Genomewide Analysis and Biological Characterization of Cathelicidins with Potent Antimicrobial Activity and Low Cytotoxicity from Three Bat Species

Cathelicidins are potent antimicrobial peptides with broad spectrum antimicrobial activity in many vertebrates and an important component of the innate immune system. However, our understanding of the genetic variations and biological characteristics of bat cathelicidins is limited. In this study, we performed genome-level analysis of the antimicrobial peptide cathelicidins from seven bat species in the six families, listed 19 cathelicidin-like sequences, and showed that the number of functional cathelicidin genes differed among bat species. Based on the identified biochemical characteristics of bat cathelicidins, three cathelicidins, HA-CATH (from Hipposideros armiger), ML-CATH (from Myotis lucifugus), and PD-CATH (from Phyllostomus discolor), with clear antimicrobial signatures were chemically synthesized and evaluated antimicrobial activity. HA-CATH showed narrow-spectrum antibacterial activity against a panel of 12 reference bacteria, comprising 6 Gram-negative and 6 Gram-positive strains. However, ML-CATH and PD-CATH showed potent antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria with minimum inhibitory concentration (MIC) of 1 and 3 μg/mL, respectively, against Staphylococcus aureus. ML-CATH and PD-CATH also showed antifungal activities against Candida albicans and Cryptococcus cuniculi with MIC of 5 to 40 μg/mL, respectively, and 80% inhibition of the metabolism of Mucor hiemalis hyphae at 80 μg/mL, while displaying minimal cytotoxicity to HaCaT cells. Taken together, although the spectrum and efficacy of bat cathelicidins were species-dependent, the antimicrobial activity of ML-CATH and PD-CATH was comparable to that of other highly active cathelicidins in vertebrates while having negligible cytotoxicity to mammalian cells. ML-CATH and PD-CATH can be exploited as promising candidates for the development of antimicrobial therapeutics.


Introduction
Chiroptera (bats) are the second largest order of mammals following rodents [1], and they possess unique biological features, such as laryngeal echolocation [2], vocal learning [3], the ability to fly [4], and exceptional longevity [5], that are the rarest among all mammals. Additionally, bats are known as reservoir hosts of many zoonotic viruses, including the Nipah virus [6], Ebola virus [7] and coronavirus [8]. With no observed pathological signs of the disease, they can better tolerate viral infections than most other mammals because of their adaptation to antiviral immune responses [9]. In particular, the similarity between severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the causative agent of current coronavirus disease 2019 outbreaks, and bat-borne coronavirus (Bat CoV RATG13) illuminated bats as special reservoirs for viral pathogens that cause

Identification of 27 AMP Family Genes via the In Silico Analysis of R. ferrumequinum Genome
To understand the genetic characteristics of the AMP subgenome in bats, we conducted in silico identification of classical AMP encoding genes from the reference genome (mRhiFer1_v1.p, GCF_004115265.1) of R. ferrumequinum as a representative species. We identified a total of 27 putative AMP genes from R. ferrumequinum, including three alphadefensins, 21 beta-defensins, one cathelicidin, one hepcidin, and one LEAP-2, using National Center for Biotechnology Information (NCBI) genome blast with query coverage > 50% and e-value < 0.001 (Table S1). The number of AMP genes identified from our analysis was consistent with the gene numbers reported in R. ferrumequinum for each AMP family in the NCBI protein database without finding additional AMP genes.

