Antimicrobial cyclic peptides effectively inhibit multiple forms of Borrelia and cross the blood-brain barrier model

Mochnáčová, Evelína; Bhide, Katarína; Kucková, Katarína; Jozefiaková, Jana; Maľarik, Tomáš; Bhide, Mangesh

doi:10.1038/s41598-025-90605-z

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Published: 20 February 2025

Antimicrobial cyclic peptides effectively inhibit multiple forms of Borrelia and cross the blood-brain barrier model

Evelína Mochnáčová¹,
Katarína Bhide¹,
Katarína Kucková¹,
Jana Jozefiaková¹,
Tomáš Maľarik¹ &
…
Mangesh Bhide^1,2

Scientific Reports volume 15, Article number: 6147 (2025) Cite this article

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Abstract

Infection caused by neuroinvasive Borrelia often manifests long-term CNS disorders and is difficult to treat as most antibiotics fail to attain an effective concentration within the brain or cannot kill the persister forms of Borrelia (cysts and round bodies). Thus, this study focused on developing antimicrobial cyclic peptides (AMPs) from a combinatorial phage display library that target phosphatidylcholine of the borrelial cell membrane. Isolated cyclic peptides with anti-Borrelia properties were then fused with the CNS homing peptide developed in this study (designated as O-BBB) to facilitate AMP transport across the blood-brain barrier. Among all O-BBB fused AMPs, Bor-18 had half maximal effective concentration (EC₅₀) 0.83 µM when tested against spirochetal Borrelia. Bor-16, Bor-18, and Bor-26 inhibited the cystic form with EC₅₀ 0.83 µM, while Bor-11 had EC₅₀ 0.41 µM. Within an hour, all four peptides caused a permeability breach in the borrelial cell membrane, causing depolarization of the membrane. Bor peptides did not inhibit eukaryotic cell metabolism or proliferation, nor did they cause erythrocyte lysis. Peptides were stable in serum, could cross the BBB in-vitro, and remained effective against Borrelia. Cyclic AMPs fused with a CNS homing moiety, the Bor peptides, deserve further investigation for their potential use in neuroborreliosis therapy.

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Introduction

Lyme borreliosis (LB) is the most common vector-borne disease prevalent in Europe and the United States, with an annual rising incidence^1,2. Early-stage LB symptoms include fatigue, headache, fever, and, in some cases, erythema migrans³, whereas pathological conditions in other organs such as joints, eyes, the heart, and the central nervous system can be observed in later stages, depending on the Borrelia genospecies involved⁴. Borrelia garinii and B. bavariensis can cross the blood-brain barrier (BBB) and invade the immune-privileged central nervous system causing Lyme neuroborreliosis⁵. Neuroborreliosis has a favorable prognosis if treated early, however the long-term administration of antibiotics over many weeks for chronic neuroborreliosis carries the risk of serious adverse effects⁶. Furthermore, in chronic neuroborreliosis, a long latent period and persistence of Borrelia infection even after repeated antibiotic therapy can be noted, which may be accompanied by the presence of drug resistant atypical extracellular and intracellular pleomorphic and cystic forms in the brain⁷.

Several alternative therapeutic agents have been developed and tested to address the antibiotic resistance. Antimicrobial peptides (AMPs), particularly short cationic peptides, stand out as alternative therapeutic agents because they can have both antimicrobial and immunomodulatory properties. The mode of antimicrobial action of these peptides is related to either cell membrane disruption or interaction with intracellular components⁸, resulting in inhibition of cell wall, nucleic acid, and protein synthesis or inhibition of enzymatic activity⁹. Antimicrobial peptides have the advantage of being nonspecific defense weapons, making it difficult for microbial pathogens to develop resistant mutants. Previous studies report testing of naturally occurring antimicrobial peptides or their derivates against Borrelia. In particular, cathelicidin-derived synthetic peptides SMAP-29, LL-37, PG-1, CRAMP, and BMAP-28 had minimum inhibitory concentrations (MIC) ranging from 307 to 449.4 mg/l¹⁰. Anti-Borrelia activity of defensin, melittin and peptides from bee venom was also tested and compared with commonly used antibiotics¹¹. The bee venom and melittin had a significant effect on the spirochetal form of Borrelia as well as biofilm, as compared to antibiotics, which had limited effects on biofilm¹². It implies that AMPs might be intriguing therapeutic candidates; nevertheless, large-scale production of naturally occurring AMPs is difficult. No synthetic AMPs have been developed so far against Borrelia, despite the fact that synthetic peptides show promise as a less costly large-scale production option. A PubMed search for synthetic peptides developed against Borrelia produced no relevant results, demonstrating the complexity in development of AMPs against Borrelia and its various forms. The only credible report demonstrating the borreliacidal effect of synthetic peptides is a patent application in which peptides containing epsilon lysine residues were tested¹³. The development of antimicrobial drugs that target outer membrane proteins or the membrane itself is challenging since Borrelia frequently alters the expression of surface proteins¹⁴. To this point, membrane phospholipids seems to be attractive target for development of AMPs as borrelial membrane contains two major membrane phospholipids: phosphatidylglycerol and phosphatidylcholine. Using a murine model, it was shown that Borrelia infection causes the production of antibodies against phosphatidylcholine¹⁵. Therefore, our goal was to target phospholipid of the borrelial cell membrane for development of AMPs selected from combinatorial phage display library.

Antibiotics and antimicrobial peptides have limited biodistribution in the CNS, making neuroinfections difficult to treat. The absence of fenestrations and the nearly impermeable cell-cell tight junction between endothelial cells of brain microvasculature prevent paracellular transport of drugs across the BBB¹⁶. Glucose and oxygen can cross the BBB via paracellular and transcellular transport. However, larger molecules require receptor-mediated endocytosis (RME)¹⁷. The CNS homing peptides, such as angiopep-2, TGN and TfrL are developed based on RME¹⁸. Several pathogens including Borrelia exploit RME to cross the BBB¹⁹. We have shown that borrelial OspA-CD40 interaction initiates translocation of the spirochete through the endothelial barrier²⁰, and the interaction is mediated through endothelial-cell binding domain of OspA²¹. Our second goal was to use endothelial-cell binding domain of OspA or its truncated fragments as CNS homing peptide and fuse it with AMPs to promote AMP transport across BBB model.

