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.

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.

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.

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.

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.

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.

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.