ABSTRACT
Lyme disease, a tickborne illness caused by Borrelia burgdorferi, is an emerging, significant public health concern. B. burgdorferi infections are challenging to study because of their complex life cycle that requires adaptation to both ticks and mammalian hosts for long-term survival and transmission. Bacterial adaptation is accomplished through extensive gene expression alterations in response to environmental cues that remain to be more fully explored. Mouse models of infection serve as valuable tools for studying B. burgdorferi adaptation to the mammalian host and the spirochete's ability to cause persistent infections and thus to interact with and evade the immune system. This article details three mouse models that differ in their primary methods of infection: infestation with B. burgdorferi infected ticks, intradermal inoculation of culture-grown spirochetes, and infection via subcutaneous transplantation of infected tissue. Each method offers unique advantages and limitations. Tick infestation is the route of natural transmission but presents logistical challenges. Syringe inoculation is easy and provides precise control over the infectious dose, but infection is with culture-adapted bacteria. Transplantation of infected tissue introduces mammalian-host-adapted B. burgdorferi in precise anatomical locations, but misses the transfer of tick factors affecting immunity. Detailed protocols are provided for each of the three infection routes, and pros and cons of each method are outlined to help researchers identify the best approach for a research question to be addressed. A protocol is also provided for the treatment of mice with antibiotics that reliably eliminates detectable spirochetes from the animals. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Syringe inoculation of mice with cultured B. burgdorferi and collection of necropsy tissues Basic Protocol 2: Infection of mice with B. burgdorferi via tick infestation Basic Protocol 3: Infection of mice with host-adapted B. burgdorferi via tissue transplant Support Protocol: Clearance of B. burgdorferi by antibiotic treatment.
Subject(s)
Borrelia burgdorferi , Disease Models, Animal , Lyme Disease , Animals , Borrelia burgdorferi/physiology , Borrelia burgdorferi/pathogenicity , Lyme Disease/microbiology , Lyme Disease/transmission , Lyme Disease/immunology , Mice , Ticks/microbiologyABSTRACT
Ixodes ricinus is a vector of several pathogens of public health interest. While forests are the primary habitat for I. ricinus, its abundance and infection prevalence are expected to vary within forest stands. This study assesses the spatio-temporal variations in tick abundance and infection prevalence with three pathogens in and around a peri-urban forest where human exposure is high. Ticks were sampled multiple times in 2016 and 2018 in multiple locations with a diversity of undergrowth, using the consecutive drags method. Three zoonotic pathogens were screened for, Borrelia burgdorferi s.l., Coxiella burnetii, and Francisella tularensis. The influence of season, type of site and micro-environmental factors on tick abundance were assessed with negative binomial generalized linear mixed-effects models. We collected 1642 nymphs and 181 adult ticks. Ticks were most abundant in the spring, in warmer temperatures, and where undergrowth was higher. Sites with vegetation unaffected by human presence had higher abundance of ticks. Forest undergrowth type and height were significant predictors of the level of tick abundance in a forest. The consecutive drags method is expected to provide more precise estimates of tick abundance, presumably through more varied contacts with foliage. Borrelia burgdorferi s.l. prevalence was estimated from pooled ticks at 5.33%, C. burnetii was detected in six pools and F. tularensis was not detected. Borrelia afzelii was the dominant B. burgdorferi genospecies. Tick abundance and B. burgdorferi s.l. infection prevalence were lower than other estimates in Belgian forests.
