Rickettsia rickettsii virulence determinants RARP2 and RapL mitigate IFN- β signaling in primary human dermal microvascular endothelial cells

Abstract

We compared the growth characteristics of a virulent Rickettsia rickettsii strain (Sheila Smith) to an attenuated R. rickettsii stain (Iowa) and a non-pathogenic species (R. montanensis) in primary human dermal microvascular endothelial cells (HDMEC). All replicated in Vero cells, however, only the Sheila Smith strain productively replicated in HDMECs. The Iowa strain showed minimal replication over a 24-h period, while R. montanensis lost viability and induced lysis of the HDMECs via a rapid programmed cell death response. Both the virulent and attenuated R. rickettsii strains, but not R. montanensis, induced an interferon-1 response, although the response was of lesser magnitude and delayed in the Sheila Smith strain. IFN-β secretion correlated with increased host cell lysis, and treatment with anti-IFNAR2 antibody decreased lysis from Iowa-infected but not Sheila Smith-infected cells. Both Sheila Smith- and Iowa-infected cells eventually lysed, although the response from Sheila Smith was delayed and showed characteristics of apoptosis. We, therefore, examined whether reconstitution of the Iowa strain with two recently described putative virulence determinants might enhance survival of Iowa within HDMECs. Reconstitution with RARP2, which is inhibitory to anterograde trafficking through the Golgi apparatus, reduced IFN-β secretion but had no effect on cell lysis. RapL, which proteolytically processes surface exposed autotransporters and enhances replication of Iowa in Guinea pigs, suppressed both IFN-β production and host cell lysis. These findings suggest distinct mechanisms by which virulent spotted fever group rickettsiae may enhance intracellular survival and replication.IMPORTANCEWe examined a naturally occurring non-pathogenic rickettsial species, R. montanensis, a laboratory-attenuated R. rickettsii strain (Iowa), and a fully virulent R. rickettsii strain (Sheila Smith) for growth in human dermal microvascular endothelial cells. The two avirulent strains replicated poorly or not at all. Only the virulent Sheila Smith strain replicated. IFN-β production correlated with the inhibition of R. rickettsii Iowa. Reconstitution of Iowa with either of two recently described putative virulence determinants altered the IFN-β response. A rickettsial ankyrin repeat protein, RARP2, disrupts the trans-Golgi network and inhibits IFN-β secretion. An autotransporter peptidase, RapL, restores proteolytic maturation of outer membrane autotransporters and diminishes the IFN-β response to enhance cell survival and permit replication of the recombinant strain. These studies point the way toward discovery of mechanisms for innate immune response avoidance by virulent rickettsia.

Keywords: Rickettsia; innate immunity; interferon-beta; primary cells; virulence.

Fig 1

HDMECs, but not Vero cells, restrict non-pathogenic rickettsiae. (A) Rickettsial intracellular burdens in Vero cells after 2 and 24 h of infection. Rickettsial titers were determined by plaque assaying lysates (mean ± S.D., N = 12). (B) Lysis of Vero host cells was monitored by determining LDH activity from culture supernatants at regular intervals (mean ± S.D., N = 6). (C) Rickettsial intracellular burdens, as described in A, were repeated using HDMECs as host cells (mean ± S.D., N = 14). (D) Lysis of HDMECs was also determined using LDH activity (mean ± S.D., N = 4). Significance in A and C was determined by t-test; P < 0.05, *; P < 0.001, ***; P < 0.0001, ****.

Fig 2

Virulent rickettsia eventually induce apoptosis, while non-pathogenic rickettsia do not. (A) HDMECs were infected with rickettsia or treated with 2 µM Staurosporine (Stauro), and Caspase-3/7 activity was measured after 4, 6, 12, and 24 h (mean ± S.D., N = 6–10). (B) Western blots with anti-PARP, cleaved PARP, GSDME, and GAPDH of HDMECs infected with rickettsia (MOI 2.5) for 6, 12, or 24 h (representative results are shown, N = 4).

