Peromyscus leucopus , Mus musculus , and humans have distinct transcriptomic responses to larval Ixodes scapularis bites.

Ixodes scapularis ticks are an important vector for at least six tick-borne human pathogens, including the predominant North American Lyme disease spirochete Borrelia burgdorferi . The ability for these ticks to survive in nature is credited, in part, to their ability to feed on a variety of hosts without excessive activation of the proinflammatory branch of the vertebrate immune system. While the ability for nymphal ticks to feed on a variety of hosts has been well-documented, the host-parasite interactions between larval I. scapularis and different vertebrate hosts is relatively unexplored. Here we report on the changes in the vertebrate transcriptome present at the larval tick bite site using the natural I. scapularis host Peromyscus leucopus deermouse, a non-natural rodent host Mus musculus (BALB/c), and humans. We note substantially less evidence of activation of canonical proinflammatory pathways in P. leucopus compared to BALB/c mice and pronounced evidence of inflammation in humans. Pathway enrichment analyses revealed a particularly strong signature of interferon gamma, tumor necrosis factor, and interleukin 1 signaling at the BALB/c and human tick bite site. We also note that bite sites on BALB/c mice and humans, but not deermice, show activation of wound-healing pathways. These data provide molecular evidence of the coevolution between larval I. scapularis and P. leucopus as well as expand our overall understanding of I. scapularis feeding. Significance: Ixodes scapularis tick bites expose humans to numerous diseases in North America. While larval tick feeding enables pathogens to enter the tick population and eventually spread to humans, how larval ticks interact with mammals has been understudied compared to other tick stages. Here we examined the transcriptomic response of a natural I. scapularis rodent host ( Peromyscus leucopus ), a non-native I. scapularis rodent host ( Mus musculus ), and an incidental host (humans). We find that there are differences in how all three species respond to larval I. scapularis , with the natural host producing the smallest transcriptomic signature of a canonical proinflammatory immune response and the incidental human host producing the most robust signature of inflammation in response to the larval tick. These data expand our understanding of the pressures on ticks in the wild and inform our ability to model these interactions in laboratory settings.

Borrelia burgdorferi initiates early transcriptional re-programming in macrophages that supports long-term suppression of inflammation.

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, establishes a long-term infection and leads to disease manifestations that are the result of host immune responses to the pathogen. Inflammatory manifestations resolve spontaneously despite continued bacterial presence, suggesting inflammatory cells become less responsive over time. This is mimicked by in vitro repeated stimulations, resulting in tolerance, a phenotypic subset of innate immune memory. We performed comparative transcriptional analysis of macrophages in acute and memory states and identified sets of Tolerized, Hyper-Induced, Secondary-Induced and Hyper-Suppressed genes resulting from memory induction, revealing previously unexplored networks of genes affected by cellular re-programming. Tolerized gene families included inflammatory mediators and interferon related genes as would be predicted by the attenuation of inflammation over time. To better understand how cells mediate inflammatory hypo-responsiveness, we focused on genes that could mediate maintenance of suppression, such as Hyper-Induced genes which are up-regulated in memory states. These genes were notably enriched in stress pathways regulated by anti-inflammatory modulators. We examined one of the most highly expressed negative regulators of immune pathways during primary stimulation, Aconitate decarboxylase 1 (Acod1), and tested its effects during in vivo infection with Bb. As predicted by our in vitro model, we show its inflammation-suppressive downstream effects are sustained during in vivo long-term infection with Bb, with a specific role in Lyme carditis.

Single-cell RNA sequencing of murine ankle joints over time reveals distinct transcriptional changes following Borrelia burgdorferi infection.

Lyme disease is caused by the bacterial pathogen Borrelia burgdorferi, which can be readily modeled in laboratory mice. In order to understand the cellular and transcriptional changes that occur during B. burgdorferi infection, we conducted single-cell RNA sequencing (scRNA-seq) of ankle joints of infected C57BL/6 mice over time. We found that macrophages/monocytes, T cells, synoviocytes and fibroblasts all showed significant differences in gene expression of both inflammatory and non-inflammatory genes that peaked early and returned to baseline before the typical resolution of arthritis. Predictions of cellular interactions showed that macrophages appear to communicate extensively between different clusters of macrophages as well as with fibroblasts and synoviocytes. Our data give unique insights into the interactions between B. burgdorferi and the murine immune system over time and allow for a better understanding of mechanisms by which the dysregulation of the immune response may lead to prolonged symptoms in some patients.

Metabolic modeling predicts unique drug targets in Borrelia burgdorferi.