In this study, we aimed to isolate Cx₇C peptides from combinatorial phage library that bind to the phosphatidylcholine of borrelial cell membrane. We confirmed binding of individual phage clones (displaying Cx₇C peptides) to different forms of Borrelia (spirochetes, cystic form and Borrelia devoid of outer membrane proteins). Simultaneously, we aimed to test ability of the endothelial-cell binding domain of OspA and its truncated fragments to traverse the BBB model. Then we fused the N-terminal fragment of endothelial-cell binding domain (which showed significant BBB crossing) with Cx₇C peptides. Finally, we performed a series of tests to check concentration-dependent borreliacidal activity of fusion peptides, ability to cause membrane depolarization, non-toxicity to mammalian cells, hemocompatibility, etc. We were able to develop four fusion peptides, Bor-11, Bor-16, Bor-18, and Bor-26, that demonstrated cidal activity against both spirochetal and cystic forms of Borrelia. All four peptides altered the membrane polarity of Borrelia, their activity remained stable in the normal human serum. Bor peptides were able to cross the BBB model in-vitro and exhibit borreliacidal activity. The anti-Borrelia peptides developed in this study deserve further investigation in order to be translated into functional therapeutics against neuroborreliosis.

Results

Cx7C peptides against borrelial cell membrane (isolated from combinatorial phage display peptide library)

Small-sized seven-mer peptides flanked by cysteine residues (CxxxxxxxC, here x is any amino acid, Cx₇C) that bind to the phospholipid of borrelial cell membrane were isolated from a combinatorial phage display peptide library (Fig. 1, panel A). The binding of isolated phage clones to borrelial phospholipid was tested using a dot blot, in which phages were incubated with various forms of Borrelia (spiral, cystic, and trypsin-shaved forms are depicted in Fig. 1, panel B-D, supplementary information 1). The binding of phages to Borrelia, as shown in Fig. 1 (panel E), demonstrated that phages enriched by pannings are against phospholipids rather than outer membrane proteins, as they demonstrated binding affinity to shaved Borrelia, devoid of outer membrane proteins.

The DNA from individual phage clones, separated from the elution of the 3rd panning, was amplified (amplicons depicted in supplementary information 2), sequenced and aligned to make clusters based on amino acid sequence homology in the Cx₇C motif. A total of 19 clusters were formed (Fig. 1, panel F), which were screened through in silico predictors for various parameters such as (1) antimicrobial potential, (2) net charge, (3) water solubility, and (4) probable cell toxicity. Fourteen clones appear to possess antimicrobial potential (Supplementary Table 1). The majority of the predicted antimicrobial peptides were cationic and eight of them were predicted to be poorly soluble in water. All peptides except ACTNANHYFCGGGS (clone Ph.Bor-15) were predicted to be non-toxic (Supplementary Table 1). None of the peptides from 19 clusters had a match in Peptide search tool of UniProt database. Five peptides (namely Ph.Bor-4, Ph.Bor-7, Ph.Bor-9, Ph.Bor-20 and Ph.Bor-24) were predicted as non-antimicrobial peptide (non-AMP). All phage clones, when assessed for their affinity to borrelial membrane using dot blot, most of the clones showed good binding ability to spirochetes fixed on membrane (Fig. 1, panel G).

To select the clones for further testing (production in recombinant form), ten best binders from dot blot (Ph.Bor-1, 8, 9, 11, 16, 18, 23, 24, 26, 29) were shortlisted. Among those, Ph.Bor-9 and Ph.Bor-24 were predicted as non-AMP, thus clone 24 was excluded from further study, however owing to binding affinity of clone 9, it was retained.

BBB homing peptide derived from OspA and its fusion with Cx7C

The N-terminal fragment of endothelial cell binding domain of OspA (the O-BBB) showed significantly higher crossing than the other two forms (full length and C-terminal fragment of endothelium binding domain, t-test p < 0.05). A significant difference in angiopep-2 and O-BBB crossing was observed at the 1st hr (P = 0.0175). At the 2nd hr, O-BBB crossing was comparable to that of angiopep-2, but at the 3rd hr, O-BBB crossing was significantly higher than that of angiopep-2 (Fig. 2, panel B). Thus, the O-BBB, which contains antiparallel beta sheets (Fig. 2, panel C), was used in the current study to fuse with Cx₇C. The fusion constructs consist of O-BBB at N-terminus of the Cx₇C, flanked by GGGS at both ends (Fig. 2, panel D). DNA fragment coding Cx₇C, amplified from shortlisted phage clones (Fig. 1, panel G; amplicons are in Fig. 3, panel A) were ligated in pQE-30 N-tag-II vector²², and overexpressed in E. coli. Purified peptides had the expected molecular weights, as seen in LDS-PAGE and MALDI-TOF (Fig. 3, panels B-C, Table 1). Fusion O-BBB-Cx₇C peptides were designated as Bor peptides (Bor-1, Bor-8, and so on).

Table 1 Amino acid sequences and molecular masses of peptides produced in recombinant forms.

Full size table

Functional analysis of O-BBB-Cx7C fusion peptides (bor peptides)

The binding of Bor peptides to Borrelia was verified by ELISA, in which all peptides, except Bor-1, showed affinity to Borrelia and no non-specific interaction with empty wells was observed. O-BBB alone showed no affinity for Borrelia (Fig. 3, panel D). The Bor peptides were tested further for cidal activity against the spirochetal form of Borrelia. Bor-11, 16, 18, and 26 demonstrated significant cidal activity even at 1.67 µM concentration (viability reduced < 50%, Fig. 4 panel A). At 6.67 µM, all other peptides possessed borreliacidal activity, which was mostly lost after further dilution (3.3 µM). The O-BBB exhibited no borreliacidal effect even at 6.67 µM. Bor-11, 16, 18, and 26 were then tested to determine the MIC (EC₅₀). Apart from Bor-16 and Bor-26, the other two peptides inhibited the spiral form of Borrelia by more than 50% at 0.41 µM (Fig. 4, panel B).

Since many antibiotics fail to kill the cystic form of Borrelia^23,24, we wanted to see if Bor-11, 16, 18, and 26 would be effective against it. The doxycycline which is effective against spirochetal form but unable to kill round bodies²⁵, was used as control. Exposing cystic Borrelia to peptides (1.66 µM) for 16 h significantly reduced their recovery and transformation into the spirochetal form (Fig. 4, panel C). Bor-11 was most effective against cystic form, inhibiting growth significantly (t-test p < 0.05) even at 0.41 µM. Incubation of cysts with 0.05 µM of the doxycycline for 16 h did not inhibit the transformation and recovery into the spirochetal form.

Bor-11, 16, 18, and 26 were examined for concentration-dependent binding to Borrelia. Bor-18 binding was significantly higher even at 0.16 µM than other peptides at the same concentration, but decreased significantly at 0.08 µM (Absorbance₄₅₀ 0.8). Other peptides exhibited concentration-dependent linear decreases in absorbance (Fig. 5, panel A). The ELISA was also performed to check the binding ability of peptides to cystic form and Borrelia devoid of outer membrane proteins (spirochetes shaved with trypsin). All four peptides showed affinity for all three forms (Fig. 5, panel B), indicating that peptides interact with the borrelial membrane (most probably with phosphatidylcholine), and that the binding is not mediated by outer membrane proteins.