Differences in the Number of Functional Cathelicidin Genes among Seven Bat Species Belonging to Different Families
To determine the number of functional cathelicidin genes in bat genomes, 64 unique cathelicidin protein sequences from 36 vertebrate species were blasted using the tblastn mode against the genomes of seven different bat species, R. aegyptiacus, H. armiger, M. natalensis, M. lucifugus, R. ferrumequinum, P. discolor and D. rotundus. Consequently, 12 cathelicidin-like sequences were identified in the analysis with query coverage > 50% and e-value < 0.001 (Table S2). In addition, BLASTP analysis using the same queries against nonredundant protein sequences of each bat species (taxid:9407, 186990, 291302, 59463, 59479, 89673, and 9430) resulted in the identification of 19 matches of cathelicidin-like sequences (Table S3). Further analysis showed that the difference in the number of matches was due to the discrepancy in the annotation of cathelicidin genes between the current genome assemblies and available sequences of bat cathelicidins in the NCBI protein database (Table S4). When the cathelicidin sequences of bats in public databases were taxonomically classified, the number of cathelicidin genes varied across bat species. A single cathelicidin-like gene has been detected in the genomes of H. armiger (LOC109375616) and M. natalensis (LOC107529421). Two putative cathelicidin isoforms, XP_036075573.1 and KAF6473429.1, have been reported in R. aegyptiacus, but they seem to result from two different annotations of an identical gene. In D. rotundus, the antimicrobial activitycontaining region was predicted only from one (XP_024421797.1) of the two cathelicidinlike sequences (XP_024421797.1 and XP_024421798.1). Similarly, two cathelicidin-like sequences, KAF6312594.1 and XP_032988513.1, were encoded from an identical locus in R. ferrumequinum, and the structure of KAF6312595.1 lacks an exon. Therefore, five out of the seven species seem to have a single functional cathelicidin gene in their genomes, and more than one cathelicidin could be present in M. lucifugus and P. discolor considering the presence of multiple sequences with antimicrobial signatures, as described in Table S4.

In Silico Determination of the Antimicrobial Activity Core Regions of Three Bat Cathelicidins
We conducted an in silico prediction of the antimicrobial activity core region for the 19 bat cathelicidin-like sequences to define the signal peptide, conserved CLD domain, and mature peptide region using two different prediction tools, antimicrobial sequence scanning system (AMPA) and database of antimicrobial activity and structure of peptides (DBAASP) (Table S4). Consequently, five putatively functional sequences, KAF6473429.1, XP_019486615.1, XP_006108360.2, XP_035886276.1, and XP_028374415.1, with the complete pre-pro protein structure of cathelicidins and predicted presence of core antimicrobial activity regions were determined (Table S5). Among them, three predicted antimicrobial activity core regions corresponding to the cathelicidins of H. armiger (XP_019486615.1), M. lucifugus (XP_006108360.2) and P. discolor (XP_028374415.1) which were named as ∆HA-CATH, ∆ML-CATH, and ∆PD-CATH where "∆" indicate a deletion of several amino acids from the nascent mature peptide region, namely HA-CATH, ML-CATH, and PD-CATH, respectively, were selected and subjected to the analysis of biochemical features using Antimicrobial Peptide Database (APD3). The three antimicrobial activity core regions or short form peptide regions (∆HA-CATH, ∆ML-CATH, and ∆PD-CATH) consist of 18 to 25 amino acids with 2.08 to 2.93 kDa in molecular weight, whereas the entire mature or long form peptides, HA-CATH, ML-CATH, and PD-CATH, were composed of 35 to 40 residues with 3.98 to 4.64 kDa in molecular weight ( Table 1). The ratios of hydrophobic residues and net charges for the short-and long-form peptides were 31 to 50% and +3 to +6, and 30 to 40% and +4 to +8, respectively. Their amino acid sequence similarity to known cathelicidins was highest with cathelicidins from sheep (SMAP-29), dogs (K9CATH), frogs (Temporin-CPb, Palustrin-2CG1), and fish (TP4, HKPLP) and the level ranged from 38.64 to 51.22%.

