Abstract
Manipulation of host cell death is an important determinant of the outcome of an infection. Here, we investigate whether Rickettsia rickettsii-infected host endothelial cells resist the effects of staurosporine, a potent inducer of apoptosis, and we explore the mechanisms underlying the anti-apoptotic effect of infection. Human microvascular endothelial cells infected with R. rickettsii for 24 or 48 h were challenged with staurosporine. The extent of apoptosis was evaluated with flow cytometry. mRNA and protein expression levels were determined by use of microarray or polymerase chain reaction and immunoblotting, respectively. Staurosporine-induced apoptosis in endothelial cells infected for 24 and 48 h was significantly reduced, compared with simultaneously treated uninfected cells. A microarray of human genes involved in apoptosis and polymerase chain reaction analyses revealed increased steady-state mRNA expression of cIAP2 (a member of the inhibitor-of-apoptosis family of proteins) at 24 h after infection. The levels of cIAP2 protein (+/-SD) in infected cells were 3.5 +/- 1.7 -fold and 2.3 +/- 1.2 -fold higher than that in uninfected control cells at 24 and 48 h after infection. Nucleofection of human-specific cIAP2-targeted siRNA resulted in inhibition of protein expression by > or = 50% but had no effect on infection-induced protection against apoptosis. R. rickettsii-induced expression of cIAP2 in host endothelial cells is likely not a major contributor to protection against staurosporine-induced cell death.
Highlights
Manipulation of host cell death is an important determinant of the outcome of an infection
Human microvascular endothelial cells infected with R. rickettsii for 24 or 48 h were challenged with staurosporine
Rocky Mountain spotted fever is caused by Rickettsia rickettsii, which is transmitted in nature via the bite of an infected tick, and leads to discomfort, disability, and death
Summary
Human microvascular endothelial cells infected with R. rickettsii for 24 or 48 h were challenged with staurosporine. Human microvascular endothelial cells (HMECs) were cultured in MCDB 131 medium (Gibco) supplemented with L-glutamine (10 mmol/L; Gibco), mouse epidermal growth factor (10 ng/mL; BD Bioscience), hydrocortisone (1 g/mL; Sigma), and 10% heat-inactivated fetal bovine serum [11]. Cells were grown in a humidified incubator with 5% CO2 at 37°C. A plaque-purified seed stock (1 ϫ 107–5 ϫ 107 pfu/mL) of R. rickettsii (Sheila Smith strain) was prepared from infected Vero cells [12]. Intracellular infection was continued for another 21 or 45 h (total duration, 24 or 48 h) before the introduction of staurosporine (275 nmol/mL; Sigma) for 24 h before cells were harvested
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