Antibodies against egf-like domains in ixodes scapularis bm86 orthologs impact tick feeding and survival of borrelia burgdorferi

ABSTRACT _Ixodes scapularis_ ticks transmit multiple pathogens, including _Borrelia burgdorferi_ sensu stricto, and encode many proteins harboring epidermal growth factor (EGF)-like domains.

We show that _I. scapularis_ produces multiple orthologs for Bm86, a widely studied tick gut protein considered as a target of an anti-tick vaccine, herein termed as Is86. We show that Is86

antigens feature at least three identifiable regions harboring EGF-like domains (termed as EGF-1, EGF-2, and EGF-3) and are differentially upregulated during _B. burgdorferi_ infection.

Although the RNA interference-mediated knockdown of _Is86_ genes did not show any influences on tick engorgement or _B. burgdorferi_ sensu stricto persistence, the immunization of murine

hosts with specific recombinant EGF antigens marginally reduced spirochete loads in the skin, in addition to affecting tick blood meal engorgement and molting. However, given the borderline

impact of EGF immunization on tick engorgement and pathogen survival in the vector, it is unlikely that these antigens, at least in their current forms, could be developed as potential

vaccines. Further investigations of the biological significance of Is86 (and other tick antigens) would enrich our knowledge of the intricate biology of ticks, including their interactions

with resident pathogens, and contribute to the development of anti-tick measures to combat tick-borne illnesses. SIMILAR CONTENT BEING VIEWED BY OTHERS INTERACTIONS BETWEEN _BORRELIA

BURGDORFERI_ AND TICKS Article 10 July 2020 _HAEMAPHYSALIS LONGICORNIS_ SUBOLESIN CONTROLS THE INFECTION AND TRANSMISSION OF SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME VIRUS Article Open

access 24 January 2025 IMMUNIZATION AGAINST ARTHROPOD PROTEIN IMPAIRS TRANSMISSION OF RICKETTSIAL PATHOGEN FROM TICKS TO THE VERTEBRATE HOST Article Open access 30 May 2023 INTRODUCTION Lyme

disease, a prevalent arthropod-borne disease in North America and Europe, is caused by a bacterial pathogen, _Borrelia burgdorferi_, and is transmitted by infected _Ixodes scapularis_ and

other closely-related ticks via feeding on animals, including humans1. Once transmitted to hosts, spirochetes can colonize a variety of organs, causing Lyme arthritis, carditis, and an array

of neurological syndromes2. Antibiotic therapy resolves clinical symptoms, in most cases, during the early stages of infection. However, persistent or relapsing symptoms (e.g., fatigue,

musculoskeletal pain, and cognitive difficulties) can later develop in a subset of patients; these symptoms are collectively referred to as chronic Lyme disease, otherwise known as

post-treatment Lyme disease syndrome (PTLDS)3. The underlying mechanisms, pathogenesis, and treatment of PTLDS remain unknown4,5. Therefore, it is of great importance to develop a vaccine

that will prevent the incidence of serious tick-borne infections such as Lyme borreliosis. Most research efforts focus on the identification of either _B. burgdorferi_ antigens or tick

proteins that are required for the survival of spirochetes within ticks, in an attempt to interfere with pathogen transmission from ticks or infectivity in the hosts, thereby preventing Lyme

disease6. In fact, a human vaccine based on a recombinant form of a _B. burgdorferi_ outer surface protein, OspA7, was developed and approved in 1998 by the Federal Drug Administration, but

it was later withdrawn because of sales issues and patient-related complications. Other strategies, such as controlling tick infestations, might serve as alternative preventive methods to

reduce the incidence of Lyme disease. _Ixodes_ ticks can transmit pathogens to humans and cause a range of serious diseases8. Given the lack of effective vaccines, tick-borne diseases

continue to spread, impacting human and animal health on a global scale. A traditional way to combat tick-borne diseases is to control the tick population using acaricides, which are

becoming increasingly less effective due to the emergence of resistant tick strains. Other preventive measures, including tick avoidance, protective clothing, and tick repellents, are only

20–40% effective9. Hence, current efforts have been geared towards the development of highly efficacious anti-tick vaccines to control tick infestations10,11,12. Generally, anti-tick vaccine

development is based on tick antigens that are accessible to host-derived antibodies, such as surface-exposed gut antigens. A ‘concealed’ midgut protein from _Rhipicephalus_ (formerly

_Boophilus_) _microplus_ cattle ticks, termed as Bm86, is expressed specifically in the tick midgut with upregulation during feeding13; it has been used as a commercially available anti-tick

vaccine to induce effective protection in cattle against _R. microplus_ infestation. Although the precise function of Bm86 has not been described, the vaccine impairs the survival of ticks

on immunized cattle and substantially reduces engorgement weights, which is caused by a disruption of the tick midgut epithelium, suggesting Bm86′s role in the development of tick midgut

tissue during the engorgement process14,15. The Bm86-based vaccine can also affect the egg-laying capacities of surviving ticks. Notably, besides reducing tick populations, Bm86-based

vaccines have the potential to partially block the transmission of tick-borne pathogens like _Babesia ovis_16,17. Despite their promise as anti-tick vaccines for cattle, vaccination with

Bm86 orthologs in _I. ricinus_ ticks (Ir86-1 and Ir86-2) did not show obvious effects on the feeding parameters of _I. ricinus_18,19, suggesting that certain species of ticks may be

refractory to the Bm86 vaccine, and that immunization with the whole protein (which was the case with the _I. ricinus_ orthologs) may induce immunodominant but non-neutralizing antibodies.

