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. 2018 Apr 17;115(16):E3788-E3797.
doi: 10.1073/pnas.1718595115. Epub 2018 Apr 2.

Plasticity in Early Immune Evasion Strategies of a Bacterial Pathogen

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Free PMC article

Plasticity in Early Immune Evasion Strategies of a Bacterial Pathogen

Quentin Bernard et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Borrelia burgdorferi is one of the few extracellular pathogens capable of establishing persistent infection in mammals. The mechanisms that sustain long-term survival of this bacterium are largely unknown. Here we report a unique innate immune evasion strategy of B. burgdorferi, orchestrated by a surface protein annotated as BBA57, through its modulation of multiple spirochete virulent determinants. BBA57 function is critical for early infection but largely redundant for later stages of spirochetal persistence, either in mammals or in ticks. The protein influences host IFN responses as well as suppresses multiple host microbicidal activities involving serum complement, neutrophils, and antimicrobial peptides. We also discovered a remarkable plasticity in BBA57-mediated spirochete immune evasion strategy because its loss, although resulting in near clearance of pathogens at the inoculum site, triggers nonheritable adaptive changes that exclude detectable nucleotide alterations in the genome but incorporate transcriptional reprograming events. Understanding the malleability in spirochetal immune evasion mechanisms that ensures their host persistence is critical for the development of novel therapeutic and preventive approaches to combat long-term infections like Lyme borreliosis.

