Papers by Thomas P Karbanowicz
Tick populations are controlled through the application of chemical pesticides. However, the rise... more Tick populations are controlled through the application of chemical pesticides. However, the rise in chemical resistance has prompted the investigation of other control methods such as the use of tick vaccines. Proteomic analysis provides valuable information about the possible function and localization of proteins, as candidate vaccine proteins are often either secreted or localized on the cell-surface membrane. Progress in the utilization of proteomics for the identification of novel treatment targets has been significant. However, their use in tick-specific investigations is still quite novel, with the continual development of tick-specific methodologies essential. In this study, an innovative sample preparation method was utilized to isolate epithelial cells from tick midguts to identify the membrane-bound proteins. Proteomic analysis was conducted comparing crude and innovative sample preparation methods with 692 and 1242 tick-specific proteins, 108 and 314 surface proteins respectively, isolated from the midguts of semi-engorged Rhipicephalus microplus adult female ticks. This research reports a novel preparation protocol for the analysis of tick midgut proteins which reduces host protein contamination.
36 The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that... more 36 The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host 37 paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks col-38 lected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced 39 using Illumina HiSeq 100 bp pair end read technologies. Unique and non-redundant holocyclotoxin 40 sequences were designated as HT2–HT19, as none were identical to the previously described HT1. 41 Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity 42 was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particu-43 larly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first 44 known genomic database developed for the Australian paralysis tick. The database contributed to the 45 identification and subsequent characterization of the holocyclotoxin family that will inform the develop-46 ment of novel anti-paralysis control methods.
Surface display libraries (SDL) have predominantly been utilized for the screening of peptides, a... more Surface display libraries (SDL) have predominantly been utilized for the screening of peptides, and single-chain variable IgG fragments, however, the use of SDL for the expression and purification of proteins is gaining interest. Prokaryote SDL express proteins within the periplasm, limiting the application of common screening techniques, such as ELISA and FACS, to assess the viability of recombinant toxin before purification. A previous attempt to express a functional holocyclotoxin-1 (HT1) from the Australian paralysis tick (Ixodes holocyclus) using a prokaryotic system was unsuccessful. In this study, the coding sequence (CDS) of HT1 was cloned into the pYD1 plasmid and transformed by electroporation into IMTV014 and EBY100 yeast cell lines. Post induction, recombinant HT1 was identified on the cell surface of IMTV014/ht1 and EBY100/ht1 transformants by FACS, Western blot, and ELISA utilizing dog anti-paralysis tick IgG. The recombinant HT1 was purified, and functionality confirmed by an in vitro synaptosome-binding assay. This research reports for the first time the extracellular expression and display of a functional HT1 on the surface of the S. cerevisiae. It also provides evidence that yeast display libraries provide a viable technology to produce recombinant toxins, and their screening using high throughput methodologies such as FACS.
Rhipicephalus microplus-the cattle tick-is the most significant ectoparasite in terms of economic... more Rhipicephalus microplus-the cattle tick-is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis. Video Link The video component of this article can be found at https://www.jove.com/video/55747/
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Papers by Thomas P Karbanowicz