Many tick proteins, such as vitellogenin receptor20, _I. scapularis_ Bm86 orthologs, and ATAQ proteins13, feature epidermal growth factor (EGF)-like domains, although their functions in

vector biology remain elusive. The Bm86 antigens in particular feature multiple EGF-like domains13. Proteins harboring EGF-like domains have demonstrated the evolution of multiple distinct

functions21. They are associated with stimulating cell growth and restoring membrane damage, in addition to supporting microbial virulence, such as the invasion of _Plasmodium falciparum_

into erythrocytes22 and _Neisseria meningitidis_23 into endothelial cells. Recent studies have shown that a monoclonal antibody against an EGF-like domain of a _Plasmodium_ protein prevented

parasite invasion via inhibition of the pathogen’s erythrocyte-binding capacity24. In the fruit fly, the EGF receptor pathways control stem cell proliferation and gut remodeling following

infection25. While multiple proteins with EGF-like domains from hard and soft ticks were identified20,26, their roles in vector physiology or development remain enigmatic. The tick gut

presents a pivotal microbial entry point and serves as the major organ for pathogen colonization and survival within the vector, especially for the Lyme disease pathogen, as it resides

exclusively in the tick gut27,28. Here we report that Bm86 orthologs in _I. scapularis_ (Is86) are expressed in the tick gut and contain three EGF-like domains, and that immunization with

recombinant EGF-like domains influences optimal blood meal engorgement and the molting of _I. scapularis_, in addition to partially blocking pathogen transmission from tick to host. These

studies may help in the development of anti-tick vaccines to combat Lyme disease. RESULTS IDENTIFICATION AND EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF)-LIKE DOMAINS IN _I. SCAPULARIS_ As

the Bm86 glycoprotein in cattle ticks has been used as a commercially available vaccine against tick infestation in cattle17,29,30, we sought to know if vaccination with the Bm86 ortholog in

_I. scapularis_ exerts similar effects. Using the NCBI BLAST program to compare sequences with _R_. _microplus_ Bm86 and _I. ricinus_ orthologs (Ir86), we noticed that there were multiple

Bm86 transcript variants in _I. scapularis_, as shown in sequence alignments (Fig. 1A). Using specific primers (Table S1) and the _I. scapularis_ cDNA template, two homologs termed as

_Is86-1_ and _Is86-2_ were PCR amplified. Furthermore, Bm86 features at least three identifiable epidermal growth factor (EGF)-like domains, designated herein as EGF-1, EGF-2, and EGF-3

(Fig. 1A). The sequence analysis indicates that EGF-3 is identical for both _Is86_ homologs, whereas EGF-1 and EGF-2 have sequence specificities for _Is86-1_ and _Is86-2_, respectively, with

a sequence similarity of 47.9% between the two domains (Fig. S1A). Next, we generated the recombinant versions of the EGF-1, -2, and -3 proteins in a bacterial expression system (Fig. S1B,

upper panel). Antisera were also raised in mice that specifically recognized the corresponding recombinant EGF proteins (Fig. S1B, lower panel). The phylogenetic tree analysis (Fig. 1B),

comparing Is86 with Bm86 orthologs from a representative set of other hard and soft tick species, demonstrated a clear orthology with Ir86 of _I. ricinus_, which is the closest relative

among the tested tick species, and showed protein homologies of 88% and 44% for Is86-1 and Is86-2, respectively. The Bm86 of _R. microplus_ clustered with other _Rhipicephalinae_ tick

species, such as _Dermacentor_ and _Amblyomma_. Interestingly, a putative Bm86 ortholog from the soft tick species _O. savignyi_ was subgrouped with Is86-1. _BORRELIA BURGDORFERI_ INFECTION

INDUCES _IS86_ EXPRESSION IN TICKS To explore the biological function of Is86 in _I. scapularis_ and its roles in _B. burgdorferi_ infection, we first investigated the expression of _Is86_

in ticks with or without spirochete infection. Consistent with previous reports that the _Bm86_ gene was expressed specifically in the tick gut with upregulation during feeding13, _Is86_ was

expressed predominantly in the _Ixodes_ tick gut and was completely absent in the salivary glands. Furthermore, although not statistically significant, the mean _Is86_ level in infected

tick guts was 1.8-fold higher than in naïve ticks (Fig. 2A). We also assessed whether _B. burgdorferi_ infection influences _Is86_ expression kinetics during the course of tick feeding. The

results showed that _Is86_ expression was upregulated after the onset of tick feeding, with the level of expression steadily elevating and peaking at day three. Decreased expression was

observed in post-fed ticks in a level comparable to that of unfed ticks (Fig. 2B), potentially because there is a reduced requirement for the protein following repletion, as it is possibly

