title: Recombinant Arthrobacter β-1, 3-glucanase as a potential effector molecule for paratransgenic control of Chagas disease.
abstract: BACKGROUND
  Chagas disease is most often transmitted to humans by Trypanosoma cruzi infected triatomine bugs, and remains a significant cause of morbidity and mortality in Central and South America. Control of Chagas disease has relied mainly on vector eradication. However, development of insect resistance has prompted us to develop a paratransgenic strategy to control vectorial transmission of T. cruzi. Here, the potential role of recombinant endoglucanases as anti-trypanosomal agents for paratransgenic application is examined. The surface of T. cruzi is covered by a thick coat of mucin-like glycoproteins that have been proposed to play a role in the binding of T. cruzi to the membrane surface of the vector gut. We hypothesize that disruption of these glycoconjugates could arrest parasite development in the vector and abort the transmission cycle. In this work, we examine the effects of recombinant Arthrobacter luteus β-1, 3-glucanase expressed via Rhodococcus rhodnii on T. cruzi Sylvio II strain.
  
  
  RESULTS
  The coding sequence for β-1, 3-glucanase was cloned in-frame to a heterologous promoter/signal sequence from the Mycobacterium kansasii alpha antigen gene resident in an E. coli/R. rhodnii shuttle vector. The resulting construct was confirmed by sequencing, and electroporated into R. rhodnii. Expression products from positive clones were purified from log phase cultures followed by dialysis into physiological buffers. Lysates and media were quantitated by ELISA against rabbit antibody specific to β-1,3-glucanase. Glucanase-positive samples were applied to live T. cruzi parasites in culture and viability accessed by spectrophotometric and fluorescent microscopic measurements. R. rhodnii-expressed β-1,3-glucanase exhibited toxicity against T. cruzi compared to controls when applied at 5 and 10% of the total culture volume. The decrease in cell viability ranged from a maximum of 50% for the media treatments to 80% for the filtered lysates.
  
  
  CONCLUSIONS
  These results suggest that recombinant β-glucanase could be a powerful addition to the arsenal of effector molecules for paratransgenic control of Chagas disease. In future studies, the ability of β-glucanase to function in combination with other effector molecules will be explored. Dual targeting of T. cruzi should not only slow resistance but also permit synergistic or additive lethal effects on T. cruzi.
authors.affiliation: Center for Global Health, Department of Internal Medicine, University of New Mexico and New Mexico VA Health Care System, Albuquerque, New Mexico, USA.
authors.forename: Christo
authors.initials: C
authors.lastname: Jose
authors.forename: Nicole
authors.initials: N
authors.lastname: Klein
authors.forename: Sarah
authors.initials: S
authors.lastname: Wyss
authors.forename: Annabeth
authors.initials: A
authors.lastname: Fieck
authors.forename: Ivy
authors.initials: I
authors.lastname: Hurwitz
authors.forename: Ravi
authors.initials: R
authors.lastname: Durvasula
mesh_terms: D000818:Animals; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014355:Chagas Disease / Q000469:parasitology / Q000517:prevention & control* / Q000635:transmission; D041981:Gastrointestinal Tract / Q000382:microbiology; D043326:Glucan 1,3-beta-Glucosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007303:Insect Vectors / Q000382:microbiology* / Q000469:parasitology; D019909:Mycobacterium kansasii / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015800:Protozoan Proteins / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins; D012239:Rhodnius / Q000382:microbiology* / Q000469:parasitology; D012240:Rhodococcus / Q000235:genetics / Q000502:physiology; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000254:growth & development
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D043326:Glucan 1,3-beta-Glucosidase