Bacterial reprogramming of tick metabolism impacts vector fitness and susceptibility to infection.

Arthropod-borne pathogens are responsible for hundreds of millions of infections in humans each year. The blacklegged tick, Ixodes scapularis, is the predominant arthropod vector in the United States and is responsible for transmitting several human pathogens, including the Lyme disease spirochete Borrelia burgdorferi and the obligate intracellular rickettsial bacterium Anaplasma phagocytophilum, which causes human granulocytic anaplasmosis. However, tick metabolic response to microbes and whether metabolite allocation occurs upon infection remain unknown. Here we investigated metabolic reprogramming in the tick ectoparasite I. scapularis and determined that the rickettsial bacterium A. phagocytophilum and the spirochete B. burgdorferi induced glycolysis in tick cells. Surprisingly, the endosymbiont Rickettsia buchneri had a minimal effect on bioenergetics. An unbiased metabolomics approach following A. phagocytophilum infection of tick cells showed alterations in carbohydrate, lipid, nucleotide and protein metabolism, including elevated levels of the pleiotropic metabolite ß-aminoisobutyric acid. We manipulated the expression of genes associated with ß-aminoisobutyric acid metabolism in I. scapularis, resulting in feeding impairment, diminished survival and reduced bacterial acquisition post haematophagy. Collectively, we discovered that metabolic reprogramming affects interspecies relationships and fitness in the clinically relevant tick I. scapularis.

An atlas of human vector-borne microbe interactions reveals pathogenicity mechanisms.

Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.

Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness.

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.

The Development of a Rabies Virus-Vectored Vaccine against Borrelia burgdorferi, Targeting BBI39.

Lyme disease (LD) is the most common tick-borne illness in the United States (U.S.), Europe, and Asia. Borrelia burgdorferi, a spirochete bacterium transmitted by the tick vector Ixodes scapularis, causes LD in the U.S. If untreated, Lyme arthritis, heart block, and meningitis can occur. Given the absence of a human Lyme disease vaccine, we developed a vaccine using the rabies virus (RABV) vaccine vector BNSP333 and an outer surface borrelial protein, BBI39. BBI39 was previously utilized as a recombinant protein vaccine and was protective in challenge experiments; therefore, we decided to utilize this protective antigen in a rabies virus-vectored vaccine against Borrelia burgdorferi. To incorporate BBI39 into the RABV virion, we generated a chimeric BBI39 antigen, BBI39RVG, by fusing BBI39 with the final amino acids of the RABV glycoprotein by molecular cloning and viral recovery with reverse transcription genetics. Here, we have demonstrated that the BBI39RVG antigen was incorporated into the RABV virion via immunofluorescence and Western blot analysis. Mice vaccinated with our BPL inactivated RABV-BBI39RVG (BNSP333-BBI39RVG) vaccine induced high amounts of BBI39-specific antibodies, which were maintained long-term, up to eight months post-vaccination. The BBI39 antibodies neutralized Borrelia in vaccinated mice when challenged with Borrelia burgdorferi by either syringe injection or infected ticks and they reduced the Lyme disease pathology of arthritis in infected mouse joints. Overall, the RABV-based LD vaccine induced more and longer-term antibodies compared to the recombinant protein vaccine. This resulted in lower borrelial RNA in RABV-based vaccinated mice compared to recombinant protein vaccinated mice. The results of this study indicate the successful use of BBI39 as a vaccine antigen and RABV as a vaccine vector for LD.

A systemic approach to identify non-abundant immunogenic proteins in Lyme disease pathogens.