involved in the development and remodeling of the tick gut during feeding events. The level of _Is86_ in _B. burgdorferi-_infected tick guts was constantly higher compared to naïve ticks

during feeding (Fig. 2B). Consistently, a specific reaction around 100 kDa was detected in tick gut proteins using the pooled Is86 EGF antisera, and the protein level in infected tick guts

was higher than in naïve ticks (Fig. 2C, Fig.S6). In order to support the specificity of the reaction, we isolated native Is86 from unfed adult tick guts using immunoprecipitation (Fig. S3,

left panel), which specifically reacted with EGF antisera at a molecular weight of about 100 kDa (Fig. S3, right panel). Similarly, immunofluorescent staining showed that a specific reaction

was detected in both naïve and _B. burgdorferi_-infected tick guts (Fig. 2D). Although our study used permeabilized gut samples, the predominant distribution of immunofluorescent signals

towards the luminal side of the _Ixodes_ gut is consistent with the prevailing notion that _Bm86_ is expressed on the surface of the tick gut epithelial cells13,31. Furthermore, an apparent

higher fluorescence was observed in infected tick guts, as compared to naïve ticks (Fig. 2D). Taken together, these results indicate that _B. burgdorferi_ infection could upregulate _Is86_

expression. Next, we tested which Is86 homologs (Is86-1 or Is86-2) were expressed temporally in ticks at various stages of the life cycle. Since EGF-1 and EGF-2 are predominantly represented

in Is86-1 and Is86-2, respectively, we used antiserum against each EGF domain. EGF-2 was detected in unfed nymphal ticks (Fig. 3A), while both Is86 homologs were detected in unfed (Fig. 3B)

and partially fed (Fig. 3C) adult tick guts. Protein levels were upregulated after _B. burgdorferi_ infection (Fig. 3A–C). The homologs displayed the same migrating patterns and sizes in

both nymphal and adult ticks. In silico analysis revealed various patterns and types of posttranslational modifications (Fig. S2). Interestingly, analysis of the unfed naïve larvae revealed

the presence of both Is86 homologs migrating with the predicted molecular masses of 44.2 kDa (Is86-1) and 50.5 kDa (Is86-2) (Fig. 3D), suggesting an absence of posttranslational

modifications of the protein homologs in larvae prior to blood meal engorgement. The testing of Is86 homolog expression in fully replete naïve and infected larvae failed, likely due to the

blood content in the tested samples or the lack of protein expression (data not shown). Additionally, we did not detect any specific reactions in any stage of ticks using EGF-3 antiserum,

suggesting that either the level of EGF-3 is low, or that the antigen does not contain an immunogenic epitope that could be recognized by the specific antiserum (Fig. 3, Fig. S7). EGF-LIKE

DOMAIN ANTIBODIES MARGINALLY INTERFERE WITH TICK PHYSIOLOGY AND AFFECT _B. BURGDORFERI_ TRANSMISSION BY TICKS Previous studies have shown that specific antibodies against Bm86 affect tick

feeding on cattle17,32. Despite promising activity as anti-tick vaccines, vaccination with the Bm86 orthologs in _I. ricinus_ (Ir86-1 and Ir86-2) did not show obvious effects on tick

feeding18. We hypothesize that instead of immunization with the whole protein, which may induce immunodominant but non-neutralizing antibodies, identifying and focusing on the most conserved

region of Is86 may produce the most useful and effective neutralizing antibodies. We sought to know whether EGF-like domain-specific antibodies targeting the Is86 homologs could impair tick

physiology and consequently _B. burgdorferi_ persistence and transmission by ticks. Groups of mice were immunized with individual Is86 EGF-1, -2, or -3, which elicited high titers of

antibodies against the Is86 homologs, in contrast to the controls (Fig. S4A). The mice were then infested with infected nymphs (10 ticks/mouse), and feeding parameters were observed.

Compared to the controls, significantly less fully replete ticks were collected from mice immunized with Is86 EGF-1 (Fig. 4A, p < 0.01), and the engorgement weights of the ticks were

substantially lower (Fig. 4B, p < 0.01). Such data were not observed after immunization with Is86 EGF-2 or -3, suggesting that antibodies against Is86 EGF-1 could interfere with tick

physiology and result in a delay of tick feeding. Additionally, several unattached live and/or dead ticks were observed after feeding on mice in the experimental groups, which did not occur

in the controls (Fig. 4B). The collected ticks were then stored in the incubator and allowed to molt. The percentage of molted ticks that had fed on mice immunized with EGF-2 was

significantly decreased, with a molting rate of 40 ± 8.5%, as compared to the controls (81 ± 9.5%) (Fig. 4C, p < 0.05). As Bm86-based vaccines have been reported to partially block the

transmission of tick-borne pathogens like _B. ovis_17, we also assessed the effects of EGF antibodies on _Borrelia_ transmission by ticks. Ten days after tick feeding, murine skin samples

were collected. The spirochete burden in mice immunized with EGF-1 was significantly decreased compared to the controls (Fig. 4D, p < 0.05). This was not caused by disparities in

spirochete burdens within the ticks (Fig. 4E), indicating that antibodies against the Is86 homologs do not impact the majority of spirochetes that persist in ticks, but potentially influence

the dissemination of a fraction of _B. burgdorferi_ through ticks. We then assessed whether antibodies against the EGF domains interfere with spirochete acquisition by ticks. Mice were

immunized with individual EGF-1, -2, or -3 (Fig. S4B), and then infected with _B. burgdorferi_ via needle inoculation. Twelve days after inoculation, naïve ticks were allowed to feed on the

mice. There were no significant differences in tick feeding duration (Fig. 5A) and tick engorgement weights (Fig. 5B) among the experimental and control groups. Although not statistically

significant, there was an apparent effect on molting success, as the mean percentage of adult ticks that molted from engorged nymphs was reduced after feeding on EGF-2 immunized mice (50 ± 