Keywords: Borrelia burgdorferi; Lyme disease; antimicrobial peptide; immune evasion; microbial persistence.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
BBA57 is required for mammalian infection but redundant for tick persistence. (AC) Acquisition and persistence of bba57 mutants in ticks. Larvae were allowed to parasitize mice infected with wild-type B. burgdorferi (WT, white bars), bba57− mutant (bba57−, gray bars), or bba57 complemented isolates (bba57+, black bars). The burden in fed larvae (A), in freshly molted and unfed nymphs (B), and in infected nymphs fed on naive mice (C) was assessed by RT-qPCR. (D) B. burgdorferi burden in mice after transmission via infected nymphs. Mice were parasitized with nymphs infected with WT (white bars), bba57− (gray bars), or bba57+ (black bars). Infection was assessed by RT-qPCR analysis of pathogen burden in skin, joint, heart, and bladder samples obtained 2 wk after infection. Asterisk, not detectable. (E) B. burgdorferi migration to tick salivary glands. Salivary glands were isolated from wild-type or bba57− mutant–infected ticks, as shown in C, which parasitized naive mice for 3 d. Samples were fixed and stained with FITC-labeled anti-B. burgdorferi goat IgG and DAPI. (F) Tick engorgement on an artificial membrane feeding system. Top view of the capsules showing partly (arrowhead) or fully (arrow) engorged nymphs infected with either wild type (WT, Top) or mutants (bba57−, Bottom). Fed ticks were collected after complete feeding. (G) Assessment of B. burgdorferi exodus from ticks, during dissemination from infected ticks to feeder chamber containing naive blood. After tick engorgement, blood samples were collected and processed to analyze the levels of WT (white bars) and bba57− mutant (gray bars) by RT-qPCR. (H) Assessment of B. burgdorferi entry in naive ticks, from feeder chamber containing spirochete-infected blood (103 cells per mL). Ticks were collected after engorgement and assessed for burden of WT (white bars) or bba57− (gray bars) mutants by RT-qPCR targeting B. burgdorferi flaB gene. Bars represent the mean ± SEM of at least triplicate experiments. Data in AH are representative of at least three independent experiments. *P < 0.05.
Fig. 2.
Fig. 2.
BBA57 facilitates B. burgdorferi survival during early mammalian infection, whereas its absence uncovers bacteria adaptation strategy improving its persistence. (A) The B. burgdorferi burden at the dermal injection site. Mice were infected with wild-type B. burgdorferi (WT, white bars) or bba57− mutant (bba57−, gray bars). The skin injection site was collected at 2 h and days 3, 5, 7, 14, and 21 to analyze the bacteria burden by RT-qPCR. (B) B. burgdorferi burden in disseminated tissues after inoculation via a direct, host-to-host, reinfection strategy. Mice were infected with WT, bba57−, or bba57 complemented (bba57+) isolates. At 14 d postinfection, the pathogen burden in disseminated organs was assessed by RT-qPCR (primary infection, white bars) in disseminated organs. A group of mice was euthanized at 21 d of infection, and B. burgdorferi retrieved from the bladder were used to infect new sets of naive mice (reinfection, black bars). The spirochete burden was analyzed 14 d after infection in reinfected mice. (C) B. burgdorferi burden in disseminated tissues after indirect, culture-to-host reinfection strategy. Mice were infected with various spirochete isolates as shown in B with the exception of culturing isolated spirochetes in BSK media between primary and reinfection. (D) B. burgdorferi gene expression during persistence in the mammalian host. Out of 114 B. burgdorferi genes tested for expression, a set of limited genes (bb0405, bb0090, and bb0598) were differently expressed at 21 d between WT (white bars) and bba57− mutants (gray bars), as examined by RT-qPCR (Left) in the distant skin sites during primary infection. The same panel of genes expressed by bba57− mutant B. burgdorferi in the distant skin sites at 14 d between indirect reinfection (white bars) and directs reinfection (black bars) as analyzed by RT-qPCR (Right). Bars represent the mean ± SEM of at least triplicates. Data in AD are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 3.
Fig. 3.
BBA57 modulates the expression of several B. burgdorferi gene products involved in spirochete evasion of host innate immunity. (A) Differential antibody responses in mice infected with wild-type B. burgdorferi (WT) or bba57− mutant (bba57−). Serum samples from mice infected for 21 d with WT (Left) or bba57− (Right) B. burgdorferi was collected and used as a probe for immunoblotting against lysates prepared from wild-type B. burgdorferi. Protein spots present in both immunoblot samples were indicated by black squares, whereas red and blue squares denote identified and unidentified proteins, respectively, and ones up-regulated or only present in WT-infected mice. Protein identification was based on comparison with published information about electrophoretic migration of B. burgdorferi proteins and the characterization of their isoelectric points (39). (BF) Reduced transcript levels of representative B. burgdorferi genes produced by the mutants at the injection site. Mice were injected with WT (white bars) or bba57− (gray bars) isolates. After 7 d, the injection site was retrieved and processed for expression analysis of ospC (B), erpB (C), erpP (D), bbi36/38/39 (E), and bmpA (F) expression by RT-qPCR. (G) BBA57 protects B. burgdorferi against serum complement-mediated killing activity; B. burgdorferi were exposed to different concentration of normal mouse serum (NMS) or heat inactivated mouse serum (HIS) for 18 h. The percentage of living spirochetes was determined based on bacteria motility using dark-field microscopy (Left). Right denotes regrowth assay. B. burgdorferi were grown in BSK medium after exposure with NMS or HIS for 48 h. The spirochetes were counted every day for a week using dark-field microscopy. Bars represent the mean ± SEM of at least triplicates. Data in AG are representative three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4.
Fig. 4.