IMPORTANCE: Lyme disease is often treated using long courses of antibiotics, which can cause side effects for patients and risks the evolution of antimicrobial resistance. Narrow-spectrum antimicrobials would reduce these risks, but their development has been slow because the Lyme disease bacterium, Borrelia burgdorferi, is difficult to work with in the laboratory. To accelerate the drug discovery pipeline, we developed a computational model of B. burgdorferi's metabolism and used it to predict essential enzymatic reactions whose inhibition prevented growth in silico. These predictions were validated using small-molecule enzyme inhibitors, several of which were shown to have specific activity against B. burgdorferi. Although the specific compounds used are not suitable for clinical use, we aim to use them as lead compounds to develop optimized drugs targeting the pathways discovered here.

A putative xanthine dehydrogenase is critical for Borrelia burgdorferi survival in ticks and mice.

Borrelia burgdorferi is a pathogenic bacterium and the causative agent of Lyme disease. It is exposed to reactive oxygen species (ROS) in both the vertebrate and tick hosts. While some mechanisms by which B. burgdorferi ameliorates the effects of ROS exposure have been studied, there are likely other unknown mechanisms of ROS neutralization that contribute to virulence. Here, we follow up on a three gene cluster of unknown function, bb_0554, bb_0555, and bb_0556, that our prior unbiased transposon insertional sequencing studies implicated in both ROS survival and survival in Ixodes scapularis. We confirmed these findings through genetic knockout and provide evidence that these genes are co-transcribed as an operon to produce a xanthine dehydrogenase. In agreement with these results, we found that B. burgdorferi exposure to either uric acid (a product of xanthine dehydrogenase) or allopurinol (an inhibitor of xanthine dehydrogenase) could modulate sensitivity to ROS in a bb_0554-bb_0556 dependent manner. Together, this study identifies a previously uncharacterized three gene operon in B. burgdorferi as encoding a putative xanthine dehydrogenase critical for virulence. We propose renaming this locus xdhACB.

An AP-3-dependent pathway directs phagosome fusion with Rab8 and Rab11 vesicles involved in TLR2 signaling.

Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.

The PD-1/PD-L1 pathway is induced during Borrelia burgdorferi infection and inhibits T cell joint infiltration without compromising bacterial clearance.

The Lyme disease bacterial pathogen, Borrelia burgdorferi, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. T cells with limited effector functions and the inability to become activated by antigen, termed exhausted T cells, are present in many long-term infections. These exhausted T cells mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Exhausted T cells express a variety of immunoinhibitory molecules, including the molecule PD-1. Following B. burgdorferi infection, we found that PD-1 and its ligand PD-L1 are significantly upregulated on CD4+ T cells and antigen presenting cell subsets, respectively. Using mice deficient in PD-1, we found that the PD-1/PD-L1 pathway did not impact bacterial clearance but did impact T cell expansion and accumulation in the ankle joint and popliteal lymph nodes without affecting B cell populations or antibody production, suggesting that the PD-1/PD-L1 pathway may play a role in shaping the T cell populations present in affected tissues.

Antiphospholipid autoantibodies in Lyme disease arise after scavenging of host phospholipids by Borrelia burgdorferi.

A close association with its vertebrate and tick hosts allows Borrelia burgdorferi, the bacterium responsible for Lyme disease, to eliminate many metabolic pathways and instead scavenge key nutrients from the host. A lipid-defined culture medium was developed to demonstrate that exogenous lipids are an essential nutrient of B. burgdorferi, which can accumulate intact phospholipids from its environment to support growth. Antibody responses to host phospholipids were studied in mice and humans using an antiphospholipid ELISA. Several of these environmentally acquired phospholipids including phosphatidylserine and phosphatidic acid, as well as borrelial phosphatidylcholine, are the targets of antibodies that arose early in infection in the mouse model. Patients with acute infections demonstrated antibody responses to the same lipids. The elevation of antiphospholipid antibodies predicted early infection with better sensitivity than did the standardized 2-tier tests currently used in diagnosis. Sera obtained from patients with Lyme disease before and after antibiotic therapy showed declining antiphospholipid titers after treatment. Further study will be required to determine whether these antibodies have utility in early diagnosis of Lyme disease, tracking of the response to therapy, and diagnosis of reinfection, areas in which current standardized tests are inadequate.

A selective antibiotic for Lyme disease.

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

Blocking Borrelia burgdorferi transmission from infected ticks to nonhuman primates with a human monoclonal antibody.

Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.

Genetic Background Amplifies the Effect of Immunodeficiency in Antibiotic Efficacy Against Borrelia burgdorferi.

