J Bacteriol Virol.  2010 Dec;40(4):151-157. 10.4167/jbv.2010.40.4.151.

Cleavage of p65 Subunit of NF-kappaB by Orientia tsutsugamushi

• Kim MJ
• Lee SM
• Kil SH
• Kang JS

• KMID: 2168578
• DOI: http://doi.org/10.4167/jbv.2010.40.4.151

Abstract

Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular parasite and usually propagates in the cytoplasm of host endothelial cells and macrophages. Macrophages are the first defense line against bacterial infection and NF-kappaB is activated upon contact with bacteria, resulting in the transcription of inflammatory cytokine to control bacterial infection. In this study, we investigated whether O. tsutsugamushi modulates NF-kappaB activation in the macrophages. We examined the changes of NF-kappaB proteins upon infection with O. tsutsugamushi and found that NF-kappaB is activated at a slow rate as judged with EMSA and immunoblot analysis. Interestingly, we found that p65 was cleaved generating a 45 kDa fragment. In addition, fragment of p65 is generated only by the virulent serotype strain of O. tsutsugamushi, suggesting this cleavage may be associated with the mouse virulence. It is still unknown whether this is a direct result of O. tsutsugamushi proteins or enzymes of host cell. Further exploration of the mechanism that modulates NF-kappaB activity by O. tsutsugamushi could contribute to a better understanding of the molecular pathogenesis of O. tsutsugamushi infection.

Keyword

O. tsutsugamushi; NF-kappaB; p65; Fragment; Macrophage

MeSH Terms

Animals
Bacteria
Bacterial Infections
Cytoplasm
Endothelial Cells
Macrophages
Mice
NF-kappa B
Orientia tsutsugamushi
Parasites
Proteins
Scrub Typhus
Sprains and Strains
NF-kappa B
Proteins

Figure

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  Figure 1. The activation of NF-κB in J774A.1 cells infected with O. tsutsugamushi. NF-κB activation was examined by EMSA for nuclear extracts prepared from J774A.1 cells for the indicated times. J774A.1 cells were infected with O. tsutsugamushi (OT) or treated ECV cell lysate for the mock-infection (ECV).

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  Figure 2. The cleavage of p65 in J774A.1 cells infected with O. tsutsugamushi. The p65 was visualized with the immunoblot analysis of protein extracts from the cytosol and nuclei of the J774A.1 cells treated ECV cell lysate (upper panel), infected with O. tsutsugamushi (middle panel), or treated with LPS (lower panel) for the indicated times.

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  Figure 3. The cleavage of p65 observed only in western blot using p65 C- and N-terminal antibodies. The p65 was visualized with the immunoblot analysis of protein extracts from the nuclei of J774A.1 cells treated ECV cell lysate (ECV) and infected with O. tsutsugamushi (OT) using the indicated primary antibodies.

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  Figure 4. The p65 cleavage by different serotypic strains of O. tsutsugamushi. The p65 was visualized with the immunoblot analysis of protein extracts from the nuclei of the J774A.1 cells infected with Kato and Gilliam strains of O. tsutsugamushi.

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