preparation, indicating that a β-amylase was present. However, after prolonged incubation, small quantities of larger products including maltotriose were obtained. Liquid chromatography and tandem mass spectrometry showed that the glucosidase preparation contained three proteins, the major component being a putative β-amylase encoded by the TVAG_080000 open reading frame. Lesser amounts of two putative α-amylases, encoded by the TVAG_178580 and TVAG_205920 open reading frames, were also present. We cloned and expressed the TVAG_080000 open reading frame and found that the recombinant protein was capable of digesting glycogen, releasing exclusively maltose. We conclude that T. vaginalis releases a variety of amylases into its growth environment and is well equipped to utilize the glycogen found in the vagina as a source of essential carbohydrates.
authors: Department of Microbiology & Immunology, Des Moines University, Des Moines, IA 50312, USA.
authors: Ronald W
authors: RW
authors: Smith
authors: Department of Microbiology & Immunology, Des Moines University, Des Moines, IA 50312, USA.
authors: Andrew
authors: A
authors: Brittingham
authors: Department of Biochemistry & Nutrition, Des Moines University, Des Moines, IA 50312, USA. Electronic address: wayne.wilson@dmu.edu.
authors: Wayne A
authors: WA
authors: Wilson
mesh_terms: D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D005260:Female; D015870:Gene Expression; D005959:Glucosidases / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014247:Trichomonas Vaginitis / Q000469:parasitology; D014246:Trichomonas vaginalis / Q000201:enzymology / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D006003:Glycogen; D000681:Amylases; D005959:Glucosidases
keywords: Glucosidase; Trichomonad; Vagina