Subject(s)
Coxiella burnetii , Forests , Francisella tularensis , Ixodes , Animals , Belgium/epidemiology , Ixodes/microbiology , Ixodes/growth & development , Francisella tularensis/isolation & purification , Coxiella burnetii/isolation & purification , Coxiella burnetii/physiology , Nymph/microbiology , Nymph/growth & development , Borrelia burgdorferi/isolation & purification , Borrelia burgdorferi/physiology , Seasons , Population Density , FemaleABSTRACT
Borrelia burgdorferi, a Lyme disease spirochete, causes a range of acute and chronic maladies in humans. However, a primary vertebrate reservoir in the United States, the white-footed deermouse Peromyscus leucopus, is reported not to have reduced fitness following infection. Although laboratory strains of Mus musculus mice have successfully been leveraged to model acute human Lyme disease, the ability of these rodents to model B. burgdorferi-P. leucopus interactions remains understudied. Here, we compared infection of P. leucopus with B. burgdorferi B31 with infection of the traditional B. burgdorferi murine models-C57BL/6J and C3H/HeN Mus musculus, which develop signs of inflammation akin to human disease. We find that B. burgdorferi was able to reach much higher burdens (10- to 30-times higher) in multiple M. musculus skin sites and that the overall dynamics of infection differed between the two rodent species. We also found that P. leucopus remained transmissive to larval Ixodes scapularis for a far shorter period than either M. musculus strain. In line with these observations, we found that P. leucopus does launch a modest but sustained inflammatory response against B. burgdorferi in the skin, which we hypothesize leads to reduced bacterial viability and rodent-to-tick transmission in these hosts. Similarly, we also observe evidence of inflammation in infected P. leucopus hearts. These observations provide new insight into reservoir species and the B. burgdorferi enzootic cycle.IMPORTANCEA Lyme disease-causing bacteria, Borrelia burgdorferi, must alternate between infecting a vertebrate host-usually rodents or birds-and ticks. In order to be successful in that endeavor, the bacteria must avoid being killed by the vertebrate host before it can infect a new larval tick. In this work, we examine how B. burgdorferi and one of its primary vertebrate reservoirs, Peromyscus leucopus, interact during an experimental infection. We find that B. burgdorferi appears to colonize its natural host less successfully than conventional laboratory mouse models, which aligns with a sustained seemingly anti-bacterial response by P. leucopus against the microbe. These data enhance our understanding of P. leucopus host-pathogen interactions and could potentially serve as a foundation to uncover ways to disrupt the spread of B. burgdorferi in nature.
Subject(s)
Borrelia burgdorferi , Disease Reservoirs , Lyme Disease , Mice, Inbred C3H , Mice, Inbred C57BL , Peromyscus , Animals , Peromyscus/microbiology , Mice , Lyme Disease/microbiology , Lyme Disease/transmission , Lyme Disease/veterinary , Borrelia burgdorferi/physiology , Borrelia burgdorferi/genetics , Disease Reservoirs/microbiology , Disease Models, Animal , Ixodes/microbiologyABSTRACT
Emerging and re-emerging pathogens often stem from zoonotic origins, cycling between humans and animals, and are frequently vectored and maintained by hematophagous arthropod vectors. The efficiency by which these disease agents are successfully transmitted between vertebrate hosts is influenced by many factors, including the host on which a vector feeds. The Lyme disease bacterium Borrelia burgdorferi sensu lato has adapted to survive in complex host environments, vectored by Ixodes ticks, and maintained in multiple vertebrate hosts. The versatility of Lyme borreliae in disparate host milieus is a compelling platform to investigate mechanisms dictating pathogen transmission through complex networks of vertebrates and ticks. Squamata, one of the most diverse clade of extant reptiles, is comprised primarily of lizards, many of which are readily fed upon by Ixodes ticks. Yet, lizards are one of the least studied taxa at risk of contributing to the transmission and life cycle maintenance of Lyme borreliae. In this review, we summarize the current evidence, spanning from field surveillance to laboratory infection studies, supporting their contributions to Lyme borreliae circulation. We also summarize the current understanding of divergent lizard immune responses that may explain the underlying molecular mechanisms to confer Lyme spirochete survival in vertebrate hosts. This review offers a critical perspective on potential enzootic cycles existing between lizard-tick-Borrelia interactions and highlights the importance of an eco-immunology lens for zoonotic pathogen transmission studies.
