Polysaccharide synthesis operon modulates Rickettsia-endothelial cell interactions

Abstract

Pathogenic Rickettsia species target vascular endothelial cells and cause systemic vasculitis. As obligate intracellular bacterial pathogens, Rickettsia must secure nutritional resources within the cytoplasm of endothelial cells while simultaneously subverting the innate immune defense system. With advances in rickettsial and host genetics, recent studies have identified novel molecular mechanisms involved in the complex interactions between Rickettsia and endothelial cells. However, it remains unclear how Rickettsia shields pathogen-derived immune stimulants, such as lipopolysaccharides (LPS) and peptidoglycan fragments, from immune recognition during intracellular replication. Prior work described two Rickettsia conorii variants with kkaebi transposon insertions in the polysaccharide synthesis operon (pso). Biochemical and immunological analyses revealed that pso is responsible for the biosynthesis of O-antigen (O-Ag) and the proper assembly of surface proteins. In the present work, we document that pso variant HK2 exhibits reduced capacities to adhere to and invade microvascular endothelial cells. Despite the low intracellular abundance, HK2 induced significantly higher levels of proinflammatory cytokines and chemokines, leading to premature cell death. Notably, HK2 exhibited defective intracellular survival in bone marrow-derived macrophages. This inability to dampen endothelial cell-mediated immune stimulation and resist macrophage-induced bactericidal activities resulted in the rapid elimination of viable Rickettsia in the mouse model of spotted fever. Further, when tested as a live-attenuated vaccine, HK2 elicited robust protective immunity against lethal spotted fever pathogenesis. Our work highlights the crucial role of pso in enabling Rickettsia to evade immune surveillance during intracellular replication within endothelial cells, ultimately delaying pathogen-induced programmed cell death and escaping immune defense mechanisms.

Fig 1. pso contributes to rickettsial invasion in Vero cells.

(A) Vero cells (N = 3, mean ± standard deviation) were infected with R. conorii WT or pso variants (HK2 and HK15) at an MOI of 0.01 to determine rickettsial replication at timed intervals. (B) Representative DIC and fluorescent microscopic images of Vero cells infected with R. conorii WT or pso variants on day 4 post-infection. Two-way ANOVA with Tukey’s multiple comparisons test was performed, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 2. pso contributes to rickettsial invasion and intracellular survival in endothelial cells.

(A) HMEC-1 cells (N = 3, mean ± standard deviation) were infected with R. conorii WT or pso variants at an MOI of 0.01 to determine rickettsial replication at timed intervals and (B) LDH release in the supernatant. (C) Representative DIC and fluorescent microscopic images of HMEC-1 cells infected with R. conorii WT or pso variants on day 4 post-infection. Two-way ANOVA with Tukey’s multiple comparisons test was performed, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 3. Differential endothelial cell responses to pso infections.

(A, B) HMEC-1 cells (N = 3, mean ± standard deviation) were infected with R. conorii WT or pso variants at an MOI of 3 for 6 hours to identify genes differentially regulated upon rickettsial infections. ELISA was performed on the culture supernatant to determine the abundance of (C) CCL2, (D) CXCL1, and (E) IL-6. One-way ANOVA with Tukey’s multiple comparisons test was performed, **** P < 0.0001.

Fig 4. pso variants exhibit defective invasion and immune modulation in primary endothelial cells.

(A) HDMECs (N = 3, mean ± standard deviation) were infected with R. conorii WT or pso variants at an MOI of 0.01 to determine rickettsial replication at timed intervals. (B) Representative DIC microscopic images of HDMECs infected with R. conorii WT or pso variants on day 4 post-infection. Areas exhibiting cytopathology are marked with yellow dotted lines and quantified [N = 25 (WT), 17 (HK2), and 32 (HK15) plaques from 9, 10, and 12 DIC images, mean ± standard error of the mean]. To determine the abundance of (C) CCL2, (D) CXCL1, and (E) IL-6, ELISA was performed on the culture supernatants of HDMECs infected with R. conorii WT or pso variants at an MOI of 3 for 6 hours. One-way or Two-way ANOVA with Tukey’s multiple comparisons test or nonparametric Kruskal-Wallis with Dunn’s multiple comparison test were performed, ** P < 0.01, **** P < 0.0001.

Fig 5. pso variant HK2 is defective for attachment and survival in BMDMs.

(A) BMDMs (N = 3, mean ± standard deviation) were infected with R. conorii WT or pso variants at an MOI of 0.1 to determine rickettsial replication at timed intervals. (B) Representative DIC microscopic images of BMDMs infected with R. conorii WT or pso variants on days 2 and 3 post-infection. Two-way ANOVA with Tukey’s multiple comparisons test was performed, * P < 0.05, ** P < 0.01.

Fig 6. pso contributes to rickettsial pathogenesis in mice.

Cohorts of C3H mice (N = 10) were intravenously injected with 1 × 10³ PFU R. conorii WT or pso variants and monitored for 14 days to document (A) body weight changes (mean ± standard error of the mean) and (B) mortality. On days 0, 2, and 4 of infection, rickettsial burdens in the (C) lungs and (D) spleen were assessed by plaque assay (N = 4). To determine the abundance of (E) CCL2, (F) CXCL1, and (G) IL-6, ELISA was performed with hyperimmune sera collected from Rickettsia-infected mice (N = 10) on day 3 of infection. One-way or Two-way ANOVA with Tukey’s multiple comparisons and Log-rank test were performed. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 7. pso variant HK2 is avirulent and functions as a live-attenuated vaccine.

Cohorts of C3H mice (N = 5) were intravenously injected with 5 × 10⁶ PFU HK2 or SPG buffer (Mock) and monitored for 21 days to document (A) body weight changes (mean ± standard error of the mean) and (B) mortality. On day 21 post-infection, surviving mice were challenged with 1 × 10³ PFU R. conorii WT to determine (C) body weight changes (mean ± standard error of the mean) and (D) mortality. Log-rank test was performed. **** P < 0.0001.