Xin Li

Transition metal ions play important structural, regulatory, and catalytic roles in all biological systems by serving as cofactors for proteins. Due to their relatively low levels in the cell compared to abundant metal ions such as potassium and magnesium, transition metals are often considered micronutrients and referred to as trace elements. Manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are the most prevalent transition metals in the Lyme disease spirochete Borrelia burgdorferi. Here, we describe a method for the accurate measurement of these trace elements in B. burgdorferi utilizing inductively coupled plasma-sector field mass spectrometry (ICP-SFMS).

The ferric uptake regulator (Fur) family of DNA-binding proteins represses and/or activates gene transcription via divalent metal ion-dependent signal sensing. The Borrelia burgdorferi Fur homologue, also known as Borrelia oxidative stress regulator (BosR), promotes spirochetal adaptation to the mammalian host by directly repressing the lipoproteins required for tick colonization and indirectly activating those required for establishing infection in the mammal. Here, we examined whether the DNA-binding activity of BosR was regulated by any of the four most prevalent transition metal ions in B. burgdorferi , Mn, Fe, Cu, and Zn. Our data indicated that in addition to a structural site occupied by Zn(II), BosR had two regulatory sites that could be occupied by Zn(II), Fe(II), or Cu(II) but not by Mn(II). While Fe(II) had no effect, Cu(II) and Zn(II) had a dose-dependent inhibitory effect on the BosR DNA-binding activity. Competition experiments indicated that Cu(II) had a higher affinity for BosR than Zn(II) or Fe(II). A BosR deficiency in B. burgdorferi resulted in a significant increase in the Cu level but no significant change in the levels of Mn, Fe, or Zn. These data suggest that Cu regulates BosR activity, and BosR in turn regulates Cu homeostasis in B. burgdorferi . While this regulatory paradigm is characteristic of the Fur family, BosR is the first one shown to be responsive to Cu(II), which may be an adaptation to the potentially high level of Cu present in the Lyme disease spirochete. IMPORTANCE Transition metal ions serve an essential role in the metabolism of all living organisms. Members of the ferric uptake regulator (Fur) family play critical roles in regulating the cellular homeostasis of transition metals in diverse bacteria, and their DNA-binding activity is often regulated by coordination of the cognate divalent metal ions. To date, regulators with metal ion specificity to Fe(II), Mn(II), Zn(II), and Ni(II) have all been described. In this study, we demonstrate that BosR, the sole Fur homologue in Borrelia burgdorferi , is responsive to Cu(II) and regulates Cu homeostasis in this bacterium, which may be an adaption to potentially Cu-rich milieu in the Lyme disease spirochete. This study has expanded the repertoire of the Fur family's metal ion specificity.

This corrects the article DOI: 10.1038/nrdp.2016.90.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro. The screens identified 66 genes whose disruption resulted in increased susceptibility to at least one of the stressors. These genes include previously characterized mediators of ROS and RNS resistance (including components of the nucleotide excision repair pathway and a subunit of a riboflavin transporter), as well as novel putative resistance candidates. DNA repair mutants were among the most sensitive to RNS in the Tn-seq screen, and survival assays with individual Tn mutants confirmed that the putative ribonuclease BB0839 is involved in resistance to nitric oxide. In contrast, mutants lacking predicted inner membrane proteins or transporters were among the most sensitive to ROS, and the contribution of three such membrane proteins (BB0017, BB0164, and BB0202) to ROS sensitivity was confirmed using individual Tn mutants and complemented strains. Further analysis showed that levels of intracellular manganese are significantly reduced in the Tn::bb0164 mutant, identifying a novel role for BB0164 in B. burgdorferi manganese homeostasis. Infection of C57BL/6 and gp91^phox-/- mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the in vitro screens are required for infectivity in mice. Collectively, our data provide insight into how B. burgdorferi responds to ROS and RNS and suggests that this response is relevant to the in vivo success of the organism. Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne disease in the United States. B. burgdorferi is maintained in the environment in a complex cycle in which it is transmitted between a tick vector and small vertebrate reservoir hosts. Because of this, B. burgdorferi must be able to adapt to changing environmental conditions. Historically, this organism has been difficult to manipulate genetically in the lab. We have taken advantage of recent technological advances to conduct a global search for genes that enable the spirochete to defend itself against reactive oxygen and nitrogen species produced by the host immune system. Using transposon insertion sequencing (Tn-seq), we exposed a pool of B. burgdorferi mutants to reactive oxygen or nitrogen species and used high-throughput sequencing to quantify the frequency of each individual mutant before and after the stress exposure. We found that genes encoding DNA repair enzymes and predicted inner membrane proteins are important for resistance to RNS and ROS, respectively. Understanding how B. burgdorferi responds to the stresses and changing environmental conditions it experiences as it transitions between its tick vector and vertebrate hosts potentially will identify new targets for interrupting its life cycle.