Since Bor peptides showed affinity for borrelial cell membrane, we sought to investigate if they cause a smaller breach of the permeability barrier of the outer and cytoplasmic membranes of Borrelia. In membrane depolarization assay, we clearly observed that Bor peptides caused a permeability breach, resulting membrane depolarization and a significant increase in fluorescence when compared to the negative control (Fig. 5, panel C). The breach in cytoplasmic membrane that may cause borreliacidal effect was also monitored using propidium iodide (live/dead staining). This test may also reveal how long it takes for the peptide to exert borreliacidal effect (time to kill). Bor peptides caused an increase in fluorescence after 1 h of incubation, indicating that borreliacidal activity began at this timepoint, which was approximately 90 min. for doxycycline (Fig. 5, panel D).

Toxicity and hemocompatibility of peptides

Bor-11, 16, 18, and 26 were tested for cytotoxicity and if they cause lysis of red blood cells. At 3.33 µM, Bor peptides and O-BBB had no adverse effects on cell metabolic activity (Fig. 6, panel A). None of the peptides significantly inhibited cell proliferation (at 1.67 µM and 3.3 µM), indicating their safety (Fig. 6, panels B–E, Pearson correlation coefficient 0.98 to 0.99 between negative and treated cells).

All of the peptides appeared to be hemocompatible, with none causing hemolysis within 5 h, even at the highest concentration (300, 100, and 50 ng, Fig. 6, panel F).

BBB crossing and borreliacidal activity of peptides

The primary mode of transport for peptides across the BBB is receptor-mediated endocytosis, thus protein attachment to endothelial cells is critical. Cell-ELISA used to test the binding of Bor peptides on brain microvascular endothelial cells (BMECs), showed that all four peptides (Bor-11, 16, 18, and 26) as well as O-BBB bind endothelial cells (Fig. 7, panel A). Peptides were also tested for their serum stability (for 1, 3 and 6 h). All peptides caused significant reduction (viability of Borrelia < 2% in all treatments) in the number of Borrelia even after 6 h of incubation in human serum, indicating that peptides maintained anti-Borrelia activity (Fig. 7, panel B).

Finally, the ability of peptides to cross the BBB model and exert a borreliacidal effect was assessed using BBB cultivated in cell-inserts. Bor peptide did not affected integrity of the BBB model as no significant difference in TEER values before and after the addition of peptides or doxycycline was observed. The crossing of dextran was observed between 4.0 and 5.3% (dextran crossing ≤ 6% indicates intactness of BBB). The abluminal content after 5 h, which contained Borrelia and possibly peptides (if they were able to cross the BBB), was transferred to a new tube and incubated for 5 days to allow borrelial growth. A significant reduction (t-test, P < 0.05, % survival ranged between 4.0 and 6.1) in growth of Borrelia was observed in case of Bor peptides, compared to a control in which only PBS was added in the luminal chamber (Fig. 7, panel C), indicating that the Bor peptides can cross the BBB and maintain their borrelicidal properties. Doxycycline, although was able to cross the BBB in-vitro, 24.9% of the Borrelia were able to survive in the abluminal content (Fig. 7, panel C).

Discussion

Therapeutic management of borreliosis relies on per oral doxycycline or intravenous ceftriaxone²⁶. However, patients can show recurrence of residual symptoms such as pain, fatigue, or cognitive difficulty known as post-treatment Lyme disease syndrome ^27,26. One possible explanation for this clinical observation is that Borrelia shields itself by generating cystic forms, biofilm, or biofilm-like protective structure that are resistant to antibiotics^{23,24,29,30,31} and can be transformed back into normal spirochetes³². Despite having a functional cell wall and intact peptidoglycan, β-lactam antibiotics are ineffective against cystic forms due to low metabolic activity²³. The Cx₇C-O-BBB fusion constructs developed by us, particularly Bor-11, 16, 18, and 26, killed not only the spirochetes, but also significantly inhibited the cystic form (Fig. 4). Doxycycline, on the other hand, was unable to inhibit cystic Borrelia in our study (Fig. 4, panel C), consistent with earlier findings²⁵. We presume that the inhibition of cystic Borrelia was caused by membrane destabilisation, as evidenced in depolarisation and propidium iodide assays (Fig. 5, panels C,D). Membrane depolarisation and permeability breach is one of the key modes of action for AMPs (reviewed by Epand and Vogel³³). Interestingly, the destabilisation of the borrelial cell membrane was within 1 h (Fig. 5, panel D), shorter than melittin (one of the widely studied natural AMPs), which destabilises the bacterial cell wall in roughly 75 min³⁴.

AMPs have drawn attention as an alternative strategy to combat antibiotic-resistant bacteria, including cystic forms, as they can destroy dormant forms and bacteria rarely develop resistance to AMPs. Natural cationic antibacterial peptides show high affinity for the negatively charged surfaces of Gram-negative bacteria, however many of them are predicted to be toxic^35,36. The Antimicrobial Peptide Database³⁶ lists nearly 3200 AMPs that are not approved for therapeutic purpose due to their cell toxicity^37,38,39. Previously it was shown that the antimicrobial activity of short cationic peptides is strongly dependent on the net charge, wherein a higher antibacterial activity was displayed by peptides with a net charge from + 2 to + 4, however at the same time peptides endowed with a greater positive charge were more toxic to human erythrocytes⁴⁰. This clearly suggests that an AMP molecule needs proper balance between a net charge and antimicrobial activity. In addition to these features, the conformation of peptide, primarily their linear versus cyclic nature, should be taken into account. We used a combinatorial phage library that displays cyclic peptides because structurally constrained molecules have several advantages over linear molecules. The cyclic peptides are more resistant to proteolytic degradation under physiological conditions, particularly in serum⁴¹. The stability of our cyclic Bor peptide was demonstrated by the fact that the binding ability of all tested peptides remained constant even after 6 h of incubation in normal human serum. Another reason for exploiting cyclic peptides against Borrelia is that their binding to bacterial cells is governed not only by electrostatic interaction (a higher positive charge), but also by molecular interactions based on the peptides’ conformation. With a net charge of + 0.9 (well below the + 2 to + 4 range), all four Bor peptides (Bor-11, 16, 18, and 26) demonstrated strong binding affinities to both the spiral and cystic forms of Borrelia. In particular, Bor-18 showed binding affinity to Borrelia at concentration as low as 0.16 µM (Fig. 5, panel A). It has previously been demonstrated that cyclic peptides containing cysteine residues have a higher binding affinity for the target than their linear counterparts⁴². The third reason for exploiting the potential of cyclic peptides was their low cytotoxicity⁴³. In line with previous finding, none of our peptides inhibited cell metabolism in the XTT assay, hindered cell proliferation, or caused hemolysis (Fig. 6).