Unrecognized immunodeficiency has been proposed as a possible cause of failure of antibiotics to resolve symptoms of Lyme disease. Here, we examined the efficacy of doxycycline in different immunodeficient mice to identify defects that impair antibiotic treatment outcomes. We found that doxycycline had significantly lower efficacy in the absence of adaptive immunity, specifically B cells. This effect was most pronounced in immunodeficient C3H mice compared with C57BL/6 mice, suggesting a role for genetic background beyond immunodeficiency. Addition of a single dose of ceftriaxone to doxycycline treatment effectively cleared infection in C3H mice with severe combined immunodeficiency.

Interactions between Borrelia burgdorferi and its hosts across the enzootic cycle.

The bacterial pathogen Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted to humans through an Ixodes tick vector. B. burgdorferi is able to survive in both mammalian and tick hosts through careful modulation of its gene expression. This allows B. burgdorferi to adapt to the environmental and nutritional changes that occur when it is transmitted between the two hosts. Distinct interactions between the spirochete and its host occur at every step of the enzootic cycle and dictate the ability of the spirochete to survive until the next stage of the cycle. Studying the interface between B. burgdorferi, the Ixodes tick vector and the natural mammalian reservoirs has been made significantly more feasible through the complete genome sequences of the organisms and the advent of high throughput screening technologies. Ultimately, a thorough investigation of the interplay between the two domains (and two phyla within one domain) is necessary in order to completely understand how the pathogen is transmitted.

Lyme Disease Pathogenesis.

Lyme disease Borrelia are obligately parasitic, tick- transmitted, invasive, persistent bacterial pathogens that cause disease in humans and non-reservoir vertebrates primarily through the induction of inflammation. During transmission from the infected tick, the bacteria undergo significant changes in gene expression, resulting in adaptation to the mammalian environment. The organisms multiply and spread locally and induce inflammatory responses that, in humans, result in clinical signs and symptoms. Borrelia virulence involves a multiplicity of mechanisms for dissemination and colonization of multiple tissues and evasion of host immune responses. Most of the tissue damage, which is seen in non-reservoir hosts, appears to result from host inflammatory reactions, despite the low numbers of bacteria in affected sites. This host response to the Lyme disease Borrelia can cause neurologic, cardiovascular, arthritic, and dermatologic manifestations during the disseminated and persistent stages of infection. The mechanisms by which a paucity of organisms (in comparison to many other infectious diseases) can cause varied and in some cases profound inflammation and symptoms remains mysterious but are the subjects of diverse ongoing investigations. In this review, we provide an overview of virulence mechanisms and determinants for which roles have been demonstrated in vivo, primarily in mouse models of infection.

Innate Immune Memory to Repeated Borrelia burgdorferi Exposure Correlates with Murine In Vivo Inflammatory Phenotypes.

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the bite of an infected tick. Once inoculated into the host dermis, it disseminates to various organs including distant skin sites, the heart, the joint and the nervous system. Most humans will develop an early skin manifestation called erythema migrans at the tick bite site. This can be followed by symptoms such as carditis, neuritis, meningitis, or arthritis if not treated. A specific mouse strain, C3H/HeN develops arthritis with B. burgdorferi infection whereas another strain, C57BL/6, develops minimal to no arthritis. Neither strain of mice show any skin signs of rash or inflammation. Factors that determine the presence of skin inflammation and the joint arthritis susceptibility in the host are only partially characterized. We show in this study that murine fibroblast-like synoviocytes display trained immunity, a program in some cells that results in increased inflammatory responses if the cell has previously come in contact with a stimulus, and that trained immunity in fibroblast-like synoviocytes tested ex vivo correlates with Lyme arthritis susceptibility. Conversely, skin fibroblasts do not exhibit trained immunity, which correlates with the absence of skin symptoms in these mice. Moreover, we demonstrate that the trained phenotype in FLS is affected by the cell environment, which depends on the host genetic background. Future studies expanding this initial report of the role of trained immunity on symptoms of B. burgdorferi infection may provide insight into the pathogenesis of disease in murine models.

Hydrogen peroxide-producing pyruvate oxidase from Lactobacillus delbrueckii is catalytically activated by phosphotidylethanolamine.

