Rickettsia rickettsii infection of cultured human endothelial cells induces NF-kappaB activation

Abstract

Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, is an obligate intracellular bacterial organism that infects primarily the vascular endothelial cells (EC). A component of the EC response to infection is transcriptional activation, which may contribute to the thrombotic and inflammatory consequences of disease. In this study, we explore R. rickettsii-induced activation of the nuclear factor-B/Rel (NF-B) family of transcription factors involved in early transcriptional responses to injurious stimuli. Two NF-B species were activated by infection and reacted with a double-stranded oligonucleotide probe corresponding to the B binding domain of the murine kappa light-chain gene enhancer. Gel supershift analysis demonstrated the reactivity of these complexes with antibodies against p65 and p50, and the induced species were tentatively identified as p50-p50 homodimers and p50-p65 heterodimers. Semiquantitative reverse transcription-PCR analysis revealed dramatic increases in the steady-state levels of mRNA coding for the inhibitory subunit of NF-B (IB␣), transcription of which is enhanced by the binding of NF-B within the IB␣ promoter region. NF-B activation was first detected 1.5 h following infection and was biphasic, with an early peak of activation at approximately 3 h, a return to baseline levels at 14 h, and even higher levels of activation at 24 h. It is likely that NF-B activation requires cellular uptake of R. rickettsii, since treatment of EC with cytochalasin B during infection to block entry inhibited activation by only 70% at 3 h. R. rickettsii-induced activation of NF-B may be an important controlling factor in the transcriptional responses of EC to infection with this obligate intracellular organism.

Activation of p38 stress-activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response

“…The present study clearly demonstrates activation of p38 MAP kinase, as evidenced by increased phosphorylation and enzymatic activity, but not of JNK pathway and thus, provides the first description of selective regulation of stress-induced MAP kinases during R. rickettsii infection of EC. Although unique in that both p38 and JNK are simultaneously activated in a variety of other situations, for example, host cell response to Salmonella typhimurium (Hobbie et al, 1997), Helicobacter pylori (Keates et al, 1999), Escherichia coli (Dahan et al 2002) and Neisseria meningitidis (Sokolova et al, 2004), non-involvement of JNK stress kinase early during the infection lends further support to our earlier finding that the activating protein-1 (AP-1), a transcription factor regulated by JNK in most cell types, likely does not participate in promoting transcriptional activation of EC after interaction with R. rickettsii (Sporn et al, 1997;Clifton et al, 2005b). p38 is a typical serine and threonine (Thr) MAP kinase which is activated by phosphorylation of Thr and tyrosine (Tyr) in a Thr-Gly-Tyr motif.…”

“…As an initial approach to examine whether attachment to the cell surface alone is the stimulus, EC were incubated with viable R. rickettsii at 4 ∞ C to arrest their internalization (Teysseire et al ., 1992). Figure 3B clearly shows that the level of phospho-p38 in the lysates of cells infected at 4 ∞ C for 90 min was much lower than those infected at 37 ∞ C. We also used cytochalasin D treatment of EC to inhibit actin polymerization and render the cells incapable of rickettsial uptake (Sporn et al ., 1997;Martinez and Cossart, 2004). Before these studies, however, experiments were conducted to ensure that the concentration of cytochalasin D used in the system inhibited the invasion of adhered rickettsiae, but had no effect on basal p38 phosphorylation (data not shown).…”

“…The standard dose of viable organisms to achieve a relatively synchronous infection of EC was approximately 6 ¥ 10 4 pfu per square centimetre of culture area. At 6 h after infection, ≥80% of cells were infected with three to four organisms per cell as observed by indirect immunofluorescent staining (Sporn et al, 1997). In experiments wherein infection was performed for durations of >3 h, non-adherent extracellular rickettsiae were removed by aspiration of initial inoculum, cell monolayers were quickly washed with warm culture medium and further incubation was carried out in freshly prepared culture medium.…”

Rickettsial Infections

“…In eukaryotic cells, rickettsiae activate NF-κB and one of the results of this activation is the inhibition of apoptosis. [47][48][49] This effect is mediated via inhibition of apical caspases-8 and -9 and by controlling intracellular levels of the bcl-2 family of proteins, specifically Bid and Bad. 50 Inhibition of apoptosis in vitro by intracellular rickettsiae appears logical since they are obligate intracellular organisms and therefore need the cell's nutrients to proliferate.…”

The Pathogen Resource Integration Center: Implications for Rickettsial Research

“…These resources will be freely available for public use. Specific objectives set forth by NIAID for the BRC initiative are: (1) to provide the scientific community with a point of entry into a comprehensive database with access to genomic and related data designed to maximize ease of use; (2) to have each center focus on the organisms listed in the NIAID Category A-C priority list of pathogens causing emerging or re-emerging diseases relevant to biodefense; and (3) ultimately to aid in the discovery of therapeutic targets, vaccines, diagnostics, and epidemiological markers.…”