Several synthetic and phage display library-derived AMPs have been generated so far, especially against antibiotic-resistant bacteria. One notable AMP, EC5, was able to permeabilise the outer membrane of E. coli and P. aeruginosa within 5 min of treatment⁴⁴. EC5, like the Bor peptides produced by us, caused depolarisation of the cytoplasmic membrane, most likely through membrane-lipid interaction. Another AMP, comprised of 10 amino acids QEKIRVRLSA, was effective against several multidrug-resistant members of the Enterobacteriaceae family⁴⁵. Many of the AMPs developed to date are from phage libraries displaying longer peptide, such as the 12-mer linear peptide library, which is available commercially (Ph.D.-12 from New England Biolabs). It was used to isolate AMPs against Listeria monocytogenes and E. coli^46,47, while the 15-mer library was used to develop AMPs against Haemophilus influenzae and Campylobacter jejuni^48,49. The combinatorial library used in our study was composed of only 7-mers flanked by cysteine residues. Several researchers prefer libraries displaying more than 10 amino acids in order to increase sequence diversity of the library and increase the likelihood of obtaining a better peptide candidate with the desired antibacterial capabilities.

Many therapeutic peptides and proteins discovered to date have proven ineffective in treating CNS infections. Poor transportation of the peptides across the BBB, as well as inadequate pharmacokinetics, are significant barriers to this progress. Thus, while developing the recombinant AMPs against Borrelia, we sought to improve their transport across the BBB. The presence of spirochetes and their cystic forms in the cerebral cortex of a patient with chronic neuroborreliosis⁷ emphasizes the need of AMPs that can cross the BBB. To date, several CNS homing peptides have been identified. Some are derived from human proteins that interact with brain microvascular endothelial cell receptors^50,51,52, while others are derived from combinatorial libraries^{53,54,55,56,57}, or pathogen-expressed proteins that help to evade the microvasculature^58,59,60,61. The OspA protein of Borrelia is known to bind endothelial and epithelial cells via distinct domains (Fig. 2, panel A). In particular, brain microvascular endothelial cell binding domain of OspA of neuroinvasive Borrelia binds to the cell receptors and evokes borrelial translocation, mimicking the events that occur during RME²⁰. We have also shown that blocking of this domain significantly reduces borrelial translocation across the BBB²⁰. Based on this knowledge, we wanted to develop CNS homing peptide that could be fused with Cx₇C for better transportation of antibacterial moiety through the BBB. The endothelial cell binding domain of OspA is composed of seven antiparallel beta sheets. Results from present study show that N-terminal region encompassing 10th and 11th beta sheets (designated as O-BBB) was able to cross the barrier readily than the C-terminal region that consists of 12th to 16th beta sheets²¹ (supplementary information 3).

There are several advantages for selecting O-BBB as a shuttle molecule, such as: (1) It contains no cysteine residue, which ensures no interference in the correct cyclisation of AMP mediated by the C–C disulphide bond; (2) It contains 85.79% hydrophilic amino acids (PEPTIDE 2.0 server), which could facilitate better solubilization of the final fusion construct as well as better production in the E.coli expression system; (3) The net charge of O-BBB is predicted to be -3.4 at pH 7.0, which may help in reducing the nonspecific electrostatic interaction to the negatively charged domains of eukaryotic and borrelial cell membrane and thus maintaining the specific interaction of Cx₇C with borrelial phosphatidylcholine; and (4) Reducing the net charge may also help to reduce toxicity. We also assumed that angiopep-2 (TFFYGGSRGKRNNFKTEEY), despite being much smaller than O-BBB, has a net charge of + 2 at pH 7, which could result in non-specific electrostatic interaction of the fusion peptide with endothelial and borrelial cell membrane. With all these advantages, Cx₇C fused with O-BBB exhibited ability to cross the BBB model significantly higher than the doxycycline (Fig. 7 panel C).

Conclusion

Using a combinatorial phage display library and phospholipase D for specific elution of peptides that bind to the borrelial membrane, we isolated and idenfied new anti-Borrelia peptides. Combining the potential of AMPs with a CNS homing peptide, we produced recombinant molecules that can cause destabilisation of the borrelial cell membrane within a1 hr, which results in a borreliacidal effect. Bor-11, Bor-16, Bor-18, and Bor-26 exhibited cidal activity against both spirochetal and cystic forms. Four peptides had no deleterious effect on eukaryotic cell metabolic activity or cell proliferation, and caused no lysis of sheep red blood cell. Bor peptides’ serum stability and ability to retain borreliacidal activity even after crossing the BBB model make them attractive molecules for the developing novel borrelicidal drug, especially against neuroborreliosis.

Materials and methods

Borrelia garinii culture and Borrelia with GFP epifluorescence

Neuroinvasive B. garinii (strain SKT-7.1, Serotype 4, GenBank accession number - GU906888.1) was cultured in complete BSK-II medium (Sigma Aldrich, USA) enriched with 6% rabbit serum at 33 °C for approximately 2 weeks to achieve early log phase. Cultures were examined under dark field microscopy (40x magnification) to assess the shape and motility of Borrelia. Early log phase was defined when borrelial count was approximately 4 × 10⁶/ml and 99% Borrelia were seen as individual bacteria (no aggregations and cysts) under the dark field microscopy.

SKT-7.1 expressing GFP was prepared as described before⁶². Shortly, pTM61 plasmid harboring constitutively expressing green fluorescent protein (GFP) under the control of the B. burgdorferi flaB promoter (P_flaB), determinants for replication in both E. coli and B. burgdorferi, and gentamycin resistance (aacC1) cassette was electroporated in electrocompetent SKT7.1 exactly as described previously⁶³. SKT7.1 expressing GFP was selected by adding 100 µg/ml of gentamycin. The presence of GFP was checked with epifluorescence microscopy.

To obtain the cystic form, culture in early log phase was diluted in fresh BSK-II medium without serum (1:1 V: V) and incubated for 2 weeks at 15 °C. Formation of cysts and absence of spiral form was checked with epifluorescence microscopy (Supplementary information 1).

Counting of Borrelia using flow cytometry

50 µl of culture was mixed with 150 µl of PBS (containing 1x SYBR Green I; Molecular probes, USA) and used to enumerate bacteria using CytoFLEX flow cytometer (Beckman Coulter, USA). For Borrelia with GFP fluorescence, the SYBR green was omitted. Conditions for acquisition of data were: Volume measurement – 15 µl, Flow rate – medium (30 µl/min). Bacteria were differentiated from debris by adjusting the threshold (FSC 10 000/SSC 10 000) and polygonal gate (SSC vs. FITC) as shown in supplementary information 1.

To count cysts, all conditions were similar as described above; however different gates were applied to differentiate cystic form from the spirochetal Borrelia as shown in supplementary information 1.