Lyme borreliosis is a tick-borne disease that predominantly occurs in temperate regions of the northern hemisphere and is primarily caused by the bacterium Borrelia burgdorferi in North America and Borrelia afzelii or Borrelia garinii in Europe and Asia. Infection usually begins with an expanding skin lesion, known as erythema migrans (referred to as stage 1), which, if untreated, can be followed by early disseminated infection, particularly neurological abnormalities (stage 2), and by late infection, especially arthritis in North America or acrodermatitis chronica atrophicans in Europe (stage 3). However, the disease can present with any of these manifestations. During infection, the bacteria migrate through the host tissues, adhere to certain cells and can evade immune clearance. Yet, these organisms are eventually killed by both innate and adaptive immune responses and most inflammatory manifestations of the infection resolve. Except for patients with erythema migrans, Lyme borreliosis is diagnosed based on a characteristic clinical constellation of signs and symptoms with serological confirmation of infection. All manifestations of the infection can usually be treated with appropriate antibiotic regimens, but the disease can be followed by post-infectious sequelae in some patients. Prevention of Lyme borreliosis primarily involves the avoidance of tick bites by personal protective measures.

The Lyme disease spirochete, Borrelia burgdorferi, must abundantly produce outer surface lipoprotein A (OspA) in the tick vector but downregulate OspA in mammals in order to evade the immune system and maintain its natural enzootic cycle. Here, we show that BosR binds two regulatory elements of the ospAB operon and that increasing BosR expression leads to downregulation of OspA. Both regulatory sequences, cisI and cisII, showed strong BosR-binding and cisII bound much tighter than cisI. A promoterless bosR gene fused with an inducible promoter was introduced into an rpoS mutant and a wild-type strain to assess RpoS-independent and -dependent downregulation of OspA by BosR. With the induction of BosR expression, OspA expression was reduced more significantly in the RpoS-deficient than wild-type background, but not completely repressed. In the presence of constitutive expression of OspC, DbpA and DbpB, increasing BosR production resulted in complete repression of OspA in the RpoS mutant. Taken together, the study clearly demonstrated BosR serves as a repressor that binds both regulatory elements of the ospAB operon and shuts off expression.