Subject(s)
Ixodes , Lizards , Lyme Disease , Animals , Lizards/microbiology , Lyme Disease/microbiology , Lyme Disease/transmission , Ixodes/microbiology , Humans , Borrelia burgdorferi Group/physiology , Borrelia burgdorferi Group/genetics , Borrelia burgdorferi/genetics , Borrelia burgdorferi/physiologyABSTRACT
Lyme disease is the most common wildlife-to-human transmitted disease reported in North America. The study of this disease requires an understanding of the ecology of the complex communities of ticks and host species involved in harboring and transmitting this disease. Much of the ecology of this system is well understood, such as the life cycle of ticks, and how hosts are able to support tick populations and serve as disease reservoirs, but there is much to be explored about how the population dynamics of different host species and communities impact disease risk to humans. In this study, we construct a stage-structured, empirically-informed model with host dynamics to investigate how host population dynamics can affect disease risk to humans. The model describes a tick population and a simplified community of three host species, where primary nymph host populations are made to fluctuate on an annual basis, as commonly observed in host populations. We tested the model under different environmental conditions to examine the effect of environment on the interactions of host dynamics and disease risk. Results show that allowing for host dynamics in the model reduces mean nymphal infection prevalence and increases the maximum annual prevalence of nymphal infection and the density of infected nymphs. Effects of host dynamics on disease measures of nymphal infection prevalence were nonlinear and patterns in the effect of dynamics on amplitude in nymphal infection prevalence varied across environmental conditions. These results highlight the importance of further study of the effect of community dynamics on disease risk. This will involve the construction of further theoretical models and collection of robust field data to inform these models. With a more complete understanding of disease dynamics we can begin to better determine how to predict and manage disease risk using these models.
Subject(s)
Lyme Disease , Population Dynamics , Lyme Disease/epidemiology , Animals , Humans , Ixodes/microbiology , Ixodes/physiology , Models, Theoretical , Ticks/microbiology , Ticks/physiology , Models, Biological , Borrelia burgdorferi/physiology , Borrelia burgdorferi/pathogenicity , Host-Parasite Interactions , NymphABSTRACT
Warming winters will change patterns of behaviour in temperate and polar arthropods, but we know little about the drivers of winter activity in animals such as ticks. Any changes in behaviour are likely to arise from a combination of both abiotic (e.g. temperature) and biotic (e.g. infection) drivers, and will have important consequences for survival and species interactions. Blacklegged ticks, Ixodes scapularis, have invaded Atlantic Canada and high proportions (30-50%) are infected with the bacteria causing Lyme disease, Borrelia burgdorferi. Infection is correlated with increased overwintering survival of adult females, and ticks are increasingly active in the winter, but it is unclear if infection is associated with activity. Further, we know little about how temperature drives the frequency of winter activity. Here, we exposed wild-caught, adult, female Ixodes scapularis ticks to three different winter temperature regimes (constant low temperatures, increased warming, and increased warming + variability) to determine the thermal and infection conditions that promote or suppress activity. We used automated behaviour monitors to track daily activity in individual ticks and repeated the study with fresh ticks over three years. Following exposure to winter conditions we determined whether ticks were infected with the bacteria B. burgdorferi and if infection was responsible for any patterns in winter activity. Warming conditions promoted increased activity throughout the overwintering period but infection with B. burgdorferi had no impact on the frequency or overall number of ticks active throughout the winter. Individual ticks varied in their levels of activity throughout the winter, such that some were largely dormant for several weeks, while others were active almost daily; however, we do not yet know the drivers behind this individual variation in behaviour. Overall, warming winters will heighten the risk of tick-host encounters.
Subject(s)
Borrelia burgdorferi , Ixodes , Seasons , Animals , Ixodes/microbiology , Ixodes/physiology , Borrelia burgdorferi/physiology , Female , Lyme Disease/transmission , Lyme Disease/microbiology , Temperature , Behavior, AnimalABSTRACT
Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.