Shaving of Borrelia with trypsin: Borrelia devoid of outer surface proteins

50 ml of Borrelia growing in log phase were centrifuged (4000 rpm for 10 min), washed with Dulbecco’s phosphate-buffered saline (DPBS; KCl 200 mg/L, KH₂PO₄ 200 mg/L, NaCl 8,000 mg/L, Na₂HPO₄ 1150 mg/L) with 20% sucrose and then resuspended in DPBS containing 5 µg/ml of trypsin gold (Promega, Milan, Italy), keeping the solution at 37 °C, 10 min with gentle shaking. Borrelia were centrifuged at 6000 rpm for 10 min and the supernatant was discarded, the pellet was washed with DPBS to obtain Borrelia devoid of surface proteins.

Selection of phages displaying Cx7C peptides with affinity for the borrelial cell membrane

Twenty milliliters of Borrelia culture in early log phase were centrifuged (7000× g, 15 min, 22 °C), the pellet was resuspended in 1 ml of DMEM (Biowest, France) and 2 × 10¹¹ phages from Cx₇C combinatorial phage library were added (Ph.D.-Cx₇C; New England Biolabs, NEB, USA). Cysteine on both sides of Cx₇C peptides constrains the cyclic form, thanks to the disulfide bond. Phages were incubated for 2 h at 22 °C with constant shaking. Non-interacting phages were removed by centrifugation (12000× g, 8 min, 16 °C) followed by 5 times washing with 1 ml of PBS (12000× g, 8 min, 16 °C). Phages interacting with borrelial cell membrane phosphatidylcholine moiety were eluted by the addition of 60 units of phospholipase D (Sigma Aldrich, USA) diluted in Tris-buffered saline (TBS, pH 7.4) for 2 h at 37 °C followed by centrifugation at 12,000× g, 8 min, 22 °C. Note that, phospholipase D was used to elute phage particles bound on Borrelia, resulting in specific enrichment of Cx₇C with a high affinity for phosphatidylcholine. Phospholipase D hydrolyzes phosphatidylcholine to produce phosphatidic acid and soluble choline. Phosphatidylcholine is one of only two major membrane phospholipids, along with phosphatidylglycerol of borrelial membrane (Fig. 1, panel A¹⁴), . Phages in supernatant were amplified in E.coli ER2738 (NEB), purified, and quantified by titration exactly as described in our publication⁶⁴ and used in the second round of borrelial cell membrane specific phages selection (panning). During the second panning, phages were negatively panned against plasticware, culture medium proteins, and BMECs to rule out enrichment of non-specific phage clones. In brief, phages (2 × 10¹⁰ phages resuspended in 100 µl of PBS) were incubated in an empty tube for 1 h with constant shaking at 37 °C. The phage solution was then transferred to a tube coated with BSK-II medium (coating is achieved by incubating BSK-II medium at 4 °C overnight) and incubated for 1 h with constant shaking at 37 °C. The solution containing phages was then transferred to the BMECs fixed in an ELISA plate well and incubated for 1 h at 37 °C with constant shaking. After that, phage-containing solution (negatively panned phages) was used in the second round of panning against Borrelia. The second round of panning was carried out while maintaining all of the conditions described for the first round. The third round of panning was carried out maintaining the same settings as the second.

To confirm the binding affinity of phages (eluted after 3rd round of panning) to different forms of Borrelia, a dot blot was performed. Briefly, 2 µl of Borrelia in early log phase or cystic form (washed and resuspended in PBS, OD₆₀₀ 1.2) or shaved with trypsin were spotted on an activated PVDF membrane, air dried and 10 µl 4% paraformaldehyde was added on the spot. After 10 min membrane was washed with PBS, dried overnight and incubated in blocking solution (LI-COR Biosciences, USA) for 1 h. Membrane was then incubated with 10¹¹ phages resuspended in blocking solution for 1 h with constant shaking. Membrane was washed 5 times with PBS containing 0.05% Tween 20 (PBS-T) each for 5 min. Bound phages were detected with anti-M13 pIII monoclonal antibody (1:1000, 1 h; NEB) and by donkey anti-mouse antibody conjugated with IRDye 770 (1:15000, 1 h; LI-COR Biosciences). Signal was captured at 800 nm (Odyssey CLx, LI-COR Biosciences). For negative control, phages were omitted from the reaction. For input control, phages were spotted on the membrane and detected by primary and secondary antibody as described above.

To separate phage clones, 0.5 µl of phage elute obtained from 3rd panning was mixed with 200 µl of E. coli ER2738 culture (OD₆₀₀), incubated for 5 min, mixed with molten agar (50 °C) and overlaid on LB agar plates containing 1 mM IPTG (Fermentas, Lithuania) and 1 mM X-gal (Sigma, USA). After overnight incubation, 30 plaques were separately picked and individual phage clones were amplified in 1 ml of E. coli ER2738 culture following the manufacturer´s protocol (NEB, USA). Genomic DNA from each clone was isolated by heating at 95 °C for 10 min and subjected to amplification of fragment encompassing sequence of 7-mer peptide flanked by cysteine residues (designated as Cx₇C) and C-terminal GGGS. Primer sequences and PCR conditions are presented in (Supplementary Table 2). Amplicons were gel purified and sequenced (supplementary information 2). As a positive control, DNA isolated from Cx₇C library was used. The amino acid sequences of Cx₇C peptides were deduced from nucleotide sequences and grouped based on sequence homology (Geneious v9.1, Biomatters, USA).

In silico analysis of identified peptide sequences was performed to deduce AMP probability using Antimicrobial Peptide Scanner.2 (https://www.dveltri.com/ascan/v2/ascan.html), net charge identification, and water solubility (https://pepcalc.com). A possible match of identified sequences with the peptide uploaded to the public repository was checked with the peptide search tool of the UniProtKB database (https://www.uniprot.org/uniprotkb). Sequences were virtually scanned by ToxinPred (http://crdd.osdd.net/raghava/toxinpred/) to reveal the possible toxicity of peptides.

Individual phage clones were tested for binding affinity to Borrelia using dot blot as described above.

BBB homing peptide

Two truncated forms of OspA were produced (supplementary information 3) encompassing 10–11 and 12–16 beta sheets, exactly as described previously²¹. Both truncated forms and angiopep-2 (used as control as it is known BBB homing peptide, BOC Sciences, China) were labelled with Cyanine5.5 NHS ester (Lumiprobe, USA) following the manufacturer’s protocol. In short, required amount of Cyanine5.5 NHS ester was calculated based on formula: Amount of NH ester (mg) = 8 x amount of peptide (mg) x NH ester molar weight (Da) / molar weight of peptide (Da). The calculated amount of Cyanine5.5 NHS ester was incubated with 0.5 mg of peptides overnight at 4 °C. After incubation dialysis against PBS was performed overnight at 4 °C to remove unbound dye, and the concentration of conjugated peptide was determined by Bradford assay.

