J Bacteriol Virol.  2008 Mar;38(1):11-17. 10.4167/jbv.2008.38.1.11.

Identification of the Proteinase K-resistant Antigen of Orientia tsutsugamushi by Monoclonal Antibodies

• Kim MK
• Kim MJ
• Lee SM
• Kang JS

• KMID: 2168514
• DOI: http://doi.org/10.4167/jbv.2008.38.1.11

Abstract

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we characterized two monoclonal antibodies, NT19 and WT14, against the proteinase K-resistant antigen of O. tsutsugamushi. Western blot analysis showed that MAb NT19 and WT14 strongly recognized two antigenic bands with molecular masses of 20 kDa and 24 kDa, which were resistant to proteinase K digestion. We suggest that the proteinase-resistant antigen might be polysaccharide. One patient serum reacted with a 24 kDa band that was similar to a band observed by WT14, suggesting the possibility of the role of this proteinase-resistant antigen as an antigenic molecule in human infection.

Keyword

Orientia tsutsugamushi; Proteinase K-resistant antigen; Monoclonal Antibody

MeSH Terms

Antibodies, Monoclonal
Blotting, Western
Clone Cells
Cytosol
Digestion
Endopeptidase K
Humans
Orientia tsutsugamushi
Scrub Typhus
Antibodies, Monoclonal
Endopeptidase K

Figure

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  Figure 1. Western blot analysis of antigen recognized by MAb NT19. ECV304 cells infected with O. tsutsugamushi (In) or control cells (Un) were treated with lysis buffer containing 1% SDS, and proteins were separated by PAGE. Equal amount of samples was treated with proteinase K.

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  Figure 2. Distinct reactivity of two MAbs against proteinase K-treated or control sample in western blotting. Samples were separated by PAGE and blotted with NT19 and WT14. For better visualization of minor bands, additional films were taken after longer exposure time.

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  Figure 3. MAbs NT19 and WT14 recognize the same antigen. Lysates of infected cell (In) or control cells (Un) were immuno-precipitated with NT19 or WT14 and then antigen bands were visualized with western blotting with NT19 or WT14.

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  Figure 4. The effect of proteinase K treatment on the reactivity of MAb NT19. Lysates were immunoprecipitated with NT19 or WT14 before proteinase K treatment. Immunoprecipitated proteins were analyzed with western blotting with NT19.

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  Figure 5. The change of size of antigenic bands recognized by MAb NT19 after proteinase K treatment. Lysates of ECV304 cells infected with O. tsutsugamushi were treated with proteinase K for the indicated times and then analyzed with western blotting with NT19 (upper) or FS10 (lower).

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  Figure 6. Scrub typhus patient's serum recognized the proteinase K-resistant antigen, which was similar to that observed by MAb NT19. Lysates of infected cell (In) or control cells (Un) were treated with proteinase K and the antigen bands were visualized with western blotting with patient's serum or NT19.

Reference

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