Publications

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.

Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface-localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B. burgdorferi surface lipoproteins of the OspEF-related protein family-termed ElpB and ElpQ-that inhibit antibody-mediated complement activation. In this study, we investigate the molecular mechanism of ElpB and ElpQ complement inhibition using an array of biochemical and biophysical approaches.  We propose a model whereby ElpB and ElpQ exploit activation-induced conformational changes that are normally important for C1s-mediated C4 cleavage. Our study expands the known complement evasion mechanisms of microbial pathogens and reveals a novel molecular mechanism for selective C1s inhibition by Lyme disease spirochetes.

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 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.

Spirochetal pathogens encode an abundance of lipoproteins that can provide a critical interface with the host environment. Borrelia burgdorferi, the model species for spirochetal biology, must survive an enzootic life cycle defined by fluctuations between vector (tick) and vertebrate host. While B. burgdorferi expresses over 80 surface lipoproteins—many of which likely contribute to host survival—the B. burgdorferi lipoproteome is poorly characterized. Here, we generated a platform to rapidly identify targets of B. burgdorferi surface lipoproteins and identified two paralogs that confer resistance to antibody-initiated complement killing that may promote survival in immunocompetent hosts. This work expands our understanding of complement evasion mechanisms and points toward a discovery approach for identifying host–pathogen interactions central to spirochete pathogenesis.

Borrelia miyamotoi is a relapsing fever spirochete that relatively recently has been reported to infect humans. It causes an acute undifferentiated febrile illness that can include meningoencephalitis and relapsing fever. Like Borrelia burgdorferi, it is transmitted by Ixodes scapularis ticks in the northeastern United States and by Ixodes pacificus ticks in the western United States. Despite reports of clinical cases from North America, Europe, and Asia, the prevalence, geographic range, and pattern of expansion of human B. miyamotoi infection are uncertain. To better understand these characteristics of B. miyamotoi in relation to other tickborne infections, we carried out a cross-sectional seroprevalence study across New England that surveyed B. miyamotoi, B. burgdorferi, and Babesia microti infections. Analysis of 1153 serum samples collected at 11 catchment sites showed that the average seroprevalence for B. miyamotoi was 2.8% (range, 0.6%-5.2%), which was less than that of B. burgdorferi (11.0%; range, 6.8%-15.6%) and B. microti (10.0%; range, 6.5%-13.6%). Antibody screening within county residence in New England showed varying levels of seroprevalence for these pathogens but did not reveal a vectoral geographical pattern of distribution.

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.

Ten children and 20 adults were admitted for Lyme carditis between June and October. Ninety percent were male, and 87% had no prior cardiac history. Seventeen had outpatient evaluation before admission. Of these, a minority (41%) had Lyme disease suspected in the outpatient setting, and fewer (12%) were initiated on Lyme disease treatment. The most common alternate diagnoses were viral illness and erythema multiforme. More children than adults had disseminated erythema migrans and fever. First-degree heart block was more prevalent in children, and Mobitz type 2 heart block was more prevalent in adults. Ten patients presented with syncope. Proportionately more adults needed temporary pacing. Children had shorter antibiotic durations compared with adults. Of the 30 cases, 27 had improved heart block, while 3 adults required a pacemaker at discharge. Nine children and 14 adults were discharged with a PR 200-300 ms. There was a single death in this series.  Conclusions: Cases tended to be younger males. Most patients had some heart block on discharge. Of patients evaluated as outpatients, Lyme disease was suspected in 41%. Improved early recognition and treatment of Lyme disease may decrease Lyme carditis.

Inadequate sensitivity has been the primary limitation for implementing high-throughput sequencing for studies of tick-borne agents. Here we describe the development of TBDCapSeq, a sequencing assay that uses hybridization capture probes that cover the complete genomes of the eleven most common tick-borne agents found in the United States. The probes are used for solution-based capture and enrichment of pathogen nucleic acid followed by high-throughput sequencing. We evaluated the performance of TBDCapSeq to surveil samples that included human whole blood, mouse tissues, and field-collected ticks. For Borrelia burgdorferi and Babesia microti, the sensitivity of TBDCapSeq was comparable and occasionally exceeded the performance of agent-specific quantitative PCR and resulted in 25 to > 10,000-fold increase in pathogen reads when compared to standard unbiased sequencing. TBDCapSeq also enabled genome analyses directly within vertebrate and tick hosts. The implementation of TBDCapSeq could have major impact in studies of tick-borne pathogens by improving detection and facilitating genomic research that was previously unachievable with standard sequencing approaches.

Powassan virus (POWV) is a tick-transmitted pathogen that may cause severe encephalitis; experimentally, it can be transmitted within just 15 minutes following a tick bite. The deer tick virus subtype of POWV (DTV) is transmitted by the deer tick and is the likely cause of the increase in the number of POWV cases reported in the United States. However, DTV has only been definitively documented in 6 patients by molecular analysis of the virus.

We documented neuroinvasive POWV infection in 2 children from Connecticut. Based on the results of testing the tick removed from case 2, this patient was infected by DTV, representing the 7th reported case and the first documented case of DTV infection in a child. Of note, the duration of the tick bites in both cases was very short.  We provide the first clinical and epidemiologic evidence that POWV/DTV can be rapidly transmitted to a human host, that is, within hours of tick attachment, which is distinctive when compared to other deer tick-transmitted infections such as Lyme disease.

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.

Fifty years ago, the index case of human babesiosis due to Babesia microti was diagnosed in a summer resident of Nantucket Island. Human babesiosis, once called “Nantucket fever” due to its seeming restriction to Nantucket and the terminal moraine islands of southern New England, has emerged across the northeastern United States to commonly infect people wherever Lyme disease is endemic. We review the history of babesiosis on Nantucket, analyze its epidemiology and ecology there, provide summaries of the first case histories, and comment on its future public health burden.