26%), as compared to control ticks (83 ± 17%) (Fig. 5C). Notably, after 48 h of feeding, the spirochete level in ticks that parasitized EGF-1 immunized mice was 44-fold lower (Fig. 5D, p 

< 0.01), and 4.7-fold and 8.3-fold lower in the EGF-2 and EGF-3 groups, respectively (Fig. 5D), in comparison to the controls. However, in fully replete ticks, only those that fed on

EGF-3 immunized mice displayed a significant decrease in spirochete burden, when compared to control ticks (median of 11.6-fold, p < 0.05) (Fig. 5E). The impairment of spirochete survival

in fed ticks was likely due to the neutralizing effects of anti-EGF antibodies in the ticks, but not in the murine hosts, as similar levels of _B. burgdorferi_ were detected in the mouse

dermis, from which the pathogen likely enters the vector (Fig. 5F). Despite a partial impact on spirochete persistence (Fig. 4E), _B. burgdorferi_ transmission from tick to host was not

affected, and comparable levels of spirochetes were detected in the mouse skin among all groups (Fig. 5F). As naïve and _B. burgdorferi_-infected ticks represent two different environments

physiologically, where the expression of _Is86_ (Fig. 2) and EGF immunoreactivity (Fig. 3, Fig. S6) vary, we performed an independent rEGF immunization experiment to investigate the

potential impact of anti-rEGF antibodies on the physiology of ticks without _B. burgdorferi_ infection. In contrast to infected ticks, rEGF immunization did not significantly impact the

feeding or molting parameters of naïve ticks (Fig. S5), likely due to the dramatically reduced production of Is86 antigens in naïve ticks (Fig. 3, Fig. S6). _IS86_ SILENCING FAILED TO

INTERFERE WITH _B. BURGDORFERI_ PERSISTENCE AND TRANSMISSION To directly assess the role of Is86 proteins in tick physiology and _B. burgdorferi_ infectivity, we employed an RNA

interference-mediated knockdown of _Is86_ in ticks. To efficiently knock down gene expression, two dsRNA constructs targeting different regions of _Is86-1_ and _Is86-2_ were generated (Fig. 

6A). _B. burgdorferi_-infected nymphs were microinjected with the pooled ds_Is86_ RNAs, targeting both _Is86_ homologs, and allowed to feed on naïve mice. Compared to ticks injected with

ds_GFP_ RNA, _Is86_ transcripts in fully replete ticks were significantly decreased after treatment with ds_Is86_ RNAs (Fig. 6B). However, the silencing of _Is86_ did not affect _B.

burgdorferi_ persistence in ticks (Fig. 6C). Ten days after tick feeding, mouse skin samples were collected. Comparable spirochete levels were detected in mice that had been parasitized by

ticks injected with ds_Is86_ RNAs and ds_GFP_ RNA (Fig. 6D). The results indicated that the RNAi-mediated knockdown of _Is86_ failed to influence spirochete persistence in ticks and pathogen

transmission to the host. DISCUSSION Many tick proteins, including ones from _I. scapularis_, feature one or multiple EGF-like domains, although their functions in vector biology or

pathogen persistence remain elusive. The Bm86 glycoprotein, originally isolated from _R. microplus_ ticks10,33,34, represents the only tick antigen to be commercially developed as an

anti-tick vaccine, protecting immunized cattle from tick infestation. The vaccine is also partially effective in blocking the transmission of certain tick-borne pathogens, such as _Babesia_

parasites17,35. Although this study lacks conceptual innovation, as Bm86 orthologs have been subjected to many earlier studies, including the vaccination of _Ixodes_ species13 with Bm86

homologs, which failed to influence _I. ricinus_ feeding or oviposition18, our current study identified a biological significance of the EGF domains in _Ixodes_ ticks. Particularly, we

discovered that immunization with specific EGF-like domains in the Bm86 orthologs of _I. scapularis_ (originally identified by Nijhof and colleagues13,) could impact, at least to a marginal

extent, tick feeding and molting success, as well as the survival of _B. burgdorferi_ in the vector, highlighting their roles in tick biology and suggesting their use as potential components

of anti-tick vaccines. Based on the available _I. scapularis_ genome data36,37, we identified at least two homologs of _Bm86_ (_Is86-1_ and _Is86-2_); however, according to further updates

in the NCBI database, these homologs likely incorporate additional members, including several splice variants. Nonetheless, our sequence alignment and phylogenetic analysis revealed that