Subject(s)
Anaplasma phagocytophilum , Arthropods , Borrelia burgdorferi , Ixodes , Lyme Disease , Animals , Hemocytes , Ixodes/microbiology , Borrelia burgdorferi/physiologyABSTRACT
The bacterial chemotaxis regulatory circuit mainly consists of coupling protein CheW, sensor histidine kinase CheA, and response regulator CheY. Most bacteria, such as Escherichia coli, have a single gene encoding each of these proteins. Interestingly, the Lyme disease pathogen, Borreliella burgdorferi, has multiple chemotaxis proteins, e.g., two CheA, three CheW, and three CheY proteins. The genes encoding these proteins mainly reside in two operons: cheW2-cheA1-cheB2-cheY2 (A-I) and cheA2-cheW3-cheX-cheY3 (A-II). Previous studies demonstrate that all the genes in A-II are essential for the chemotaxis of B. burgdorferi; however, the role of those genes in A-I remains unknown. This study aimed to fill this gap using the CheW2 gene, the first gene in A-I, as a surrogate. We first mapped the transcription start site of A-I upstream of cheW2 and identified a σ70-like promoter (PW2) and two binding sites (BS1 and BS2) of BosR, an unorthodox Fur/Per homolog. We then demonstrated that BosR binds to PW2 via BS1 and BS2 and that deletion of bosR significantly represses the expression of cheW2 and other genes in A-I, implying that BosR is a positive regulator of A-I. Deletion of cheW2 has no impact on the chemotaxis of B. burgdorferi in vitro but abrogates its ability to evade host adaptive immunity, because the mutant can establish systemic infection only in SCID mice and not in immunocompetent BALB/c mice. This report substantiates the previous proposition that A-I is not implicated in chemotaxis; rather, it may function as a signaling transduction pathway to regulate B. burgdorferi virulence gene expression.
Subject(s)
Borrelia burgdorferi , Chemotaxis , Animals , Mice , Chemotaxis/genetics , Virulence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mice, SCID , Borrelia burgdorferi/physiology , Escherichia coli/metabolism , Methyl-Accepting Chemotaxis Proteins/metabolismABSTRACT
Cell motility and biomechanics are critical in various (patho)physiological processes, including the regulation of vascular barrier integrity, which can be subverted by bacterial pathogens. Here, we present a protocol on how to expose endothelial cells (ECs) to vector-borne Borrelia burgdorferi (Bb) and characterize EC kinematics and dynamics during exposure to live or heat-inactivated Bb through traction force and monolayer stress microscopy. Modifications to this protocol may be necessary for studying how different cell types interact with Bb or other microorganisms. For complete details on the use and execution of this protocol, please refer to Yuste et al. (2022).1.
Subject(s)
Borrelia burgdorferi , Borrelia burgdorferi/physiology , Endothelial Cells/metabolism , Biomechanical PhenomenaABSTRACT
The analysis of publications on the websites PubMed, ClinicalKey, devoted to the pathogenesis of neuroborreliosis (NB), using keywords for search: «pathogenesis of neuroborreliosis¼, «neuroborreliosis in children¼, «pathogenesis of Lyme disease¼, as well as an analysis of the results of the published research results of the staff of the Research Institute of Pediatric Infections, St-Petersburg, Russia is presented. Syndromes of early and late NB are more often observed among the forms without migrating erythema, and their development can be caused by all representatives of the species B. burgdorferi s.l. (B.b.), but more often - B. garinii, since it most effectively suppresses the factors of innate and adaptive immune response, reducing interferon production, phagocytosis and complement synthesis. The cause of immunosuppression with the development of NB may be simultaneous infection with several genovids and borrelia species or pathogens of other infections transmitted by Ixodes ticks, for example, infection with B.b. and tick-borne encephalitis virus. The ability to move along peripheral nerves, the change of surface antigens of the VlsE protein, as well as the formation of atypical cysts and granular forms allows B.b. to affect different structures of the peripheral and central nervous system, avoid an immune response and persist for a long time, causing chronic neuroinfection. Both the B.b. themselves, capable of being outside and inside glial cells and neurons, and inflammatory reactions developing in response to their introduction and associated with the synthesis of cytokines and chemokines and mimicry, cause damage to the vascular endothelium, vasculitis and impaired blood supply to the brain, demyelination, autoimmune inflammation and degeneration, leading to the development of NB syndromes, the spectrum of which varies depending on the duration of neuroinfection. In the development of NB and its outcomes, the following are also important: early initiation of treatment, the effectiveness of antibacterial drugs, the use of immunotropic agents that optimize the patient's immune response to the fight against neuroinfection, as well as the timely use of pathogenetic drugs, such as Cytoflavin, which have a complex effect on the vascular endothelium.