BBA57 protects B. burgdorferi at the dermal inoculum site against neutrophil-derived bactericidal responses. (A) Histology of murine skin at tick bite sites 5 d after placement of naive nymphal ticks or infested nymphs shows increased cellular infiltrates at the tick-bite site in those animals exposed to bba57B. burgdorferi (yellow arrows). (B) B. burgdorferi burden at the tick bite site. The WT (white bars) or bba57− mutant B. burgdorferi (gray bars)-infected ticks were placed on naive mice. Five days later, the bite sites were collected and processed for bacterial burden analysis by RT-qPCR. (C) The levels of B. burgdorferi at the injection site in immunodeficient mice. Groups of BALB/c (white bars) or SCID mice (black bars) were injected with pathogens, and their burdens were detected at the injection site after 5 d by RT-qPCR. (D) Burden of B. burgdorferi at the injection site following macrophages depletion. Mice were treated with liposomes containing either PBS (white bars) or clodronate (black bars). Following cell depletion, mice were injected pathogens, and their burdens were detected after 5 d by RT-qPCR, as shown in C. (E) B. burgdorferi burden at the injection site after neutrophil depletion. Mice were treated with control (white bars) or anti-Ly6G (clone 1A8) antibodies (black bars) to deplete neutrophils. Following cell depletion, mice were injected pathogens, and their burdens were detected after 5 d by RT-qPCR, as shown in C. Bars represent the mean ± SEM of at least triplicates. Data in AE are representative of at least six mice and two independent experiments. *P < 0.05.
Fig. 5.
Fig. 5.
BBA57 alters the host antimicrobial response. (A) Assessment of host antibacterial inflammatory response at the dermal injection site. Equal numbers of wild-type B. burgdorferi (WT) or bba57− mutant (bba57−) (105 cells per animal) were injected into the skin. Five days following inoculum, the injection sites were retrieved and processed for a PCR array for simultaneous analysis of expression of 84 host antibacterial response genes. The table summarizes the fold change of gene expression of bba57− mutant compared with WT. Genes up-regulated during mutant infection are represented in red. Blue squares show the most dramatically induced genes, bactericidal/permeability-increasing protein (BPI), and an IFN gene (Ifna9) explored in this study. (B) Up-regulation of BPI during syringe-inoculated infection with bba57 mutants. Equal numbers of WT (white bars) or bba57− (black bars) isolates (105 cells per animal) were injected into mouse skin. Injection sites were retrieved following 5 d of inoculum and analyzed for BPI expression by RT-qPCR. (C) Induction of BPI during tick-borne infection with bba57 mutants. Naive nymphal ticks or nymphs infected with WT (gray bars) or bba57− mutants (black bars) were allowed to parasitize naive mice. After 5 d of feeding, the bite site was retrieved and BPI expression was analyzed by RT-qPCR. (D) Reduction of bba57 mutant-induced BPI expression following depletion of neutrophils. Mice were treated for cell depletion as shown in Fig. 3E. The expression of BPI was measured at the injection site by RT-qPCR after 5 d of infection. (E) BPI induction by neutrophils following exposure with spirochetes in culture. Primary neutrophils were isolated from mice and then immediately incubated in vitro with only medium (white bar), WT B. burgdorferi (gray bar), or bba57− mutants (black bar). Following spirochete exposure, neutrophils were collected, and BPI expression was measured by RT-qPCR. (F) Up-regulation of BPI during infection with an isogenic mutant lacking a key BBA57-modulated gene, ospC. Equal numbers (105 cells) of the WT B. burgdorferi (white bar) or ospC mutants (black bar) were injected into mouse skin. Injection sites at the dermis were retrieved after 5 d of inculum and processed for BPI expression by RT-qPCR. Bars represent the mean ± SEM of at least triplicates. Data in A–F are representative of at least eight mice and two independent experiments. *P < 0.05; ***P < 0.001.
Fig. 6.
Fig. 6.
BBA57 regulates IFN-stimulated genes. (A) Expression of type I IFN genes in C3H mice is influenced by BBA57. Groups of C3H mice were inoculated with equal levels (105 per animal) of WT B. burgdorferi (white bars) or bba57− mutants (gray bars). Five days after infection, skin injection sites were collected and processed for RT-qPCR to analyze expression of ifna9 (Left) and several other IFN-stimulated genes (Right). (B) Expression of type I IFN genes in BALB/c mice is influenced by BBA57. The animals were infected and analyzed as detailed in A. (C) Modulation of expression of cxcl10 chemokine by BBA57 in primary dermal and myeloid human cells. Human fibroblasts, keratinocytes, macrophages, and neutrophils were incubated with medium only (black bars), WT B. burgdorferi (white bars), or bba57− mutant (gray bars) at a MOI of 50. After 24 h of incubation with fibroblasts (Top Left), keratinocytes (Top Right), or macrophages (Bottom Left) or after 12 h of incubation with neutrophils (Bottom Right), cells were collected and processed for cxcl10 expression using RT-qPCR. Bars represent the mean ± SEM of at least triplicates. Data in AC are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 7.
Fig. 7.
A proposed model showing BBA57 modulation of host innate immune response to promote spirochete persistence. During early host infection process, B. burgdorferi dramatically up-regulated its surface lipoprotein BBA57 that subsequently governs the expression of other surface lipoprotein gene products such as OspC, BBI36/38/39, ErpB, and ErpP. These events facilitate suppression of antimicrobial peptide expression including BPI, largely expressed by neutrophils. The BPI suppression is most likely mediated by OspC. The BBA57 function also assists the pathogen to protect from the host complement system, possibly through the capture of factor H via Erp proteins (at least by Erp B and ErpP). Therefore, bba57− mutants with impaired expression of OspC and Erp proteins are more susceptible to BPI and complement killing. Besides, expression of surface proteins like BBA57 also favors induction of host IFN-stimulated genes such as cxcl10. This chemokine might promote the dissemination of the bacteria to disseminated organs. After evasion of early innate immunity response, residual spirochetes disseminate and colonize distant organs (skin, heart, joint, or bladder), where they eventually establish persistent infection. bba57 mutants, despite their near clearance at the dermal inoculum site, are also able to accomplish persistent infection following a nonheritable adaptive process that likely involves up-regulation of specific microbial genes, notably, bb0598, bb0405, and bb0090.

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