Construction of BBB model in-vitro

BMECs were cultured exactly as described in our earlier publication⁶⁵. Briefly, 1 × 10⁶ BMECs (cell line D3; Merck Millipore, USA) were seeded in a collagen-I (Corning, USA) coated T-75 cell culture flask (Sarstedt, Germany) in the complete endothelial medium at 37 °C, 95% humidity and 5% CO₂ until confluence. The complete endothelial medium contains DMEM-F12 (Sigma-Aldrich, USA) supplemented with 1.4 µM hydrocortisone (Sigma-Aldrich), 10 ng/ml bFGF (Sigma-Aldrich), 5 mg/ml ascorbic acid (Sigma-Aldrich), 1x penicillin-streptomycin (Jena Bioscience, Germany), 10 mM HEPES (Biowest, France), 2 mM L-glutamin (Life Technologies, USA) and 10% fetal bovine serum (Biowest).

BBB model was cultured on cell-inserts. In brief, 5 × 10⁵ BMECs were seeded in the luminal chamber of collagen-I coated 24-transwell Inserts (1 μm pores; cellQuart, Germany, transwell system depicted in supplementary information 4) containing 600 µl of complete endothelial medium in both luminal and abluminal chambers. The inserts were incubated at 37 °C in humidified CO₂ incubator until a monolayer was formed. The formation of the monolayer was verified by measuring the transendothelial electrical resistance (TEER) between the luminal and abluminal chamber of inserts⁶⁶. TEER was measured by pair of chopstick electrodes on a volt ohm meter EVOM (World Precision Instrument, USA). The resistance is inversely proportional to the growth area of the semipermeable membrane (cm²). The TEER value (Ω/cm²) was calculated using the formula:

$$ \left( {Resistance_{{insert}} {-}Resistance_{{blank}} } \right){\text{ }}x{\text{ }}growth{\text{ }}area{\text{ }}of{\text{ }}the{\text{ }}insert{\text{ }}\left( {0.3{\text{ }}cm^{2} {-}24{\text{ }}well{\text{ }}transwells{\text{ }}have{\text{ }}0.3{\text{ }}cm^{2} area} \right). $$

Wherein:

Resistance_{insert =}recorded resistance value between luminal and abluminal chambers of inserts in which the BMEC monolayer is fully grown.

Resistance_{blank =}recorded resistance value of semipermeable membrane in transwell without cells (only collagen coated).

When the observed TEER value was ~ 100 Ω/cm², the barrier properties were confirmed by diffusion of CF 770 dextran⁶⁷. Briefly, 0.1 µg/ml of CF 770 dextran was added to the luminal chamber of each insert, and its passage through the BBB model was recorded by scanning the contents of the abluminal chamber at 800 nm on Odyssey CLx Imaging System (LI-COR) at 1, 3, and 5 h. The concentration of CF 770 dextran in the abluminal chamber was determined by a standard curve prepared by a two-fold dilution of 0.2 µg/ml of CF 770 Dextran. Barrier properties were confirmed when the amount of CF 770 dextran that passed from the luminal to the abluminal chamber was lower than 6% of the amount added to the luminal chamber. Inserts with confirmed barrier properties (by both TEER and dextran) were used to test the passage of Cy5.5 labelled truncated fragments or angiopep-2-Cy5.5.

Assessment of crossing of labelled truncated fragments or angiopep-2 across BBB model

1 µg/ml of each of the truncated fragment labelled with Cy5.5 or angiopep-2-Cy5.5 or Dextran CF 770 (Biotium, USA) was added in the upper chamber (luminal) of the BBB model and incubated at 37 °C in cell culture incubator. Crossing of truncated fragments from the luminal to the abluminal chamber was measured by measuring the fluorescence at 1, 2, and 3 h of incubation. Fluorescence was measured on Odyssey CLx using wavelengths: 650/705 nm (for Cy5.5 labelled molecules) and 770/797 nm (for dextran CF 770). The concentration of each component in the abluminal chamber was calculated using standard curves. The percentage of crossing of BBB was calculated. The assay was performed in triplicate.

The N-terminal fragment of endothelial cell binding domain (O-BBB, NEKGETSEKTIVRANGTRLEYTDIKSDG), which demonstrated significant BBB crossing, was used to fuse with Cx₇C. The tertiary structure of the O-BBB was predicted on ColabFold, which uses AlphaFold2⁶⁸.

Synthesis of Cx7C fused with O-BBB

DNA encoding Cx₇C was amplified from representative phage clone from each cluster that showed affinity to Borrelia. Primers used to amplify Cx₇C fragment are listed in (Supplementary Table 2). DNA amplification, cloning into pQE-30 N-tag-II vector, electroporation in E. coli T5 SHuffle, clonal selection, protein overexpression and purification, assessment of purity by LDS-PAGE and MALDI-TOF-MS (Bruker Daltonics; Germany) were performed exactly as described in our methodological paper⁶⁹. pQE-30 N-tag-II vector allows N-terminal fusion of O-BBB tag to Cx₇C peptides. The fusion constructs consist of O-BBB at N-terminus of the Cx₇C, flanked by GGGS at both ends (Fig. 2, panel D). The GGGS between O-BBB and Cx₇C was placed to avoid any steric hindrance among two molecules, while GGGS at C-terminus was added to keep sequence similarity inherited from Ph.D.-Cx₇C library (details are available in user manual from New England Biolabs, catalog number E8212S). Nine different Cx₇C sequences were fused with O-BBB. These sequences were chosen based on the phage clones that exhibited binding affinity to Borrelia in the dot blot assay described above. Fused peptides were designated as Bor-1, Bor-8, Bor-9, Bor-11, Bor-16, Bor-18, Bor-23, Bor-26 and Bor-29.

The tertiary structures of the O-BBB fused Cx₇C were predicted on ColabFold, which uses AlphaFold2 ⁶⁸.

Assessment of binding of Cx₇C fusion peptides (bor peptides) to Borrelia

Fifty microliters of the Borrelia (washed and resuspended in PBS, OD₆₀₀ 0.3) were spotted in a 96-well plate, air dried overnight at room temperature, and fixed with 50 µl of 4% paraformaldehyde for 10 min. Nonspecific binding sites were blocked with 100 µl of 5% BSA in PBS-T and 2 µg of Bor peptides dissolved in 100 µl of PBS-T containing 1% BSA were added. After 1 h of incubation, wells were washed 5 times with 100 µl of PBS-T. Bor peptides bound on Borrelia were detected with HisProbe-HRP (1:1000 in PBS-T containing 0.5% BSA, Thermo Scientific, incubation 1 h at room temperature) and 1-Step Ultra TMB-ELISA substrate (100 µl/well, 25 min at room temperature, Thermo Scientific). 100 µl of 2M H₂SO₄ was added and readings were taken at 450 nm on ELISA plate reader (PerkinElmer, Victor X3, USA). The assay was performed in triplicates. For negative controls, either Borrelia or peptides were omitted from the assay. For input control 2 µg of Bor peptides were coated in well and detected by HisProbe-HRP and TMB. O-BBB was also included in the assay to see if it shows any non-specific binding to Borrelia.