Subject(s)
Borrelia burgdorferi , Lyme Neuroborreliosis , Borrelia burgdorferi/physiology , Child , Complement System Proteins , Humans , Inflammation , Lyme Neuroborreliosis/microbiology , Phagocytosis , SyndromeABSTRACT
Range expansion of the vector tick species, Ixodes scapularis, has been detected in Ontario over the last two decades. This has led to elevated risk of exposure to Borrelia burgdorferi, the bacterium that causes Lyme disease. Previous research using passive surveillance data suggests that I. scapularis populations establish before the establishment of B. burgdorferi transmission cycles, with a delay of â¼5 years. The objectives of this research were to examine spatial and temporal patterns of I. scapularis and its pathogens from 2017 to 2019 in southwestern, eastern, and central Ontario, and to explore patterns of B. burgdorferi invasion. Over the 3-year study period, drag sampling was conducted at 48 sites across Ontario. I. scapularis ticks were tested for B. burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia species, including Babesia microti and Babesia odocoilei, and Powassan virus. I. scapularis was detected at 30 sites overall, 22 of which had no history of previous tick detection. B. burgdorferi was detected at nine sites, eight of which tested positive for the first time during this study and five of which had B. burgdorferi detected concurrently with initial tick detection. Tick and pathogen hotspots were identified in eastern Ontario in 2017 and 2018, respectively. These findings provide additional evidence on the range expansion and population establishment of I. scapularis in Ontario and help generate hypotheses on the invasion of B. burgdorferi in Ontario. Ongoing public health surveillance is critical to monitor changes in I. scapularis and its pathogens in Ontario.
Subject(s)
Arachnid Vectors/physiology , Borrelia burgdorferi/physiology , Ixodes/physiology , Lyme Disease/epidemiology , Anaplasma phagocytophilum/classification , Anaplasma phagocytophilum/isolation & purification , Animals , Arachnid Vectors/microbiology , Babesia/classification , Babesia/isolation & purification , Encephalitis Viruses, Tick-Borne/isolation & purification , Ixodes/microbiology , Lyme Disease/transmission , Ontario/epidemiologyABSTRACT
Redox metabolism is crucial in host defense. Previously, it was shown that Borrelia burgdorferi induces the antioxidative metabolism in primary human monocytes. In this study, we explore how B. burgdorferi affects the anti-oxidative arm of redox metabolism, i.e. the generation of reactive oxygen species (ROS). Peripheral blood mononuclear cells (PBMCs) were exposed to B. burgdorferi and generation of ROS was determined both after acute stimulation and after re-stimulation with a secondary stimulus. Though the spirochete induces very low levels of ROS itself, it dramatically decreases the long-term capacity of PBMCs to generate ROS in response to serum-opsonized zymosan (SOZ). This was followed by a compensatory overshoot in ROS generation at later time points. The PI3K/Akt pathway and intracellular levels of methionine play an important regulatory role in this process. Dysregulation of oxidative metabolism may be a novel mechanism by which the spirochete modulates the human immune system and evades killing.