Fig 3

R. rickettsii strains induce IFN-β signaling in HDMECs. (A) ELISA measurements of IFN-β secretion in HDMEC culture supernatants after 24 h of stimulation (50 mM MSA-2) or infections with rickettsia (mean ± S.D., N = 6). Comparisons were to uninfected controls. Significance was determined by one-way ANOVA; P < 0.05, *; P < 0.0001, ****. (B) Measurements of IFN-β secretion as in A, after 6, 12, 18, and 24 h of infection with R. rickettsii strains (mean ± S.D., N = 6). Significance was determined by one-way ANOVA; P < 0.05, *; P < 0.0001, ****. (C) Western blotting with anti-phospho-(p-) signal transducer and activator of transcription 1 (STAT1), STAT1, p-STAT2, STAT2, and GAPDH of samples from HDMECs stimulated with MSA-2 or infected with rickettsia for 6 and 12 h (representative results are shown, N = 4). (D) CXCL10 secretion in HDMEC culture supernatants after 12 or 24 h of infections with rickettsia (mean ± S.D., N = 6). Significance was assessed by two-way ANOVA. Shown are comparisons to uninfected controls; P < 0.0001, ****. Unlabeled bars were not significantly different from the control. (E) Western blotting with anti-OASL, RIG-I, IRF-1, TRAIL, IDO, and GAPDH of samples from HDMECs stimulated with MSA-2 or infected with rickettsiae for 12 and 24 h (representative results are shown, N = 4).

Fig 4

HDMECs restrict R. rickettsii Iowa, but not Sheila Smith, through IFN-β signaling. (A) Lysis of HDMECs was determined by LDH activity in the culture supernatants after 12, 18, and 24 h of infection. Select samples were also treated with isotype control or anti-IFNAR2 (mean ± S.D., N = 6). (B) Normalized bacterial burdens within HDMECs infected with R. rickettsii Iowa or Sheila Smith treated with isotype control or anti-IFNAR2 for 24 h (mean ± S.D., N = 6). Significance in A was assessed with two-way ANOVA and one-way ANOVA in B; P < 0.05, *; P < 0.0001, ****. (C) Western blotting with anti-p-STAT1, STAT1, p-STAT2, STAT2, and GAPDH of HDMECs infected with R. rickettsii strains treated with isotype control or anti-IFNAR2 antibodies (3 mg/mL) for 6 and 12 h (representative results are shown, N = 4). (D) Western blotting with anti-TRAIL, IDO, and GAPDH of HDMECs infected with R. rickettsii strains treated with isotype control or anti-IFNAR2 for 12, 18, and 24 h (representative results are shown, N = 3), ns = not significant.

Fig 5

Complementation of R. rickettsii Iowa with RARP2 or RapL restores virulence phenotypes in HDMECs. (A) Lysis of HDMECs was measured over 24 h of infection with R. rickettsii strains (error bars represent mean ± S.D., N = 6). No samples were significantly different at T = 0. At 24 HPI, there was no significant difference between Iowa and Iowa + RarP2; Iowa and Iowa + FC1 were significantly different (P < 0.01); as were SS and Iowa + RapL (P < 0.01). All other samples were significantly different from each other (P < 0. 0001). Data were analyzed by one-way ANOVA using Tukey’s multiple comparison adjustment. (B) ELISA measurements of IFN-β in HDMEC supernatants and monolayer lysates after 12 h of infection with R. rickettsii strains (error bars represent mean ± S.D., N = 6). Data were analyzed by two-way ANOVA with Sidak’s multiple comparison test. ns = not significant; * = P < 0.05; **** = P < 0.0001. (C) Normalized rickettsial burdens within HDMECs after 2 or 24 h of infection with R. rickettsii Iowa strains complemented with control vector (FC1) or those expressing Sheila Smith RARP2, RapL, or RapL with a point mutation (RapLS160A) in the putative active site serine (17). Shown are the means ± S.D. (n = 6). Significance was assessed by two-way ANOVA with Sidak’s multiple comparison test. ns = not significant; * = P < 0.05; **** = P < 0.0001. (D) IFN-β levels in supernatants from HDMEC cells infected with R. rickettsii Iowa strains shown in panel C were determined. Secreted IFN-β in the RARP2-expressing strain was below the limits of detection. Shown are the means ± S.D. (n = 6). Data were analyzed by one-way ANOVA using Tukey’s multiple comparison adjustment. ns = not significant; * = P < 0.05; *** = P < 0.001.