Borrelia burgdorferi, the pathogen of Lyme disease, differentially produces many outer surface proteins (Osp), some of which represent the most abundant membrane proteins, such as OspA, OspB, and OspC. In cultured bacteria, these proteins can account for a substantial fraction of the total cellular or membrane proteins, posing challenges to the identification and analysis of non-abundant proteins, which could serve as novel pathogen detection markers or as vaccine candidates. Herein, we introduced serial mutations to remove these abundant Osps and generated a B. burgdorferi mutant deficient in OspA, OspB, and OspC in an infectious 297-isolate background, designated as OspABC- mutant. Compared to parental isolate, the mutant did not reflect growth defects in the cultured medium but showed differential mRNA expression of representative tested genes, in addition to gross changes in cellular and membrane protein profiles. The analysis of differentially detectable protein contents of the OspABC- mutant, as compared to the wild type, by two-dimensional gel electrophoresis followed by liquid chromatography-mass spectrometry, identified several spirochete proteins that are dominated by proteins of unknown functions, as well as membrane transporters, chaperons, and metabolic enzymes. We produced recombinant forms of two of these represented proteins, BBA34 and BB0238, and showed that these proteins are detectable during spirochete infection in the tick-borne murine model of Lyme borreliosis and thus serve as potential antigenic markers of the infection.IMPORTANCEThe present manuscript employed a systemic approach to identify non-abundant proteins in cultured Borrelia burgdorferi that are otherwise masked or hidden due to the overwhelming presence of abundant Osps like OspA, OspB, and OspC. As these Osps are either absent or transiently expressed in mammals, we performed a proof-of-concept study in which their removal allowed the analysis of otherwise less abundant antigens in OspABC-deficient mutants and identified several immunogenic proteins, including BBA34 and BB0238. These antigens could serve as novel vaccine candidates and/or genetic markers of Lyme borreliosis, promoting new research in the clinical diagnosis and prevention of Lyme disease.

Characterization of spatial lipidomic signatures in tick-bitten guinea pig skin as a model for host-vector-pathogen interaction profiling.

IMPORTANCE: Here, we demonstrate the adaptability of spatial "omics" methods to identify interphylum processes regulated at the vector-host interface of ticks during a mammalian blood meal. This approach enables a better understanding of complex bipartite or tripartite molecular interactions between hosts, arthropod vectors and transmitted pathogens, and contributes toward the development of spatially aware therapeutic target discovery and description.

A unique borrelial protein facilitates microbial immune evasion.

IMPORTANCE: Lyme disease is a major tick-borne infection caused by a bacterial pathogen called Borrelia burgdorferi, which is transmitted by ticks and affects hundreds of thousands of people every year. These bacterial pathogens are distinct from other genera of microbes because of their distinct features and ability to transmit a multi-system infection to a range of vertebrates, including humans. Progress in understanding the infection biology of Lyme disease, and thus advancements towards its prevention, are hindered by an incomplete understanding of the microbiology of B. burgdorferi, partly due to the occurrence of many unique borrelial proteins that are structurally unrelated to proteins of known functions yet are indispensable for pathogen survival. We herein report the use of diverse technologies to examine the structure and function of a unique B. burgdorferi protein, annotated as BB0238-an essential virulence determinant. We show that the protein is structurally organized into two distinct domains, is involved in multiplex protein-protein interactions, and facilitates tick-to-mouse pathogen transmission by aiding microbial evasion of early host cellular immunity. We believe that our findings will further enrich our understanding of the microbiology of B. burgdorferi, potentially impacting the future development of novel prevention strategies against a widespread tick-transmitted infection.

A ticking time bomb hidden in plain sight.

The deer tick transmits nearly half of the known tick-borne pathogens in the United States, and its expanding geographic range increases the risk of human infection. To decrease the abundance of and infection risk from deer ticks, approaches that include vaccines for human use and for animal hosts are desired.

Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness.

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 describe immune cells or hemocytes from the clinically relevant tick Ixodes scapularis using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence in situ hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two I. scapularis hemocyte markers: hemocytin and astakine. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in I. scapularis. Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.

Croquemort elicits activation of the immune deficiency pathway in ticks.

The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.

A high-quality Ixodes scapularis genome advances tick science.

Ixodes spp. and related ticks transmit prevalent infections, although knowledge of their biology and development of anti-tick measures have been hindered by the lack of a high-quality genome. In the present study, we present the assembly of a 2.23-Gb Ixodes scapularis genome by sequencing two haplotypes within one individual, complemented by chromosome-level scaffolding and full-length RNA isoform sequencing, yielding a fully reannotated genome featuring thousands of new protein-coding genes and various RNA species. Analyses of the repetitive DNA identified transposable elements, whereas the examination of tick-associated bacterial sequences yielded an improved Rickettsia buchneri genome. We demonstrate how the Ixodes genome advances tick science by contributing to new annotations, gene models and epigenetic functions, expansion of gene families, development of in-depth proteome catalogs and deciphering of genetic variations in wild ticks. Overall, we report critical genetic resources and biological insights impacting our understanding of tick biology and future interventions against tick-transmitted infections.