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 immune deficient 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 immunodeficientC3H 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.

The white-footed mouse is considered to be the primary host for subadult deer ticks and the most important reservoir host for these and other disease agents. Local transmission is thought to be modulated by less reservoir competent hosts such as deer diverting ticks from feeding on mice. We measured the proportion of mouse fed or deer fed host-seeking nymphs from 4 sites during 2 transmission seasons by bloodmeal remnant analysis using a new retrotransposon-based qPCR assay. By directly measuring whether ticks had fed on either mice or deer using an new qPCR assay to detect remnants of host DNA leftover from the larval bloodmeal, we demonstrate that host utilization by ticks varies significantly over time and space and that mice often feed fewer ticks than expected. This has implications for our understanding of the ecology of these diseases and for the efficacy of control measures

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.

Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption.

Borrelia miyamotoi disease (BMD) is a newly recognized borreliosis that is cotransmitted by ticks wherever Lyme disease is zoonotic. To determine whether blood smears may detect B. miyamotoi in the blood of acute BMD patients, we made standard malariological thick smears from anticoagulated blood samples that were previously determined to contain this agent (by PCR) and analyzed them for morphological evidence of spirochetes. We found that in strong contrast to the diagnosis of typical relapsing fever, microscopy of blood smears is not sensitive enough for confirming a diagnosis of BMD but that SCID mouse inoculation could be a useful complement to PCR.

Subtropical lone star tick larvae typically emerge in late summer. We found clusters of host-seeking lone star tick larvae during early June 2018 in New York and Massachusetts, USA. Invasion and persistence of this tick in more northern locations may have been promoted by adaptation to an accelerated life cycle.

The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner.

Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts.  In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A.

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.

Metabolic biosignatures of patients with early Lyme disease can potentially provide diagnostic targets as well as highlight metabolic pathways that contribute to pathogenesis. Metabolic pathway analysis and chemical identification of metabolites with LC-tandem mass spectrometry (LC-MS/MS) demonstrated alterations of eicosanoid, bile acid, sphingolipid, glycerophospholipid, and acylcarnitine metabolic pathways during early Lyme disease. The findings demonstrated that infection of humans with B. burgdorferi alters defined metabolic pathways that are associated with inflammatory responses, liver function, lipid metabolism, and mitochondrial function.

Some patients have residual non-specific symptoms after therapy for Lyme disease, referred to as post-treatment Lyme disease symptoms or syndrome, depending on whether there is functional impairment. A standardized test battery was used to characterize a diverse group of Lyme disease patients with and without residual symptoms. There was a strong correlation between sleep disturbance and certain other symptoms such as fatigue, pain, anxiety, and cognitive complaints. Evaluation of how sleep disturbance, fatigue, pain and cognitive complains interrelate can potentially lead to new interventions that will improve the overall health of these patients.

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. 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.

We recently developed anti-OspA human immunoglobulin G1 monoclonal antibodies (HuMAbs) that are effective in preventing Borrelia transmission from ticks in a murine model. Here, we investigated a novel approach of DNA-mediated gene transfer of HuMAbs that provide protection against Lyme disease.

Geographical range expansions of blacklegged tick [Ixodes scapularis Say (Acari: Ixodidae)] populations over time in the United States have been attributed to a mosaic of factors including 20th century reforestation followed by suburbanization, burgeoning populations of the white-tailed deer [Odocoileus virginianus Zimmerman (Artiodactyla: Cervidae)], and, at the northern edge of I. scapularis’ range, climate change.

Introduction: Human babesiosis is reported throughout the world and is endemic in the northeastern and northern Midwestern United States and northeastern China. Transmission is primarily through hard bodied ticks. Most cases of severe disease occur in immunocompromised individuals and may result in prolonged relapsing disease or death.

Maine, USA, is a high Lyme disease incidence state, with rising incidence of Lyme disease and other tick-borne illnesses associated with increasing I. scapularis abundance and northward range expansion. Members of the public submitted ticks to a tick identification program (1990-2013). From these passive surveillance data, we characterized temporal trends in I. scapularis submission rate (an index of abundance), comparing Maine’s northern tier (seven counties) versus southern tier (nine counties). In the northern tier, the I. scapularis submission rate increased throughout the duration of the time series, suggesting I. scapularis was emergent but not established. By contrast, in the southern tier, submission rate increased initially but leveled off after 10-14 yr, suggesting I. scapularis was established by the mid-2000s.

The United Kingdom (UK) is considered to have a low incidence of LB, although this varies regionally. To determine whether an association exists between tick bite consultations and LB incidence in the UK, retrospective questionnaires were sent to general practitioners (GPs) in high (Wiltshire), medium (Cumbria) and low (Wales) incidence areas. During 2011, the greatest incidence of consultations for tick bites was reported by GPs in Cumbria (204 consultations per 100,000 inhabitants), followed by Wiltshire (160 per 100,000 population) and Wales (54 per 100,000 population). In Wiltshire and Cumbria, GPs predominantly provided advice on tick removal, whilst Welsh GPs mostly advised patients on tick bite prevention. Focusing on Cumbria during 2011-2013, 72.5% of GPs removed ticks from patients (incidence of 101 consultations per 100,000 population), and more GPs diagnosed LB based on clinical features than laboratory-confirmed diagnoses.

Incidence of human granulocytic anaplasmosis is rising in Maine, USA. This increase may be explained in part by adoption of tick panels as a frequent diagnostic test in persons with febrile illness and in part by range expansion of Ixodes scapularis ticks and zoonotic amplification of Anaplasma phagocytophilum.