Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105

Background Scrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKβ-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined. Principal findings Western blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen’s ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm. Conclusions O. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.
Author summary Scrub typhus is a neglected disease that can be fatal and occurs predominantly in the Asia-Pacific, one of the most densely populated regions of the world. Notably, cases continue to emerge outside this area. The etiologic agent is Orientia tsutsugamushi, a bacterial pathogen that infects certain leukocytes and cells that line blood vessels in animals and humans. The success of O. tsutsugamushi to colonize these cells is at least partially attributable to its ability to counter host immunity. In this study, we demonstrate that O. tsutsugamushi stabilizes p105, a mammalian inhibitor of the transcription factor, NF-κB, which is otherwise key for activating proinflammatory and antimicrobial gene expression. O. tsutsugamushi is the first example of a bacterium that inhibits NF-κB by promoting elevated levels of p105 and impairing its degradation. Our findings provide fundamental information that helps explain how this important pathogen has evolved to stealthily establish infection in host cells.