Dome1-JAK-STAT signaling between parasite and host integrates vector immunity and development.

Ancestral signaling pathways serve critical roles in metazoan development, physiology, and immunity. We report an evolutionary interspecies communication pathway involving a central Ixodes scapularis tick receptor termed Dome1, which acquired a mammalian cytokine receptor motif exhibiting high affinity for interferon-gamma (IFN-γ). Host-derived IFN-γ facilitates Dome1-mediated activation of the Ixodes JAK-STAT pathway. This accelerates tick blood meal acquisition and development while upregulating antimicrobial components. The Dome1-JAK-STAT pathway, which exists in most Ixodid tick genomes, regulates the regeneration and proliferation of gut cells-including stem cells-and dictates metamorphosis through the Hedgehog and Notch-Delta networks, ultimately affecting Ixodes vectorial competence. We highlight the evolutionary dependence of I. scapularis on mammalian hosts through cross-species signaling mechanisms that dually influence arthropod immunity and development.

Function and evolution of the aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis.

Ticks are important vectors of pathogenic viruses, bacteria, and protozoans to humans, wildlife, and domestic animals. Due to their life cycles, ticks face significant challenges related to water homeostasis. When blood-feeding, they must excrete water and ions, but when off-host (for stretches lasting several months), they must conserve water to avoid desiccation. Aquaporins (AQPs), a family of membrane-bound water channels, are key players in osmoregulation in many animals but remain poorly characterized in ticks. Here, we bioinformatically identified AQP-like genes from the deer tick Ixodes scapularis and used phylogenetic approaches to map the evolution of the aquaporin gene family in arthropods. Most arachnid AQP-like sequences (including those of I. scapularis) formed a monophyletic group clustered within aquaglycerolporins (GLPs) from bacteria to vertebrates. This gene family is absent from insects, revealing divergent evolutionary paths for AQPs in different hematophagous arthropods. Next, we sequenced the full-length cDNA of I. scapularis aquaporin 1 (IsAQP1) and expressed it heterologously in Xenopus oocytes to functionally characterize its permeability to water and solutes. Additionally, we examined IsAQP1 expression across different life stages and adult female organs. We found IsAQP1 is an efficient water channel with high expression in salivary glands prior to feeding, suggesting it plays a role in osmoregulation before or during blood feeding. Its functional properties are unique: unlike most GLPs, IsAQP1 has low glycerol permeability, and unlike most AQPs, it is insensitive to mercury. Together, our results suggest IsAQP1 plays an important role in tick water balance physiology and that it may hold promise as a target of novel vector control efforts.

Immune evasion strategies of major tick-transmitted bacterial pathogens.

Tick-transmitted bacterial pathogens thrive in enzootic infection cycles, colonizing disparate vertebrate and arthropod tissues, often establishing persistent infections. Therefore, the evolution of robust immune evasion strategies is central to their successful persistence or transmission between hosts. To survive in nature, these pathogens must counteract a broad range of microbicidal host responses that can be localized, tissue-specific, or systemic, including a mix of these responses at the host-vector interface. Herein, we review microbial immune evasion strategies focusing on Lyme disease spirochetes and rickettsial or tularemia agents as models for extracellular and intracellular tick-borne pathogens, respectively. A better understanding of these adaptive strategies could enrich our knowledge of the infection biology of relevant tick-borne diseases, contributing to the development of future preventions.

Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries.

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient, most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in histidine-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this HIS-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

Application of biophysical methods for improved protein production and characterization: A case study on an high-temperature requirement A-family bacterial protease.

The high-temperature requirement A (HtrA) serine protease family presents an attractive target class for antibacterial therapeutics development. These proteins possess dual protease and chaperone functions and contain numerous binding sites and regulatory loops, displaying diverse oligomerization patterns dependent on substrate type and occupancy. HtrA proteins that are natively purified coelute with contaminating peptides and activating species, shifting oligomerization and protein structure to differently activated populations. Here, a redesigned HtrA production results in cleaner preparations with high yields by overexpressing and purifying target protein from inclusion bodies under denaturing conditions, followed by a high-throughput screen for optimal refolding buffer composition using function-agnostic biophysical techniques that do not rely on target-specific measurements. We use Borrelia burgdorferi HtrA to demonstrate the effectiveness of our function-agnostic approach, while characterization with both new and established biophysical methods shows the retention of proteolytic and chaperone activity of the refolded protein. This systematic workflow and toolset will translate to the production of HtrA-family proteins in higher quantities of pure and monodisperse composition than the current literature standard, with applicability to a broad array of protein purification strategies.