Subject(s)
Borrelia burgdorferi , Borrelia burgdorferi/physiology , Humans , Leukocytes, Mononuclear , NADP/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Reactive Oxygen Species/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
The annual incidence of Lyme disease, caused by tick-transmitted Borreliella burgdorferi, is estimated to be at least 476,000 cases in the United States and many more worldwide. Ten to 20% of antimicrobial-treated Lyme disease patients display posttreatment Lyme disease syndrome (PTLDS), a clinical complication whose etiology and pathogenesis remain uncertain. Autoimmunity, cross-reactivity, molecular mimicry, coinfections, and borrelial tolerance to antimicrobials/persistence have been hypothesized and studied as potential causes of PTLDS. Studies of borrelial tolerance/persistence in vitro in response to antimicrobials and experimental studies in mice and nonhuman primates, taken together with clinical reports, have revealed that B. burgdorferi becomes tolerant to antimicrobials and may sometimes persist in animals and humans after the currently recommended antimicrobial treatment. Moreover, B. burgdorferi is pleomorphic and can generate viable-but-nonculturable bacteria, states also involved in antimicrobial tolerance. The multiple regulatory pathways and structural genes involved in mediating this tolerance to antimicrobials and environmental stressors by persistence might include the stringent (rel and dksA) and host adaptation (rpoS) responses, sugar metabolism (glpD), and polypeptide transporters (opp). Application of this recently reported knowledge to clinical studies can be expected to clarify the potential role of bacterial antibacterial tolerance/persistence in Lyme disease and PTLDS.
Subject(s)
Borrelia burgdorferi , Lyme Disease , Post-Lyme Disease Syndrome , Ticks , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Borrelia burgdorferi/physiology , Lyme Disease/microbiologyABSTRACT
BACKGROUND: Identifying the mechanisms driving disease risk is challenging for multi-host pathogens, such as Borrelia burgdorferi sensu lato (s.l.), the tick-borne bacteria causing Lyme disease. Deer are tick reproduction hosts but do not transmit B. burgdorferi s.l., whereas rodents and birds are competent transmission hosts. Here, we use a long-term deer exclosure experiment to test three mechanisms for how high deer density might shape B. burgdorferi s.l. prevalence in ticks: increased prevalence due to higher larval tick densities facilitating high transmission on rodents (M1); alternatively, reduced B. burgdorferi s.l. prevalence because more larval ticks feed on deer rather than transmission-competent rodents (dilution effect) (M2), potentially due to ecological cascades, whereby higher deer grazing pressure shortens vegetation which decreases rodent abundance thus reducing transmission (M3). METHODS: In a large enclosure where red deer stags were kept at high density (35.5 deer km-2), we used an experimental design consisting of eight plots of 0.23 ha, four of which were fenced to simulate the absence of deer and four that were accessible to deer. In each plot we measured the density of questing nymphs and nymphal infection prevalence in spring, summer and autumn, and quantified vegetation height and density, and small mammal abundance. RESULTS: Prevalence tended to be lower, though not conclusively so, in high deer density plots compared to exclosures (predicted prevalence of 1.0% vs 2.2%), suggesting that the dilution and cascade mechanisms might outweigh the increased opportunities for transmission mechanism. Presence of deer at high density led to shorter vegetation and fewer rodents, consistent with an ecological cascade. However, Lyme disease hazard (density of infected I. ricinus nymphs) was five times higher in high deer density plots due to tick density being 18 times higher. CONCLUSIONS: High densities of tick reproduction hosts such as deer can drive up vector-borne disease hazard, despite the potential to simultaneously reduce pathogen prevalence. This has implications for environmental pathogen management and for deer management, although the impact of intermediate deer densities now needs testing.