_Is86-1_ and _Is86-2_ represent prototypes of two major groups of _Bm86_ orthologs in black-legged ticks, which is in agreement with a previous report13 that showed a divergence between

these _Is86_ homologs from prostriata ticks to other hard tick species in the metastriata group. Notably, the relatively low sequence identity of _Is86_ homologs when aligned to the closely

related _I. ricinus Ir86_ further underscores its extensive sequence diversification, even across closely related tick species. Such sequence variability, which is also observed for _Bm86_

in _Rhipicephalus (Boophilus)_ spp. ticks38, may reflect their functional diversity among various tick species. Although the exact function of the Bm86 protein family still remains highly

enigmatic, these gene-products feature several conserved domains that are typically found in the epidermal growth factor (EGF) family of proteins13,15. The EGF domains are usually

represented as a small domain of 30–40 amino acids which regulate a diverse array of cellular functions, primarily supporting the growth and development of an organism14,21. In our present

study, we identified at least three EGF-like domains in Is86 homologs in _I. scapularis_, although additional EGF domains have been reported to occur in various _Ixodes_ spp. ticks13. An

important role of Bm86 in the physiology of _R. microplus_ ticks, as well as in the transmission of _Babesia_ species in cattle, was previously reported17. In fact, vaccines based on Bm86

antigens have proven to be a feasible control method against _R. microplus_ tick infestations in multiple countries. However, primarily due to the extensive sequence diversity across tick

species, Bm86 orthologs are likely to undergo dramatic functional divergence, thereby lacking broad vaccine efficacy in many other tick species32. In fact, an amino acid sequence divergence

of greater than 2.8% could result in a decreased vaccine efficiency of Bm86 antigens39. Accordingly, as with _I. ricinus_ ticks, vaccination with Bm86 homologs failed to influence tick

feeding or oviposition18. These findings agree with the results of our RNAi-mediated gene silencing studies, as the knockdown of Is86 homologs did not impact _I. scapularis_ physiology or

pathogen survival. The silencing of the _R. microplus Bm86_ gene via RNAi also failed to affect the efficiency of the transovarial transmission of _B. bovis_40. Nevertheless, despite an

absence of gene silencing effects, the partial impact of immunization with the truncated Is86 antigens suggests the potential roles of the EGF-like domains in tick biology. The importance of

these domains in Is86 (or similar antigens) in regards to tick biology is particularly highlighted by: (1) their induction during the tick engorgement process, and more importantly during

the _B. burgdorferi_ infection of ticks, (2) their extracellular exposure, in particular towards the luminal surfaces of the midgut cells, and consequent antibody accessibility, (3) their

copious expression throughout the tick developmental stages, including sub-adult and adult, (4) the abilities of anti-EGF antisera to impact, at least marginally, multiple aspects of tick

biology, including blood meal engorgement and molting, and finally (5) their detectable influences on spirochete survival in ticks, in terms of both pathogen acquisition and transmission

through the vector to the host. In order to expand upon these promising initial results, the effect of EGF immunization on _Borrelia_ transmission requires more elaborate studies to better

address the vaccine potential of these antigens. As Bm86 orthologs bear considerable sequence homology to closely related ticks, such as _I. ricinus_, similar studies would shed light on the

potential applicability of EGF domains as global vaccines. Despite our identification of the EGF-like domains in Is86 antigens as a possible component of anti-tick vaccine targets, we were

unable to decipher their precise biological functions. Vaccination against its ortholog, Bm86, reportedly resulted in tissue damage in the midgut of adult _B. microplus_ ticks41. Based on

our data, no such effect was apparent in _I. scapularis_ nymphal ticks, either via anti-Is86 immunization or through RNAi-mediated gene silencing. It is therefore possible that the anti-Is86

antibodies are cross-reactive to other unknown tick proteins, and that the observed vaccination effects of EGF-like domain antibodies are impacted by their recognition of other

cross-reactive antigens, rather than the specific inhibition of Is86 homologs. Although our immunoblot and immunoprecipitation studies using anti-EGF antibodies failed to identify additional

tick proteins other than Is86, these antibodies still can bind to and inhibit the activity of unidentified tick gut proteins through steric hindrance42, which could play essential

supportive roles in tick biology and pathogen survival. We noticed perplexing discrepancies in the abilities of specific EGF antisera to detect target proteins at various tick feeding or

developmental stages. Although Bm86 orthologs are known to be glycosylated, our study recorded a puzzling and substantially higher molecular weight of both Is86 orthologs. Some tick

physiological parameters were affected to some extent for both homologs, but more significant results were observed with anti-EGF-1 antibodies (Is86-1). While the expression patterns of the

homologs were comparable towards the end of tick feeding, differences were noted in unfed and early-fed ticks, with Is86-1 being absent in unfed tick guts and generally being expressed at

lower levels than Is86-2. It is also possible that, in addition to or in lieu of Is86, the EGF antibodies also detected multiple patterns of Is86 glycosylation, splice variants, or cleavage

products, or recognized additional cross-reactive tick antigens, that are differentially produced during tick feeding or the development process. Nevertheless, we observed that Is86-1

contained a broader spectrum of posttranslational modifications at a higher frequency than Is86-2. Such differences may be a contributing factor as to why the antibody-based blocking of

