PERK-mediated antioxidant response is key for pathogen persistence in ticks

Abstract

A crucial phase in the lifecycle of tick-borne pathogens is the time spent colonizing and persisting within the arthropod. Tick immunity is emerging as a key force shaping how transmissible pathogens interact with the vector. How pathogens remain in the tick despite immunological pressure remains unknown. In persistently infected Ixodes scapularis , we found that Borrelia burgdorferi (Lyme disease) and Anaplasma phagocytophilum (granulocytic anaplasmosis) activate a cellular stress pathway mediated by the endoplasmic reticulum receptor PERK and the central regulatory molecule, eIF2α. Disabling the PERK pathway through pharmacological inhibition and RNAi significantly decreased microbial numbers. In vivo RNA interference of the PERK pathway not only reduced the number of A. phagocytophilum and B. burgdorferi colonizing larvae after a bloodmeal, but also significantly reduced the number of bacteria that survive the molt. An investigation into PERK pathway-regulated targets revealed that A. phagocytophilum and B. burgdorferi induce activity of the antioxidant response regulator, Nrf2. Tick cells deficient for nrf2 expression or PERK signaling showed accumulation of reactive oxygen and nitrogen species in addition to reduced microbial survival. Supplementation with antioxidants rescued the microbicidal phenotype caused by blocking the PERK pathway. Altogether, our study demonstrates that the Ixodes PERK pathway is activated by transmissible microbes and facilitates persistence in the arthropod by potentiating an Nrf2-regulated antioxidant environment.

Figure 1 |. Tick-borne pathogens induce eIF2α-regulated stress responses in infected, unfed nymphs.

(A) Graphic representation of IRE1α-TRAF2 signaling and the Integrated Stress Response pathways in Ixodes ticks. (B-I) Gene expression in flat, unfed I. scapularis nymphs that are either uninfected (−), A. phagocytophilum-infected (A.p.), or B. burgdorferi-infected (B.b.). Each data point is representative of 1 nymph. Gene expression was quantified by qRT-PCR using primers listed in Supplemental Table 1. Student’s t-test. *P < 0.05. (J) Phosphorylated eIF2α (36 kDa) immunoblot against ISE6 tick cells that were either uninfected (−), infected for 24 hrs (A. phagocytophilum: A.p.; B. burgdorferi: B.b.; MOI 50), or treated with the eIF2α phosphorylation inhibitor ISRIB for 1 hr prior to infection (24 hrs). β-actin was probed as an internal loading control (45 kDa). Immunoblots are representative of 2 biological replicates. See also Supplemental Figure 1.

Figure 2 |. The PERK-eIF2α-ATF4 axis promotes A. phagocytophilum infection in tick cells.

(A-B) ISE6 tick cells (1 × 10⁶) were pretreated with ISRIB (A) or salubrinal (B) at the indicated concentrations for 1 hr prior to infection with A. phagocytophilum for 18 hrs (MOI 50). (C-G) IDE12 tick cells (1 × 10⁶) were treated with silencing RNAs (siRNA) against indicated genes or scrambled RNA controls (scRNA) for 24 hrs prior to infection with A. phagocytophilum (MOI 50) for 18 hrs. A. phagocytophilum burden and gene silencing for eIF2α (C), ATF4 (D), GCN2 (E), HRI (F), and PERK (G) were measured by qRT-PCR. Data are representative of at least five biological replicates with at least two technical replicates. Error bars show SEM, *P < 0.05 (Student’s t-test). scRNA, scrambled RNA; siRNA, small interfering RNA.

Figure 3 |. The PERK pathway supports A. phagocytophilum in vivo.

I. scapularis larvae were immersed overnight in siRNA targeting PERK (A-D), eIF2α (E-H), or ATF4 (I-L) and fed on A. phagocytophilum-infected mice. Silencing efficiency (A, E, I) and bacterial burden were assessed at three time intervals by qRT-PCR: immediately following repletion (B, F, J), one-week post-repletion (C, G, K), and after ticks molted to nymphs (D, H, L). Data are representative of 10–20 ticks and at least two experimental replicates. Each point represents one tick, with two technical replicates. Error bars show SEM, *p < 0.05 (Welch’s t-test). NS, non-significant. scRNA, scrambled RNA, siRNA, small interfering RNA.

Figure 4 |. In vivo B. burgdorferi colonization and persistence through the molt is supported by the PERK pathway.