Assessment of borreliacidal activity of peptides

The number of Borrelia in culture (in early log phase) was enumerated using by C6 flow cytometer exactly as described previously⁷⁰. 2 × 10³Borrelia were inoculated in 200 µl of fresh BSK-II medium containing Bor peptides or only O-BBB at varying concentrations (6.67, 3.33, 1.67 and 0 µM) and incubated at 33 °C for 5 days. After incubation, the number of Borrelia in the culture were enumerated by flow cytometry. Percent inhibition of Borrelia was calculated by taking into account the number of Borrelia in medium containing 0 µM of peptide. Doxycycline (50 µM to 5 nM) was used in the assay as a positive control. The Bor peptides with significant borreliacidal activity at 1.67 µM were tested further at lower concentrations (0.83, 0.41, 0.20, and 0.10 µM) while maintaining all other experimental parameters.

Assessment of inhibition of conversion of cystic form in to spirochetal form

Culture containing cysts was observed under the fluorescent microscope to check presence of any spirochetal form (Supplementary information 1). Borrelial cysts were counted using a C6 flow cytometery (Supplementary information 1). 2 × 10³ cysts were incubated with Bor peptides at different concentrations (two-fold serial dilutions from 3.33 to 0.05 µM) in 200 µl of BSK-II medium devoid of rabbit serum for 16 h. Please note that absence of serum maintains the cystic form of Borrelia and are unable to convert readily in to spirochetal form. After incubation cultures were centrifuged at 6000 RPM for 15 min. Supernatant containing peptide was discarded and 200 µl of fresh complete BSK-II medium (containg rabbit serum) was added into the same tube and incubated for 15 days at 33 °C. In this assay, it was necessary to remove peptides after 16 h as presence of peptides in culture may inhibit newly formed spirochete in complete BSK-II medium, which may interfere the assessment of inhibitory effect of peptides exclusively on cyctic form. Doxycycline (50 nM) was used as a control, and the assay was carried out exactly as with Bor peptides. As a live control, the peptides were replaced by PBS. The enumeration of Borrelia in each treatment and controls was performed as described above. Percent inhibition of conversion of cystic form in to spirochetal form was calculated taking into account number of Borrelia present in PBS control.

Assessment concentration dependent binding of Bor-11, Bor-16, Bor-18 and Bor-26 to spirochetal form of Borrelia

ELISA was used to determine the concentration-dependent binding of Bor peptides to Borrelia. Borrelia growing in the early log phase were fixed to the microtiter wells with paraformaldehyde exactly as described above. All steps in ELISA were identical to those described above, with the exception of the peptide concentrations used in the assay ranging from 2.6 µM to 86 nM (two-fold serial dilution). Peptides were omitted from the assay as negative controls.

Assessment binding of Bor-11, Bor-16, Bor-18 and Bor-26 to different forms of Borrelia

Borrelia growing in the early log phase, its cystic form, and Borrelia devoid of surface proteins were fixed to the microtiter wells with paraformaldehyde. All steps in ELISA were identical to those described above, with the exception of the peptide concentration, which was 2 µg/well. Peptides were omitted from the assay as negative controls. As a control, an antibody raised against OspA (Abcam, UK) was used in the assay.

Evaluation of the effect of peptides on borrelial membrane

For detection of the borrelial membrane depolarization caused by the peptides, previously published protocol⁷¹ was adopted with minor changes. 10 ml of Borrelia (non-GFP) culture was centrifuged, the pellet was washed with PBS and resuspended in PBS to obtain OD₆₀₀ 1.0. 10 µl of this suspension was added to each well of the 96-well plate and mixed with 165 µl of PBS, 25 µl of 1 M glucose, and 5 µl of 5 µM DiBAC₄(3) dye (Thermo Scientific). Immediately, Bor peptides (6.67 µg) were added to each well. The plate was immediately placed into a time-lapse imaging reader (Cytation 7, BioTek, Germany) with preset filters for DiBAC₄(3) detection (490 nm_Ex and 516 nm_Em). The measurement was carried out every 2 min during 4 h and data were processed using Gen5 V3.11.

To access effect of Bor peptides on membrane permeability a protocol published previously⁷² was modified. In brief, 10 ml of Borrelia (non-GFP) culture was centrifuged, the pellet was washed with DPBS and resuspended in DPBS to obtain OD₆₀₀ 1.0. 10 µl of this suspension was added to each well of a 96-well plate and propidium iodide (30 µM; ThermoFisher Scientific, United States) was added to each well. Bor peptides (6.67 µg) were added to each well immediately. One well was retained without the addition of any peptide to form a negative control. Whereas, Triton X-100 (0.1%, Sigma-Aldrich, Germany) was added in positive control well. Fluorescence of PI was measured on Cytation7 (BioTek, USA) every 2 min until 2 h at 535 nm_Ex and 617 nm_Em.

Assessment of cell toxicity (XTT assay)

HEK293T cells were grown in a 96-well culture plate (Sigma Aldrich) in DMEM high glucose medium (Thermo Scientific) enriched with 2mM L-glutamine (Sigma Aldrich) and 5% fetal bovine serum (BioWest) at 37 °C with 5% CO₂ until 70% confluence. Cells were either used for XTT or in cell proliferation assay.

Cell metabolism was determined through XTT (AppliChem, Germany) following the manufacturer’s instructions as described in our earlier publication⁷³. Briefly, HEK293T cells were cultivated in 96-well plate. At 70% confluence, the cells were added with peptides (Bor-11, 16, 18 and 26) at various concentrations (3.33 and 0 µM). Cells were incubated for 24 h at 37 °C and 5% CO₂. After incubation, 50 µl XTT reagent was directly added, incubated for 3 h, and measured at 450 nm. For controls, peptides were either replaced by PBS or Triton X (0.1%). The assay was conducted in six replicates. Metabolic activity was calculated using the formula: $\left[ {\left( {{\text{absorbanc}}{{\text{e}}_{{\text{sample}}}} - {\text{ absorbanc}}{{\text{e}}_{{\text{blank}}}}} \right){\text{ }} \times {\text{ 1}}00} \right]/\left( {{\text{absorbanc}}{{\text{e}}_{{\text{negative control}}}} - {\text{ absorbanc}}{{\text{e}}_{{\text{blank}}}}} \right)$. Here PBS served as negative control.