Visualization of the Dynamics of Invasion and Intravasation of the Bacterium That Causes Lyme Disease in a Tissue Engineered Dermal Microvessel Model.

Lyme disease is a tick-borne disease prevalent in North America, Europe, and Asia. Despite the accumulated knowledge from epidemiological, in vitro, and in animal studies, the understanding of dissemination of vector-borne pathogens, such as Borrelia burgdorferi (Bb), remains incomplete with several important knowledge gaps, especially related to invasion and intravasation into circulation. To elucidate the mechanistic details of these processes a tissue-engineered human dermal microvessel model is developed. Fluorescently labeled Bb are injected into the extracellular matrix (ECM) to mimic tick inoculation. High resolution, confocal imaging is performed to visualize the sub-acute phase of infection. From analysis of migration paths no evidence to support adhesin-mediated interactions between Bb and ECM components is found, suggesting that collagen fibers serve as inert obstacles to migration. Intravasation occurs at cell-cell junctions and is relatively fast, consistent with Bb swimming in ECM. In addition, it is found that Bb alone can induce endothelium activation, resulting in increased immune cell adhesion but no changes in global or local permeability. Together these results provide new insight into the minimum requirements for Bb dissemination and highlight how tissue-engineered models are complementary to animal models in visualizing dynamic processes associated with vector-borne pathogens.

Controlled Proteolysis of an Essential Virulence Determinant Dictates Infectivity of Lyme Disease Pathogens.

The Borrelia burgdorferi BB0323 protein undergoes a complex yet poorly defined proteolytic maturation event that generates N-terminal and C-terminal proteins with essential functions in cell growth and infection. Here, we report that a borrelial protease, B. burgdorferi high temperature requirement A protease (BbHtrA), cleaves BB0323 between asparagine (N) and leucine (L) at positions 236 and 237, while the replacement of these residues with alanine in the mutant protein prevents its cleavage, despite preserving its normal secondary structure. The N-terminal BB0323 protein binds BbHtrA, but its cleavage site mutant displays deficiency in such interaction. An isogenic borrelial mutant with NL-to-AA substitution in BB0323 (referred to as Bbbb0323NL) maintains normal growth yet is impaired for infection of mice or transmission from infected ticks. Notably, the BB0323 protein is still processed in Bbbb0323NL, albeit with lower levels of mature N-terminal BB0323 protein and multiple aberrantly processed polypeptides, which could result from nonspecific cleavages at other asparagine and leucine residues in the protein. The lack of infectivity of Bbbb0323NL is likely due to the impaired abundance or stoichiometry of a protein complex involving BB0238, another spirochete protein. Together, these studies highlight that a precise proteolytic event and a particular protein-protein interaction, involving multiple borrelial virulence determinants, are mutually inclusive and interconnected, playing essential roles in the infectivity of Lyme disease pathogens.

Vaccines Against Vector-Borne Diseases.

Arthropod vectors account for a number of animal and human diseases, posing substantial threats to health and safety on a global scale. Ticks are considered as one of the most prominent vectors, as they can parasitize almost any vertebrate class and transmit a multitude of infectious diseases, particularly ones that affect humans and domestic animals. While various tick species elicit different tick-borne infections in specific geographic regions, single species can have widespread effects, such as blacklegged ticks, which are widely distributed across the eastern United States and can transmit a variety of infections, including Lyme borreliosis, anaplasmosis, relapsing fever disease, ehrlichiosis, babesiosis, and Powassan virus disease. Despite increasing awareness about ticks as serious disease vectors, effective vaccines against most tick-borne infections are not available. Previously, the successful development of an anti-tick vaccine for use in veterinary animals was based on an 86-kDa midgut antigen from Rhipicephalus (formerly Boophilus) microplus ticks. Herein we describe the fundamentals of vaccine development using protein antigens as model vaccinogen candidates, beginning with the cloning, expression, and purification of recombinant proteins, host immunization, and the assessment of protective efficacy in laboratory settings using a tick-borne murine model of Lyme borreliosis.