A Chitin Deacetylase-Like Protein Is a Predominant Constituent of Tick Peritrophic Membrane That Influences the Persistence of Lyme Disease Pathogens within the Vector

Abstract

Ixodes scapularis is the specific arthropod vector for a number of globally prevalent infections, including Lyme disease caused by the bacterium Borrelia burgdorferi. A feeding-induced and acellular epithelial barrier, known as the peritrophic membrane (PM) is detectable in I. scapularis. However, whether or how the PM influences the persistence of major tick-borne pathogens, such as B. burgdorferi, remains largely unknown. Mass spectrometry-based proteome analyses of isolated PM from fed ticks revealed that the membrane contains a few detectable proteins, including a predominant and immunogenic 60 kDa protein with homology to arthropod chitin deacetylase (CDA), herein termed I. scapularis CDA-like protein or IsCDA. Although IsCDA is primarily expressed in the gut and induced early during tick feeding, its silencing via RNA interference failed to influence either the occurrence of the PM or spirochete persistence, suggesting a redundant role of IsCDA in tick biology and host-pathogen interaction. However, treatment of ticks with antibodies against IsCDA, one of the most predominant protein components of PM, affected B. burgdorferi survival, significantly augmenting pathogen levels within ticks but without influencing the levels of total gut bacteria. These studies suggested a preferential role of tick PM in limiting persistence of B. burgdorferi within the vector. Further understanding of the mechanisms by which vector components contribute to pathogen survival may help the development of new strategies to interfere with the infection.

A Dityrosine Network Mediated by Dual Oxidase and Peroxidase Influences the Persistence of Lyme Disease Pathogens within the Vector

“…For analysis of Duox and peroxidase expression, the naïve nymphal ticks or nymphs microinjected with dsRNA were allowed to feed on naïve or B. burgdorferiinfected C3H/HeN mice (25 ticks/mouse) as detailed (29,30). The ticks were collected at various time points of feeding or as postfed nymphs.…”

“…RNA Interference-RNA interference of target tick genes was accomplished using our published procedures (30,32). Tick cDNA was prepared as described above and used as a template to amplify various fragments of the open reading frames of the corresponding target genes: Duox (accession number ISCW007865) or peroxidases (accession numbers ISCW017368 and ISCW002528, referred to herein as peroxidase 002528 and peroxidase 017368).…”

“…Cryosectioning was performed at Ϫ20°C using a cryotome, and 5-m-thick sections were incu-bated in PBS with 5% goat serum and 0.5% Tween 20, as detailed (30). The sections were then labeled with mouse monoclonal antibodies against dityrosine (Genox Corporation), rabbit antiperoxidase antibodies (Sigma), or isotype control (mouse or rabbit IgG; Sigma) followed by Alexa 488-labeled goat antimouse or Alexa 568-labeled goat anti-rabbit IgG (Invitrogen).…”

“…The sections were then labeled with mouse monoclonal antibodies against dityrosine (Genox Corporation), rabbit antiperoxidase antibodies (Sigma), or isotype control (mouse or rabbit IgG; Sigma) followed by Alexa 488-labeled goat antimouse or Alexa 568-labeled goat anti-rabbit IgG (Invitrogen). The slides were then washed using PBS with 0.05% Tween 20, stained with DAPI (Invitrogen), and imaged by a LSM 510 laser confocal microscope (Zeiss) as described (30).…”

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control

“…(B.) microplus [42,[45][46][47], A. americanum, I. scapularis, and D. variabilis adults [43], which produced significant effects on the eggs and larvae, and microinjection has also been accomplished in I. scapularis [48][49][50][51][52] and engorged O. moubata nymphs [53].…”