Is86-1 caused physiological changes in ticks, such as delayed feeding and reduced weight, indicating a dominant physiological function of Is86-1. Although marginal, as EGF-1 and EGF-2

antibodies affected distinct phases of tick biology, namely blood meal engorgement and the molting process, respectively, it is likely that both Is86 homologs, or other tick gut protein(s)

with cross-reactive EGF antibodies, have predominant functions at distinct phases of the tick life cycle. Our study also uncovered a novel mode of action for the EGF-based antigens,

suggesting that other unknown molecules are implicated in the process. This may also include tick molecules that are induced during borrelial infection, and/or the spirochete proteins that

interact with the gut surface receptors and the antibodies targeting EGF-like domains, which interrupt that interaction. There has been an increasing body of evidence that EGF-like domains

are involved in versatile functions during cellular growth and development21, including protein–protein interactions43 and cellular adhesion with concomitant intracellular signaling44. In

particular, due to the well-documented roles of EGF proteins in cell growth, proliferation, repair, or remodeling, as well as infection control22,23,25, it is likely that the EGF domains in

Is86 proteins may serve critical roles in _Ixodes_ biology and infection. We speculate that the induction of Is86 during blood meal engorgement, or more notably during infection, could

involve discrete protein–protein interactions relevant to _B. burgdorferi_, either directly via EGF domain interactions with spirochetes, or indirectly via another EGF domain-containing

protein. This possibility is bolstered by the fact that EGF-like domains in Is86, or in other cross-reactive proteins, are exposed predominantly at the luminal side of the tick gut

epithelium (a localization pattern also reported for Bm8614), where extracellular pathogens like _B. burgdorferi_ reside and express a plethora of surface antigens, many of which are known

to be involved in host–pathogen interactions27,45. A deeper understanding of the biological functions of the Is86 homologs and their roles in _I. scapularis_ tick physiology, as well as the

precise molecular mechanisms through which antibodies against EGF-like domains or other novel tick antigens of unknown functions reduce spirochete entry and exit through ticks, will not only

enrich our knowledge of tick-pathogen interactions, but will also ultimately impact the development of new strategies for the prevention of tick-transmitted infections. METHODS BACTERIA,

MICE, AND TICKS _Borrelia burgdorferi_ sensu stricto infectious isolate B31 A3, grown in Barbour-Stoenner-Kelly-H (BSK-H) medium, was used throughout this study46. Four- to six-week-old

C3H/HeN mice were purchased from the Charles River Laboratories. All experimental protocols were approved and performed in accordance with the guidelines of the Institutional Animal Care and

Use Committee and Institutional Biosafety Committee of the University of Maryland, College Park. The study was carried out in compliance with the ARRIVE guidelines. _Ixodes scapularis_ tick

egg masses were purchased from the Oklahoma State University Tick Rearing Facility. Larval ticks were allowed to feed on naïve or _B. burgdorferi_-infected mice47; upon engorgement, the

ticks were collected and maintained in an incubator at 20 °C with 95% relative humidity and a 12-h light/dark photoperiod regimen. The collected larvae were allowed to molt to nymphs, which

typically took 4 weeks. The unfed nymphs were then allowed to parasitize mice; when fully replete, the ticks were collected and allowed to molt to adult ticks in the incubator. POLYMERASE

CHAIN REACTION (PCR) The oligonucleotide sequences for each of the primers used in specific PCR reactions are listed in Supplementary Table S1. The pathogen-free naïve and _B.

burgdorferi_-infected unfed and fed nymphal ticks were collected at various time points during feeding (Days 1, 2, or 3, or as fully replete ticks). The tick specimens were immobilized, and

the midgut and salivary glands were dissected. To increase the sensitivity of _Is86_ detection in our initial experiments, we pooled tick samples from various time points of feeding and used

a generic qPCR primer pair that could detect other _Is86_ transcripts. Additionally, groups of naïve or _B. burgdorferi_-infected mice were parasitized by naïve or infected ticks, which

were allowed to feed to full repletion, and murine skin biopsies were collected ten days after the ticks dropped off. Due to challenges associated with their size, larval and nymphal samples

were processed differently. Nymphs were dissected for the isolation of specific organs (such as the gut or salivary glands), whereas larval samples were processed as entire bodies. Total

RNAs were isolated either from tick or mouse tissues using TRIzol (Invitrogen), reverse transcribed to complementary DNA (cDNA) (Invitrogen), and treated with DNase (NEB) to minimize DNA

contamination as detailed48.The gene transcripts were analyzed using quantitative PCR (qPCR). The amplification parameters for _rps4_/_Is86_ are as follows: initial denaturation at 95 °C for

5 min, followed by 40 cycles each at 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 30 s. The amplification parameters for tick _β_-actin/_flaB_ are as follows: initial denaturation at 95 °C

for 5 min, followed by 40 cycles each at 95 °C for 10 s and 60 °C for 1 min. The final step in both amplification cycles was the melt curve analysis at 55 °C for 30 s, increased by 0.5 °C

per cycle to 95 °C. The amplification was performed in an iQ5 real-time thermal cycler (Bio-Rad) using SYBR Green Master Mix (Thermo Fisher Scientific). The relative transcript levels of