Cell proliferation assay

For proliferation assay, peptides (Bor-11, 16, 18, and 26) at varying concentrations (3.33, 1.67, and 0 µM of peptides diluted in complete DMEM medium) were added on the freshly seeded HEK293T in 96-well plate. Cells without peptides served as growth control. Cells were incubated at 37 °C in a humidified CO₂ incubator for 96 h and cell counting was performed at every 24 h with high contrast bright field microscopy on Cytation7 imaging system (BioTek, USA). Cell enumeration and cell morphology were documented with Gen Image 5 ver 3.11 software. Technical parameters are presented in https://www.agilent.com/cs/library/applications/automated-kinetic-imaging-assay-cell-proliferation-5994-2593EN-agilent.pdf). Correlation between the number of cells in treatments and negative control was determined by Pearson correlation coefficient (r)⁷⁴.

Hemolysis assay

Bor-11, 16, 18, and 26 peptides were evaluated for their hemocompatibility using the method described earlier⁷⁵. In brief, 30 ml of healthy sheep blood was collected in the presence of 2500 IU heparin (Zentiva, Slovakia) by licensed veterinarian at the animal rearing house of the University of Veterinary Medicine and Pharmacy, Košice. The blood was centrifuged at 657 × g for 5 min and the separated erythrocytes were washed three times with 0.9% saline and dispersed in 150 ml of 0.9% saline. Erythrocyte suspension (1 ml) was incubated with peptides at varying concentrations (300, 100, and 50 ng) for 1 h, 3 h, and 5 h at 37 °C. 1 ml of erythrocyte suspension either mixed with 2% Triton X-100 (Sigma) or 0.9% saline serving as positive and negative controls, respectively in the assay. All the samples were gently mixed every 30 min. At the end of incubation, the samples were centrifuged (657 × g for 5 min) and the supernatant was collected in new tubes and allowed to oxidize hemoglobin at room temperature. The absorbance of oxyhemoglobin was measured at 414 nm. Hemolysis was calculated using the formula: % hemolysis = (absorbance_sample - absorbance_{negative control}) / (absorbance_{positive control} - absorbance_{negative control}) x 100. The entire assay was performed in triplicates.

Binding of Bor peptides to endothelial cells

The ability of Bor peptides to bind to endothelial surface was tested by cell-ELISA. In short, the monolayer of the BMECs cultured in 96 wells was washed once with PBS and fixed with 4% paraformaldehyde for 10 min. Blocking of nonspecific binding sites, incubation of 2 µg of Bor peptides with cells, and detection of bound peptides with HisProbe-HRP and TMB-ELISA substrate were performed as ELISA assay described above. The assay was performed in triplicates. For negative controls, either cells or peptides were omitted from the assay. O-BBB was used as a control.

Serum stability of the Bor peptides

To test the stability of peptides in serum, 1.67 µM of each Bor peptide was incubated with an equal volume of human serum (complement inactivated at 56 °C for 30 min) for 1, 3, and 6 h. After each time point, the mixture was transferred to BSK-II medium containing 2 × 10³Borrelia (in early log phase). For control reactions, peptides were replaced either with doxycycline (50 nM), or PBS, or heat inactivated normal human serum (equal volume). The cultures were incubated for 5 days at a total volume of 200 µl. Following incubation, the number of Borrelia in each treatment was measured using flow cytometry, as described before. Assay was performed in triplicates.

Crossing of Bor peptides through in-vitro BBB model and borreliacidal effect after crossing the barrier

A BBB model was cultured on 24-well cell inserts, as described above. Borrelia in early log phase were counted using flow cytometry, and 2 × 10⁴ bacteria were inoculated into the abluminal chamber of cell inserts. After 60 min (37 °C, 5%, CO₂), 4.1 µM of each peptide (approximately 6.25 µg) diluted in 200 µl of complete endothelial medium was added in each luminal chamber. 500 ng of CF770 Dextran was also added into the luminal chamber. As a negative control, peptides were replaced by PBS and as positive control doxycycline (0.11 µg/ml) was added to the luminal chamber. Please note that throughout the experiment, the luminal chamber contained 400 µl of the medium, while abluminal chamber had 600 µl. Medium was devoid of penicillin and streptomycin standardly used in cell culture. Inserts were incubated for 5 h (37 °C, 5%, CO₂), and TEER was measured to determine whether the BBB integrity was compromised during the incubation. Abluminal content was scanned at 800 nm to measure % crossing of dextran. The abluminal content (500 µl) of each insert was transferred to a 0.5 ml sterile tube and incubated for 6 days at 37 °C and 5% CO₂. The borrelicidal effect was determined by measuring the number of Borrelia grown in the tube using flow cytometry (CytoFLEX, Beckman Coulter, US), as previously described.

Data availability

Data generated in this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank Ing. Martina Cepkova and Ing. Viera Rusnakova for their technical assistance. Research was funded by grants APVV-22-0084, APVV-18-0259, VEGA1/0348/22 and EURONANOMED2021-105.

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Laboratory of Biomedical Microbiology and Immunology, The University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 04181, Košice, Slovakia
Evelína Mochnáčová, Katarína Bhide, Katarína Kucková, Jana Jozefiaková, Tomáš Maľarik & Mangesh Bhide
Institute of Neuroimmunology of Slovak Academy of Sciences, 84510, Bratislava, Slovakia
Mangesh Bhide

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Evelína Mochnáčová
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Katarína Bhide
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Katarína Kucková
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Jana Jozefiaková
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Contributions

MB and EM designed experiments. EM and MB performed phage display and panning. EM and JJ overexpressed Bor peptide and purified. SDS-PAGE, MALDI and other quality control of Bor was performed EM. EM, KK, KB and JJ performed ELISA, XTT, proliferation and hemolysis assays. EM and KK performed Borrelia culturing. KB and MB constructed BBB model in-vitro and performed translocation assays for dextan, angiopep-2, and various OspA truncated forms. MB performed protein modeling. EM performed membrane depolarization assay, time to kill assay and EC50 assays. KB, TM, JJ, KK and MB performed assays for the crossing of Bor across BBB model followed by borreliacidal test. Statistical and bioinformatic analyses were performed by EM and MB. MB and EM prepared the manuscript. All authors approved the final version of the manuscript.

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Correspondence to Mangesh Bhide.

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Mochnáčová, E., Bhide, K., Kucková, K. et al. Antimicrobial cyclic peptides effectively inhibit multiple forms of Borrelia and cross the blood-brain barrier model. Sci Rep 15, 6147 (2025). https://doi.org/10.1038/s41598-025-90605-z

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Received: 02 December 2024
Accepted: 14 February 2025
Published: 20 February 2025
DOI: https://doi.org/10.1038/s41598-025-90605-z

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