Subject(s)
Borrelia burgdorferi/genetics , Deer/parasitology , Ixodes/microbiology , Lyme Disease/epidemiology , Lyme Disease/transmission , Tick Infestations/veterinary , Animal Distribution , Animals , Borrelia burgdorferi/physiology , Female , Larva/genetics , Larva/microbiology , Male , Prevalence , Rodentia/parasitology , Scotland/epidemiology , Vector Borne Diseases/epidemiology , Vector Borne Diseases/parasitology , Vector Borne Diseases/transmissionABSTRACT
BACKGROUND: We evaluated the efficiency of an ex vivo feeding technique using a silicone membrane-based feeding chamber to (i) assess the anti-feeding and acaricidal efficacy of a spot-on combination of dinotefuran, pyriproxyfen and permethrin (DPP, Vectra® 3D) against adult Ixodes scapularis and Ixodes ricinus ticks, and to (ii) explore its effect on blocking the acquisition of Borrelia burgdorferi sensu stricto. METHODS: Eight purpose-bred dogs were randomly allocated to two equal-size groups based on body weight assessed on day 2. DPP was administered topically, as spot-on, to four dogs on day 0. Hair from the eight dogs was collected individually by brushing the whole body on days 2, 7, 14, 21, 28 and 35. On each day of hair collection, 0.05 g of sampled hair was applied on the membrane corresponding to each feeding unit (FU). Seventy-two FU were each seeded with 30 adults of I. scapularis (n = 24 FU) or I. ricinus ticks (n = 48 FU). Bovine blood spiked with B. burgdorferi sensu stricto (strain B31) was added into each unit and changed every 12 h for 4 days. Tick mortality was assessed 1 h after seeding. One additional hour of incubation was added for live/moribund specimens and reassessed for viability. All remaining live/moribund ticks were left in the feeders and tick engorgement status was recorded at 96 h after seeding, and the uptake of B. burgdorferi s.s. was examined in the collected ticks by applying quantitative real-time PCR. RESULTS: Exposure to DPP-treated hair was 100% effective in blocking B. burgdorferi s.s. acquisition. The anti-feeding efficacy remained stable (100%) against both Ixodes species throughout the study. The acaricidal efficacy of DPP evaluated at 1 and 2 h after exposure was 100% throughout the study for I. ricinus, except the 1-h assessment on day 28 (95.9%) and day 35 (95.3%). The 1-h assessment of acaricidal efficacy was 100% at all time points for I. scapularis. CONCLUSIONS: The ex vivo feeding system developed here demonstrated a protective effect of DPP against the acquisition of B. burgdorferi without exposing the animals to the vectors or to the pathogen.
Subject(s)
Dog Diseases/prevention & control , Guanidines/administration & dosage , Insecticides/administration & dosage , Ixodes/drug effects , Lyme Disease/prevention & control , Neonicotinoids/administration & dosage , Nitro Compounds/administration & dosage , Permethrin/administration & dosage , Pyridines/administration & dosage , Administration, Topical , Animals , Borrelia burgdorferi/physiology , Dog Diseases/microbiology , Dogs , Drug Combinations , Feeding Behavior , Female , Ixodes/classification , Ixodes/microbiology , Lyme Disease/transmission , MaleABSTRACT
The Lyme disease spirochete Borrelia burgdorferi relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute key virulence properties of the pathogen, yet these mechanisms remain largely unknown. In vivo expression technology applied to B. burgdorferi (BbIVET) during mammalian infection identified gene bb0562, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of bb0562, bb0563 and bb0564 individually and together demonstrated that bb0562 alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover, bb0562 promoted spirochete survival during the blood dissemination phase of infection. Gene bb0562 was also found to be important for spirochete growth in low serum media and the growth defect of Δbb0562 B. burgdorferi was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting bb0562 may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that bb0562 is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.
Subject(s)
Bacterial Proteins/metabolism , Fatty Acids/deficiency , Gene Expression Regulation, Bacterial , Host-Pathogen Interactions , Lipase/metabolism , Lyme Disease/microbiology , Virulence Factors/metabolism , Animals , Bacterial Proteins/genetics , Borrelia burgdorferi/physiology , Female , Ixodes/microbiology , Lyme Disease/immunology , Lyme Disease/metabolism , Male , Mice , Mice, Inbred C3H , Mice, Inbred NOD , Virulence Factors/geneticsABSTRACT
We developed a transwell assay to quantify migration of the Lyme disease agent, Borrelia burgdorferi sensu stricto (s.s.), toward Ixodes scapularis salivary gland proteins. The assay was designed to assess B. burgdorferi s.s. migration upward against gravity through a transwell polycarbonate membrane overlaid with 6% gelatin. Borreliae that channeled into the upper transwell chamber in response to test proteins were enumerated by flow cytometry. The transwell assay measured chemoattractant activity for B. burgdorferi s.s. from salivary gland extract (SGE) harvested from nymphal ticks during bloodmeal engorgement on mice 42 h post-attachment and saliva collected from adult ticks. Additionally, SGE protein fractions separated by size exclusion chromatography demonstrated various levels of chemoattractant activity in the transwell assay. Sialostatin L, and Salp-like proteins 9 and 11 were identified by mass spectrometry in SGE fractions that exhibited elevated activity. Recombinant forms of these proteins were tested in the transwell assay and showed positive chemoattractant properties compared to controls and another tick protein, S15A. These results were reproducible providing evidence that the transwell assay is a useful method for continuing investigations to find tick saliva components instrumental in driving B. burgdorferi s.s. chemotaxis.