PERK (A-D), eIF2α (E-H), or ATF4 (I-L) were silenced in I. scapularis larvae by immersing ticks in siRNA overnight. Recovered ticks were fed on B. burgdorferi-infected mice. Silencing efficiency (A, E, I) and bacterial burden were assessed at three time intervals by qRT-PCR: immediately following repletion (B, F, J), two weeks post-repletion (C, G, K), and after ticks molted to nymphs (D, H, L). Data are representative of 10–20 ticks and at least two experimental replicates. Each point represents one tick, with two technical replicates. Error bars show SEM, *P < 0.05 (Welch’s t-test). scRNA, scrambled RNA, siRNA, small interfering RNA.

Figure 5 |. Infection triggers an Nrf2-regulated antioxidant response in ticks that promotes pathogen survival.

(A-B) HEK293T cells (1 × 10⁴) were transfected with luciferase reporter vectors for assaying activity of ER stress transcription factors XBP1, NF-κB, CHOP, SREBP1, and NRF2 or were untransfected (−). Cells were then infected with A. phagocytophilum (A.p.) (A) or B. burgdorferi (B.b.) (B). After 24 hrs, D-luciferase was added and luminescence was measured as relative luminescence units (RLU). Measurements were normalized to uninfected controls (gray bars). Luciferase assays are representative of 3–5 biological replicates with at least two experimental replicates ± SEM. Student’s t-test. *P < 0.05. (C-D) Predicted Ixodes Nrf2 structure modeled with AlphaFold^, (blue) and overlaid with human Nrf2 (orange) using UCSF ChimeraX. The bZIP domain is indicated by a box with dashed lines. (D) Magnified region of the bZIP domain depicting residues that that are predicted to interact with ARE sequences in DNA promoter regions (R877, R880, R882, N885, A888, A889, R893, R895, K896). See also Supplemental Figure 2. (E) Nrf2 expression levels in flat, unfed nymphs that are uninfected (−), A. phagocytophilum-infected (A.p.), or B. burgdorferi-infected (B.b.). Each data point is representative of 1 nymph. Gene expression was quantified by qRT-PCR using Nrf2 primers listed in Supplemental Table 1. Student’s t-test. *P < 0.05. (F-H) IDE12 tick cells were treated with silencing RNAs (siRNA) targeting nrf2 for 24 hrs prior to infection with A. phagocytophilum (18 hrs) (F-G) or B. burgdorferi (H). Gene silencing (F-H) and bacterial burden (G-H) were quantified by qRT-PCR. ROS was measured as relative fluorescent units (RFU) after 24 hrs of infection (F). Data are representative of at least 4–5 biological replicates and two technical replicates. Error bars show SEM, *P < 0.05 (Student’s t-test). scRNA, scrambled RNA; siRNA, small interfering RNA.

Figure 6 |. Antioxidant activity of the PERK-eIF2α pathway protects pathogens in ticks.

(A-D) ROS (A, B) and RNS (C, D) measurements in ISE6 cells (1.68 × 10⁵) untreated (−), infected (A.p. or B.b.), or pretreated with 1μM ISRIB prior to infection with A. phagocytophilum (ISRIB + A.p.) (A, C) or B. burgdorferi (ISRIB + B.b.) (B, D). Fluorescence was measured at the indicated time points and is presented as RFU, normalized to untreated, uninfected controls (−). (E-F) IDE12 cells were infected with A. phagocytophilum (E) or B. burgdorferi (F) alone or in the presence of 5mM NAC for 24 hrs. (G-H) perk was silenced in IDE12 cells (1 × 10⁶). Cells were infected with A. phagocytophilum (G) or B. burgdorferi (H) alone or in the presence of 5mM NAC. Silencing levels and bacterial burdens were quantified by qRT-PCR. Data are representative of at least 4–5 biological replicates and two technical replicates. Error bars show SEM, *P < 0.05 (Student’s t-test). NAC, N-acetyl cysteine. scRNA, scrambled RNA; siRNA, small interfering RNA.

Figure 7 |. The PERK-eIF2α-ATF4 axis promotes pathogen survival in ticks through an Nrf2-mediated antioxidant response.

When colonizing the tick, A. phagocytophilum and B. burgdorferi trigger the Ixodes IMD pathway and ROS/RNS through the IRE1α-TRAF2 axis of the UPR. Tick-borne microbes persist in the tick over time by stimulating the PERK branch of the UPR, which signals through eIF2α and the transcription factors ATF4 and Nrf2 to trigger an antioxidant response that promotes microbial survival.