_Is86_ were measured against the tick house-keeping gene _rps4_ in a three-step amplification cycle using annealing temperatures of 55 °C as detailed49. The relative spirochete burdens were

assessed by measuring copies of _flaB_ transcripts as a better surrogate for live pathogens48 in a two-step amplification cycle, using annealing temperatures of 60 °C and normalizing against

the _β-actin_ gene as detailed48. GENERATION OF RECOMBINANT PROTEINS AND POLYCLONAL ANTISERA To identify the _Bm86_ ortholog in the _I. scapularis_ genome, the _Bm86_ genes from _R.

microplus_ and _I. ricinus_ were used for similarity and identity comparison against the _I. scapularis_ genome, which is available from the NCBI database (XM_029991986.1). Two _Bm86_

homologs, _Is86-1_ and _Is86-2_, were PCR amplified using a template from the _I. scapularis_ genome37, cloned into the pGEM-T Easy Vector (Promega), sequenced, and deposited in the NCBI

nucleotide database. The EGF-like domains within _Is86-1_ and _Is86-2_ were predicted according to published data15, and further validated using the PROSITE server

(https://prosite.expasy.org). Clones harboring either _Is86-1_ or _Is86-2_ were used as templates for further PCR amplification of EGF-1 and EGF-2, respectively, using primers containing

restriction sites (Table S1). EGF-3 was amplified from both clones. The PCR-amplified EGF domain products (EGF-1, -2, and -3) were subcloned into a pET28a expression vector. Heterologous

expression of the three recombinant EGF-like domains was induced in BL21 (DE3) _E. coli_ with 0.4 mM IPTG at 37 °C. The recombinant proteins, containing 6xHis-tag located at the N-terminus,

were purified using ProBond Nickel-Chelating Resin (Thermo Fisher Scientific), dialyzed, and refolded in 50 mM Tris–HCl. Polyclonal antisera against each of the three recombinant domains

were generated in mice as described50. The antibody titers were assessed using an enzyme-linked immunosorbent assay (ELISA) and the specificity was evaluated using Western blotting50.

PHYLOGENETIC ANALYSIS The protein sequences of _Bm86_ orthologs among hard and soft tick species were based on multiple sequence alignments generated with ClustalW. The phylogenetic tree was

constructed using the maximum likelihood method with 1000 bootstrap replicates in MEGA7 software51. WESTERN BLOTTING Immunoblotting was performed as described48. Briefly, the dissected guts

of unfed ticks (~ 30 μg of total larval lysate, 5 nymphs/lane, 3 females/lane) and two-day fed ticks (~ 30 μg of female gut lysate/lane, 2 nymphs/lane) were pooled, extracted and resolved

by SDS-PAGE, and immunoblotted using antiserum (1:1,000) against Is86 homologs. The blots were developed by the addition of horseradish peroxidase (HRP)-conjugated secondary antibodies

(1:5,000 to 10,000), using the chemiluminescent immunoblotting detection reagent (Thermo Scientific Inc.). Immunoblotting to confirm _B. burgdorferi_ infection in mice was performed as

described48. ACTIVE IMMUNIZATION WITH RECOMBINANT EGF DOMAINS AND INFECTION STUDIES For _B. burgdorferi_ transmission experiments, mice were immunized with individual recombinant Is86 EGF-1,

-2, or -3, as detailed42,47. Briefly, 10 μg of recombinant protein was dissolved in 50 μl of phosphate-buffered saline (PBS), emulsified with 50 μl of complete (first injection) or

incomplete (remaining two injections) Freund's adjuvant, and subcutaneously administered into each mouse at 10-day intervals. As recombinant EGF proteins are relatively pure (Fig. S1B),

mice immunized with PBS and adjuvant served as the only control group. Two weeks after the second boost, groups of mice (3 mice/group) were infested with _B. burgdorferi_-infected _I.

scapularis_ nymphs (10 ticks/mouse). A portion of ticks was forcibly detached after 48 h of feeding, and others were collected when they dropped off after full repletion. The spirochete

burdens in 48-h fed and replete ticks were assessed using qPCR. Ten days after tick feeding, mice were euthanized, and skin biopsy samples were collected to assess the spirochete burdens by

qPCR. For _B. burgdorferi_ acquisition experiments, mice were immunized with recombinant EGF-1, -2, or -3, as described in the above paragraph. The mice were then infected with _B.

