title: Host blood proteins and peptides in the midgut of the tick Dermacentor variabilis contribute to bacterial control.
abstract: Antimicrobial midgut proteins and peptides that result from blood digestion in feeding American dog ticks Dermacentor variabilis (Say) were identified. Midgut extracts from these ticks showed antimicrobial activity against Micrococcus luteus, regardless of whether they were challenged with peptidoglycan, blood meal components, rabbit blood, Bacillus subtilis, Escherischia coli or Borrelia burgdorferi. However, no peptide band co-migrating with defensin was found in midgut extracts from the challenged ticks. Partial purification of the midgut extracts using C(18) Sep Paks and gel electrophoresis showed the presence of 4 distinct bands with rMW 4.1, 5.3, 5.7 and 8.0 kDa identified by tryptic digestion-mass fingerprinting as digestive fragments of rabbit alpha-, beta-, gamma-chain hemoglobin, and rabbit ubiquitin. No evidence of varisin, a defensin previously identified in the hemolymph of D. variabilis, was found in the tryptic digest, although varisin was found in a hemocyte lysate using the same methods. However, varisin transcript was detected in midgut cell lysates. Also present in all midgut samples was a cluster of 3 overlapping bands with rMW 13.0, 14.1 and 14.7 kDa which were identified by tryptic-digestion LC-MS and MALDI-TOF as rabbit alpha- and beta-chain hemoglobin (undigested) and transtherytin. Lysozyme transcript was detected in midgut cell extracts but the peptide was not. Studies done on other tick species demonstrated that hemoglobin digestion resulted in antimicrobial fragments. Antimicrobial hemoglobin fragments (including fragments larger than any reported previously) also were found in D. variabilis, as well as ubiquitin, a peptide known to occur as part of an antimicrobial complex in vertebrate leukocytes. In addition, we noted that Borrelia burgdorferi spirochetes were not lysed in the midgut lumen, which would be expected if defensin and lysozyme were active in this location. In this respect, the midgut's response to microbial challenge differs from that of the hemolymph. In summary, the midgut's antimicrobial activity appears to be primarily a byproduct of hemoglobin digestion rather than expression of immune peptides and proteins.
authors.affiliation: Department of Biological Sciences, Old Dominion University, Norfolk, Virginia 23529, USA, dsonensh@odu.edu
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Sonenshine
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hynes
authors.forename: Shane M
authors.initials: SM
authors.lastname: Ceraul
authors.forename: Robert
authors.initials: R
authors.lastname: Mitchell
authors.forename: Tiffany
authors.initials: T
authors.lastname: Benzine
mesh_terms: D000818:Animals; D001412:Bacillus subtilis; D001798:Blood Proteins / Q000302:isolation & purification / Q000502:physiology*; D025065:Borrelia burgdorferi; D003870:Dermacentor / Q000033:anatomy & histology / Q000382:microbiology* / Q000502:physiology*; D004926:Escherichia coli; D005260:Female; D041981:Gastrointestinal Tract / Q000378:metabolism* / Q000382:microbiology*; D006790:Host-Parasite Interactions; D016982:Micrococcus luteus; D011817:Rabbits
chemical_list: D001798:Blood Proteins