Subject(s)
Arthropod Proteins/chemistry , Bacteriological Techniques/methods , Borrelia burgdorferi/physiology , Chemotaxis , Ixodes/chemistry , Parasitology/methods , Animals , Borrelia burgdorferi/growth & development , Mice , Nymph/growth & development , Nymph/physiology , Saliva/chemistryABSTRACT
Borrelia burgdorferi (Bb), the etiological agent of Lyme disease, produces a series of simple glycolipids where diacylglycerol and cholesterol serve as the precursor. The cholesterol-based glycolipids, cholesteryl 6-O-acyl-ß-D-galactopyranoside (ACGal) and cholesteryl-ß-D-galactopyranoside (CGal) are immunogenic and proposed to contribute to the pathogenesis of Lyme disease. Detailed studies of CGal and ACGal in Bb have been hampered by a lack of knowledge of their underlying biosynthetic processes. The genome of Bb encodes four putative glycosyltransferases, and only one of these, BB0572, was predicted to be an inverting family 2 glycosyltransferase (GT2 enzyme) capable of using UDP-galactose as a substrate and forming a ß-glycosidic bond. Comparison of the 42 kDa BB0572 amino acid sequence from Bb with other Borrelia spp demonstrates that this protein is highly conserved. To establish BB0572 as the galactosyltransferase capable of cholesterol glycolipid formation in Bb, the protein was produced as a recombinant product in Escherichia coli and tested in a cell-free assay with 14C-cholesterol and UDP-galactose as the substrates. This experiment resulted in a radiolabeled lipid that migrated with the cholesterol glycolipid standard of CGal when evaluated by thin layer chromatography. Additionally, mutation in the predicted active site of BB0572 resulted in a recombinant protein that was unable to catalyze the formation of the cholesterol glycolipid. These data characterize BB0572 as a putative cholesterol galactosyltransferase. This provides the first step in understanding how Bb cholesterol glycolipids are formed and will allow investigations into their involvement in pathogen transmission and disease development.
Subject(s)
Borrelia burgdorferi/metabolism , Cholesterol/metabolism , Galactosyltransferases/metabolism , Glycolipids/metabolism , Lyme Disease/microbiology , Borrelia burgdorferi/physiologyABSTRACT
The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon-exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function.
Subject(s)
Bacterial Proteins/metabolism , Borrelia burgdorferi/physiology , Cell Wall/chemistry , Chemokines, CXC/metabolism , Lyme Disease/pathology , Peptidoglycan/metabolism , Bacterial Proteins/genetics , Cell Wall/microbiology , Chemokines, CXC/genetics , Humans , Lyme Disease/metabolism , Lyme Disease/microbiologyABSTRACT
BACKGROUND: Macrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied wildtype (WT) and MyD88-/- Bb-stimulated bone marrow-derived macrophages (BMDMs). RESULTS: MyD88-/- BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88-/- BMDMs identified a large cohort of differentially expressed MyD88-dependent genes associated with re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively. CONCLUSION: Our findings show that MyD88 signaling enhances, but is not required, for bacterial uptake or phagosomal maturation and provide mechanistic insights into how MyD88-mediated phagosomal signaling enhances Bb uptake and clearance.