The Nucleotide Excision Repair Pathway Protects Borrelia burgdorferi from Nitrosative Stress in Ixodes scapularis Ticks

Abstract

The Lyme disease spirochete Borrelia burgdorferi encounters a wide range of environmental conditions as it cycles between ticks of the genus Ixodes and its various mammalian hosts. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are potent antimicrobial molecules generated during the innate immune response to infection, however, it is unclear whether ROS and RNS pose a significant challenge to B. burgdorferi in vivo. In this study, we screened a library of B. burgdorferi strains with mutations in DNA repair genes for increased susceptibility to ROS or RNS in vitro. Strains with mutations in the methyl-directed mismatch repair gene mutS1 are hypersensitive to killing by ROS, while strains lacking the nucleotide excision repair (NER) gene uvrB show increased susceptibility to both ROS and RNS. Therefore, mutS1-deficient and uvrB-deficient strains were compared for their ability to complete their infectious cycle in Swiss Webster mice and I. scapularis ticks to help identify sites of oxidative and nitrosative stresses encountered by B. burgdorferi in vivo. Both mutS1 and uvrB were dispensable for infection of mice, while uvrB promoted the survival of spirochetes in I. scapularis ticks. The decreased survival of uvrB-deficient B. burgdorferi was associated with the generation of RNS in I. scapularis midguts and salivary glands during feeding. Collectively, these data suggest that B. burgdorferi must withstand cytotoxic levels of RNS produced during infection of I. scapularis ticks.

Keywords: Borrelia; DNA repair; Lyme disease; nitric oxide; oxidative stress.

FIGURE 1

DNA repair pathways of B. burgdorferi. B. burgdorferi encodes genes of several DNA repair pathways including: BER, NER, and MMR. The BER pathway consists of monofunctional glycosylases including uracil DNA glycosylase (Ung, BB0053) and 3-methyladenine glycosylase (Mag, BB0422); a bifunctional glycosylase/endonuclease III (Nth, BB0745); and an exonuclease (XthA, BB0534). Glycosylases recognize and remove DNA bases that are oxidized (8-oxoguanine), alkylated, or deaminated leaving an apurinic or apyrimidinic site (AP site). AP endonucleases (Nth and/or XthA) cleave one nucleotide for short-patch BER, or 2–13 nucleotides for long-patch BER. The combined action of ssDNA exonuclease (RecJ, BB0254), DNA polymerase I (Dpol, BB0548), and DNA ligase (Lig, BB0552) repair the DNA lesion. The NER pathway removes bulky chemically induced lesions, lesions induced by UV light, or otherwise unrecognized DNA lesions that distort the DNA helix using the UvrABC endonuclease complex (UvrBA, BB0836-837; UvrC, BB0457). UvrA and UvrB form a dimer that scans the genome for DNA lesions, or is actively recruited to DNA lesions by the transcription coupling factor Mfd (BB0627). Following recognition of a lesion, UvrA dissociates from UvrB. Next, UvrB forms a dimer with UvrC that cuts a 12 base-pair segment of DNA, which is excised by DNA helicase II (UvrD, BB0344). The resulting gap in host DNA is then filled by Dpol and Lig. The MMR pathway in B. burgdorferi consists of the mismatch repair proteins MutS1 (BB0797), MutS2 (BB0098), and MutL (BB0211), but lacks a homolog for the weak endonuclease MutH. MutS1 binds to mismatched or damaged bases in the DNA strand, and then forms a complex with MutL. Typically, MutH binds to hemi-methylated regions of DNA, and is activated following contact with MutL. However, the B. burgdorferi MutL homolog encodes a putative endonuclease domain that may have activity that compensates for the lack of MutH, as has been described for Thermus thermophilus (Shimada et al., 2013). MutL is therefore activated upon binding to MutS1, resulting in a cut and displacement of the damaged DNA strand. Next, RecJ and UvrD excise the damaged strand of DNA. The single stranded binding protein (SSB, BB0114) protects the exposed segment of ssDNA, which is then repaired by DNA polymerase III (DpoIII, BB0438) and Lig. The cellular role of MutS2 in B. burgdorferi is unknown, although it may function as an inhibitor of homologous recombination, or the repair of oxidized DNA bases as described for Helicobacter pylori (Wang et al., 2005)

FIGURE 2

The NER and MMR pathways contribute to B. burgdorferi resistance to ROS and RNS produced chemically in vitro. Wild-type B. burgdorferi strain B31 A3 and the isogenic DNA repair mutants ΔuvrB::aadA and ΔmutS1::aadA were grown to a cell density of 5 × 10⁷ cells ml^-1 in BSK II medium under microaerobic (3% O₂, 5% CO₂) conditions. B. burgdorferi strains were treated with 1.0 mM diethylamine NONOate (DEA/NO) or 2.5 mM H₂O₂ for 2 h at 34°C in either BSK-II medium (A) or in HN (25 mM Hepes, 100 mM NaCl) buffer (B). Following incubation, cells were diluted in fresh BSK II medium and plated on solid BSK II media. Data represent the mean CFUs ± SD of six replicates collected from three separate experiments. ^∗P < 0.05 compared to wild-type controls using a two-way ANOVA.

FIGURE 3

The NER pathway promotes survival of B. burgdorferi in I. scapularis ticks. The number of viable B. burgdorferi spirochetes in ticks after feeding on Swiss-Webster mice infected with wild-type, ΔuvrB::aadA, or ΔmutS1::aadA B. burgdorferi strains was determined prior to the molt at 12–18 days post-feeding (A), and after molting to nymphs at 62–81 days (B). Individual ticks were homogenized, and serial dilutions were plated on solid BSK II medium. The number of B. burgdorferi CFUs from individual ticks are shown, along with the mean number of spirochetes per tick. ^∗P < 0.05 compared to wild-type controls using a one-way ANOVA.

FIGURE 4

RNS production is induced during feeding of I. scapularis adults. Midguts and salivary glands were harvested from unfed ticks and ticks fed on rabbits. Organs were rinsed in PBS and subsequently incubated in PBS + 25 μM DAF-2 (5,6-diaminoflorescein diacetate) for 10 min. Images were collected using digital interference contrast (DIC) microscopy, or immunofluorescent microscopy using a FITC filter (Ex_λ = 495 nm, Em_λ = 519 nm).

FIGURE 5

Expression of nos, duox, and salp25D in I. scapularis larvae and nymphs. RNA isolated from whole I. scapularis larvae (A), and salivary glands and midguts of nymphs (B) were analyzed by RT-qPCR for the transcription of nos, duox, and salp25D. The expression levels of each gene were normalized using actin (act) as a reference gene. Data represent the mean ± SD of 4–8 biological replicates. ^∗P < 0.05 compared to unfed controls using a two-way ANOVA.

FIGURE 6

Nitric oxide synthase is expressed during feeding of I. scapularis ticks. Salivary glands (SG) and midguts (MG) harvested from I. scapularis nymphs fed on Swiss Webster mice were probed with rabbit serum (-) or with a universal NOS (uNOS) antibody (+) followed by incubation with an Alexa Fluor 488 secondary antibody and DAPI.

FIGURE 7

Nitrotyrosine formation in salivary glands during feeding of I. scapularis nymphs. Salivary glands (SG) and midguts (MG) harvested from I. scapularis nymphs fed on Swiss Webster mice were probed with rabbit serum (-) or with an anti-nitrotyrosine antibody (+) followed by incubation with an Alexa Fluor 647 secondary antibody and DAPI.