Lyme disease, the most common vector-borne disease in the United States, is caused by a tick-borne infection with Borrelia burgdorferi. Currently, Ohio is considered by the Centers for Disease Control and Prevention (CDC) to be non-endemic for Lyme disease. The low incidence of Lyme disease in this state was largely attributed to the absence of the transmitting vector, Ixodes scapularis, commonly known as the blacklegged tick. However, a tick surveillance program established by Ohio Department of Health indicated that the number of I. scapularis ticks in Ohio had increased sharply in recent years, from 0 - 5 ticks per year during 1983 - 2008 to 15 in 2009, 40 in 2010, and 184 in 2011. During the fall deer hunting season, examination of deer heads submitted to Ohio Department of Agriculture found 29 I. scapularis from 7 counties in 2010 and 1,830 from 25 counties in 2011. As of 2012, the tick had been found in 57 of the 88 counties of Ohio. In addition, all three active stages (larva, nymph, and adult) of I. scapularis were found in Tiverton Township of Coshocton County, demonstrating the presence of established tick populations at this central Ohio location. Of 530 nymphal or adult I. scapularis analyzed by quantitative polymerase chain reaction (qPCR), 32 (6.1%) tested positive for the B. burgdorferi flaB gene, ranging from 36 to 390,000 copies per tick. Antibodies to B. burgdorferi antigens were detected in 2 of 10 (20%) field-captured Peromyscus leucopus from Tiverton Township, and in 41 of 355 (11.5%) dogs residing in Ohio. Collectively, these data suggest that the enzootic life cycle of B. burgdorferi has become established in Ohio, which poses risk of Lyme disease to people and animals in the area.

Differential gene expression is a key strategy adopted by the Lyme disease spirochaete, Borrelia burgdorferi, for adaptation and survival in the mammalian host and the tick vector. Many B. burgdorferi surface lipoproteins fall into two distinct groups according to their expression patterns: one group primarily expressed in the tick and the other group primarily expressed in the mammal. Here, we show that the Fur homologue in this bacterium, also known as Borrelia oxidative stress regulator (BosR), is required for repression of outer surface protein A (OspA) and OspD in the mammal. Furthermore, BosR binds directly to sequences upstream of the ospAB operon and the ospD gene through recognition of palindromic motifs similar to those recognized by other Fur homologues but with a 1 bp variation in the spacer length. Putative BosR binding sites have been identified upstream of 156 B. burgdorferi genes. Some of these genes share the same expression pattern as ospA and ospD. Most notably, 12 (67%) of the 18 genes previously identified in a genome‐wide microarray study to be most significantly repressed in the mammal are among the putative BosR regulon. These data indicate that BosR may directly repress transcription of many genes that are downregulated in the mammal.

Borrelia burgdorferi (Bb) sensu lato, the etiologic agent of Lyme borreliosis, adapts to distinct environments in the mammalian host and the tick vector by differential gene expression. As a result, infected mice are not exposed to and rarely make antibodies to the set of antigens that are preferentially expressed in the tick, including outer surface protein A (OspA), Borrelia iron and copper-binding protein A (BicA), and OspD. Surprisingly, however, antibodies to OspA and BicA have been noted in American patients with Lyme arthritis. Here, we examined serum samples from 210 American patients and 66 European patients with a range of early or late manifestations of Lyme borreliosis and found that only American patients with Lyme arthritis commonly had antibody responses to OspA, BicA, and OspD. This suggests that infection with American but not European Borrelia strains often leads to concerted upregulation or derepression of tick-specific spirochetal antigens in these patients.

TRIF is an adaptor molecule important in transducing signals from intracellularly signaling Toll-like receptor 3 (TLR3) and TLR4. Recently, TLR2 was found to signal from intracellular compartments. Using a synthetic ligand for TLR2/1 heterodimers, as well asBorrelia burgdorferi, which is a strong activator of TLR2/1, we found that TLR2 signaling can utilize TRIF. Unlike TRIF signaling by other TLRs, TLR2-mediated TRIF signaling is dependent on the presence of another adaptor molecule, MyD88. However, unlike MyD88 deficiency, TRIF deficiency does not result in diminished control of infection withB. burgdorferiin a murine model of disease. This appears to be due to the effects of MyD88 on phagocytosis via scavenger receptors, such as MARCO, which are not affected by the loss of TRIF. In mice, TRIF deficiency did have an effect on the production of inflammatory cytokines, suggesting that regulation of inflammatory cytokines and control of bacterial growth may be uncoupled, in part through transduction of TLR2 signaling through TRIF.