BACKGROUND: Pyruvate oxidase (Pox) is an important enzyme in bacterial metabolism for increasing ATP production and providing a fitness advantage via hydrogen peroxide production. However, few Pox enzymes have been characterized from bacterial species. The tetrameric non-hydrogen-peroxide producing Pox from E. coli is activated by phospholipids, which is important for its function in vivo. RESULTS: We characterized the hydrogenperoxide-producing Pox from L. delbrueckii strain STYM1 and showed it is specifically activated by phosphotidylethanolamine (16:0-18:1), but not by phosphotidylcholine or phosphotidylglycerol. This activation is a mixture of K- and V-type activation as both km and enzyme turnover are altered. Furthermore, we demonstrated that the L. delbrueckii Pox forms pentamers and either decamers or dimers of pentamers in solution, which is different from other characterized Pox enzymes. Lastly, we generated a C-terminal truncation mutant that was only weakly activated by phosphotidylethanolamine, which suggests the C-terminus is important for lipid activation. CONCLUSIONS: To our knowledge this is the first known hydrogenperoxide-producing Pox enzyme that is activated by phospholipids. Our results suggest that there are substantial differences between Pox enzymes from different bacterial species, which could be important for their role in biological systems as well as in the development of Pox-based biosensors.

Design of a broadly reactive Lyme disease vaccine.

A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate self-assembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 months. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease.

Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase.

Despite a growing interest in using probiotic microorganisms to prevent disease, the mechanisms by which probiotics exert their action require further investigation. Porphyromonas gingivalis is an important pathogen implicated in the development of periodontitis. We isolated several strains of Lactobacillus delbrueckii from dairy products and examined their ability to inhibit P. gingivalis growth in vitro We observed strain-specific inhibition of P. gingivalis growth in vitro Whole-genome sequencing of inhibitory and noninhibitory strains of L. delbrueckii revealed significant genetic differences supporting the strain specificity of the interaction. Extracts of the L. delbrueckii STYM1 inhibitory strain contain inhibitory activity that is abolished by treatment with heat, proteinase K, catalase, and sodium sulfite. We purified the inhibitory protein(s) from L. delbrueckii STYM1 extracts using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration chromatography. Pyruvate oxidase was highly enriched in the purified samples. Lastly, we showed that purified, catalytically active, recombinant pyruvate oxidase is sufficient to inhibit P. gingivalis growth in vitro without the addition of cofactors. Further, using a saturated transposon library, we isolated transposon mutants of P. gingivalis in the feoB2 (PG_1294) gene that are resistant to killing by inhibitory L. delbrueckii, consistent with a mechanism of hydrogen peroxide production by pyruvate oxidase. Our results support the current understanding of the importance of strain selection, not simply species selection, in microbial interactions. Specific L. delbrueckii strains or their products may be effective in the treatment and prevention of P. gingivalis-associated periodontal disease.IMPORTANCEP. gingivalis is implicated in the onset and progression of periodontal disease and associated with some systemic diseases. Probiotic bacteria represent an attractive preventative therapy for periodontal disease. However, the efficacy of probiotic bacteria can be variable between studies. Our data support the known importance of selecting particular strains of bacteria for probiotic use, not simply a single species. Specifically, in the context of probiotic intervention of periodontitis, our data suggest that high-level expression of pyruvate oxidase with hydrogen peroxide production in L. delbrueckii could be an important characteristic for the design of a probiotic supplement or a microbial therapeutic.

Genome-wide screen identifies novel genes required for Borrelia burgdorferi survival in its Ixodes tick vector.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction.

Identifying Vancomycin as an Effective Antibiotic for Killing Borrelia burgdorferi.

Borrelia burgdorferi is the causative agent of Lyme borreliosis. Antibiotic therapy of early acute infection is effective for most patients, but 10 to 20% go on to develop posttreatment Lyme disease syndrome (PTLDS). The nature of PTLDS remains unknown, but currently approved antibiotics for the treatment of Lyme disease do not appear to impact these symptoms after they have developed. We reason that minimizing the time the pathogen interacts with the host will diminish the probability of developing PTLDS, irrespective of its nature. This calls for an efficient eradication of the pathogen during acute infection. In search of a superior killing antibiotic, we examined approved antibiotics for their ability to kill B. burgdorferi Vancomycin proved more effective in killing the pathogen in vitro than ceftriaxone, the standard of care for disseminated B. burgdorferi infection. Both compounds were also the most effective in killing stationary-phase cells. This is surprising, given that inhibitors of cell wall biosynthesis are known to only kill growing bacteria. We found that peptidoglycan synthesis continues in stationary-phase cells of B. burgdorferi, explaining this paradox. A combination of vancomycin and gemifloxacin sterilized a stationary-phase culture of B. burgdorferi Examination of the action of antibiotics in severe combined immunodeficient (SCID) mice showed that doxycycline, a standard of care for uncomplicated acute infection, did not clear the pathogen. In contrast, both ceftriaxone and vancomycin cleared the infection. A trial examining the early use of more potent antibiotics on the development of PTLDS may be warranted.