burgdorferi_ via intradermal needle inoculation. Two weeks after inoculation, the mouse sera were collected and probed with _B. burgdorferi_ lysates and immunoblotted to confirm infection,

prior to infestation with naïve nymphs (15 ticks/mouse, 2 mice/group). Ticks were collected after 48 h of feeding and after full repletion. Spirochete burdens in the ticks were evaluated

with qPCR, as described previously42. Following the completion of tick feeding, murine skin samples were collected to assess spirochete burdens using qPCR. In both infection studies, to

monitor the progress of feeding, ticks were checked daily until all had detached from the mice. Detached ticks were collected and counted, and their engorgement weights were measured using a

laboratory scale. Ticks were then allowed to molt, and their molting rates were calculated two months after repletion. RNA INTERFERENCE AND INFECTION STUDIES RNA interference (RNAi)

experiments targeting tick _Is86-1_ and _Is86-2_ were conducted as described42. Briefly, the targeted sequences in _Is86-1_ and _Is86-2_ were PCR amplified using T7 promoter

sequence-containing primers (Table S1). A fragment of the _GFP_ gene was amplified as a control. The dsRNAs were synthesized and purified using the MEGAscript RNAi Kit (Ambion). To assess

whether the silencing of _Is86_ expression affects _B. burgdorferi_ transmission by ticks, infected nymphs were microinjected with ds_Is86_ RNA (pooled dsRNAs targeting both homologs) or

ds_GFP_ RNA (5 µg/µl) using a microinjector (Eppendorf). After overnight incubation, the ticks were allowed to feed on naïve mice until full repletion (10 ticks/mouse). The ticks were

collected and individually processed for the assessment of gene silencing, using primers that bind further upstream and downstream of the target dsRNA sequence (Table S1). Mouse skin samples

were collected 10 days after tick feeding. Pathogen levels in the individual ticks and in murine tissues were evaluated by measuring the _flaB_ transcripts with qPCR and normalizing against

tick and mouse _β-actin_, respectively. ARTIFICIAL MEMBRANE TICK FEEDING SYSTEM An artificial membrane feeder was used to generate adult ticks from naïve and _Borrelia_-infected nymphs. The

system was developed using published procedures as detailed52. Although various _B. burgdorferi_ sensu lato spirochetes can resist or remain sensitive to killing by the host complement, we

had previously shown that commercial defibrinated bovine blood can used in our membrane feeder system to study the acquisition and transmission of _B. burgdorferi_ B31 strain. Briefly,

defibrinated bovine blood was used, and the blood was changed every 12 to 14 h. Adult ticks (10 ticks/capsule) were placed on the artificial membrane feeding system, allowed to feed on

bovine blood until partial repletion (~ 48 h), and collected to process for further analysis. Adult ticks were used for Is86 expression and immunoprecipitation experiments.

IMMUNOPRECIPITATION Native Is86 was immunoprecipitated from adult tick guts using the Protein G Immunoprecipitation Kit (Sigma-Aldrich) as detailed53. Briefly, a RIPA buffer (Sigma) served

to extract a midgut lysate from 10 ticks (~ 10 µg), which was then incubated with the pooled EGF-1, -2, and -3 antisera. The immunoprecipitated products were resolved using SDS-PAGE and

either stained with Sypro Ruby or probed with pooled EGF antisera. CONFOCAL MICROSCOPY Confocal microscopy was performed as detailed42. Unfed tick guts were fixed in 4% paraformaldehyde at 4

 °C overnight, then rinsed three times with PBS and permeabilized with acetone for 10 min. The tick tissues were blocked with 5% normal goat serum in PBS for 1 h at room temperature (RT) and

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PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS The authors are thankful to Kathryn Nassar for assistance with the preparation of this manuscript. This study was

supported by the Slovak Research and Development Agency, grant number APVV-18-0201, and the National Institute of Allergy and Infectious Diseases, Award Numbers R01AI080615, R01AI116620,

and P01AI138949. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. C.K. is the recipient of

the Deborah and Mark Blackman Postdoctoral Fellowship from Global Lyme Alliance. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Veterinary Medicine, University of Maryland,

College Park, MD, 20742, USA Juraj Koči, Sandhya Bista, Payal Chirania, Xiuli Yang, Chrysoula Kitsou, Vipin Singh Rana & Utpal Pal * Institute of Zoology, Slovak Academy of Sciences,

Dúbravská cesta 9, 84506, Bratislava, Slovakia Juraj Koči * Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dúbravská cesta 9, 84505, Bratislava, Slovakia

Juraj Koči * Department of Microbiology and Clinical Microbiology, Faculty of Medicine, Istinye University, Zeytinburnu, İstanbul, 34010, Turkey Ozlem Buyuktanir Yas * Department of

Biological Sciences, Old Dominion University, Norfolk, VA, 23529, USA Daniel E. Sonenshine * Virginia-Maryland Regional College of Veterinary Medicine, College Park, MD, USA Utpal Pal

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CONTRIBUTIONS J.K. and U.P. conceived and designed the research. J.K., S.B., P.C., X.Y., C.K., V.S.R., D.S., O.B., and U.P. performed the research and analyzed the data. J.K., X.Y., D.S. and

U.P. wrote the paper. CORRESPONDING AUTHORS Correspondence to Juraj Koči or Utpal Pal. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL

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ARTICLE CITE THIS ARTICLE Koči, J., Bista, S., Chirania, P. _et al._ Antibodies against EGF-like domains in _Ixodes scapularis_ BM86 orthologs impact tick feeding and survival of _Borrelia

burgdorferi_. _Sci Rep_ 11, 6095 (2021). https://doi.org/10.1038/s41598-021-85624-5 Download citation * Received: 15 July 2020 * Accepted: 01 March 2021 * Published: 17 March 2021 * DOI:

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