Confirmation of the Antifungal Activity of Three Bat Cathelicidins
Six bat-cathelicidin peptides (∆HA-CATH, ∆ML-CATH, ∆PD-CATH, HA-CATH, ML-CATH, and PD-CATH) were evaluated for the presence of antifungal activity against three different fungal species, Candida albicans (C. albicans), Cryptococcus cuniculi (C. cuniculi), and Mucor hiemalis (M. hiemalis) ( Table 3; Figure 1). The antifungal effect of the yeast-type fungi, C. albicans and C. cuniculi, was estimated in a manner similar to the antibacterial activity method. However, the antifungal activity of M. hiemalis, a mold-type fungus, was estimated based on the inhibition of the metabolic rate against hyphae. The treatment of ML-CATH and PD-CATH showed effective antifungal activities against the two yeast forms fungi with MIC of 5 to 40 µg/mL (1.2 to 10.1 µM) and M. hiemalis with a similar rate of metabolic inhibition (~80%) to fluvastatin sodium. In addition, intermediate levels of antifungal activity were observed against C. albicans and C. cuniculi from the treatment of ∆PD-CATH which did not show antibacterial activity against our reference bacterial panel, indicating the inherently strong antimicrobial potency of PD-CATH compared to the other bat cathelicidins tested in this study and differences in the effect of ∆PD-CATH against bacterial and fungal cell walls. In addition, HA-CATH only showed slight antifungal activity (MIC = 45 µg/mL or 9.7 µM) against C. cuniculi, indicating limited activity against fungi which is consistent with the antibacterial activity of HA-CATH ( Table 2). The antifungal activity of the tested peptides seems to be higher for yeast than mold-type fungi. Consistent with the results of antibacterial analysis, ∆HA-CATH, ∆ML-CATH, and ∆PD-CATH did not show antifungal effects against the reference strains, except ∆PD-CATH against C. cuniculi.

Negligible Cytotoxicity of Bat Cathelicidins against Human Keratinocytes
The level of cytotoxicity is important for the therapeutic use of AMPs as antimicrobial agents. To estimate the cytotoxicity of HA-CATH, ML-CATH, and PD-CATH in mammalian cells, the viability of HaCaT cells was evaluated for each peptide ( Table 4). The results showed that no cytotoxicity was observed at 64 µg/mL concentration, and only less than 3% of cells were affected at 160 µg/mL for both HA-CATH and ML-CATH. For PD-CATH, the cytotoxicity was not observed at 64 µg/mL, but the cell viability was decreased to 10.4% at 160 µg/mL concentration, showing significant cytotoxicity at the high concentration. Therefore, the cytotoxicity on mammalian cells showed a positive correlation with the potency of bat cathelicidins. In addition, melittin, an AMP known to have a potent cell lytic activity, showed severe cell lysis, with 7.2% cell viability at 64 µg/mL concentration. These results indicate that the cytotoxic effect of HA-CATH and ML-CATH is at a minimal level, and given the effective bactericidal concentration of PD-CATH (3-30 µg/mL), the cytotoxicity is within the acceptable range for therapeutic use.

Discussion
Fifty genome sequences of diverse bat species with varying degrees of coverage are currently available in the public database (https://www.ncbi.nlm.nih.gov/genome, accessed on 22 June 2022). Considering the extreme phylogenetic diversity of bats, comparative analysis of their AMP genes may reveal interesting outcomes regarding the genetic and functional diversity of these molecules. The number of classical AMP genes of R. ferrumequinum was similar to that of naked mole rats when compared to other mammals (Table S6) [25,[44][45][46][47][48]. However, only limited information is available on the genetic and biological characteristics of bat AMPs, including cathelicidins. In addition, the results of our analysis to identify cathelicidin orthologs from the seven selected bat species showed few differences in annotations compared to the currently predicted protein sequences of cathelicidins in NCBI (Table S4), suggesting the need of more cDNA information, especially for isoform-like sequences. Cathelicidins are probably the most potent AMPs in mammals, with variations in their activities in cases of multiple cathelicidins within a species [49]. Although few studies reported bioinformatic prediction of bat cathelicidins based on available sequencing information and experimental characterization of their antimicrobial activity [43,50], the number of tested bacterial strains and peptides was highly limited, and other characteristics such as fungicidal and metabolically inhibitory activities against fungi and cytotoxicity have not been validated experimentally. To the best of our knowledge, this is the first report of the antimicrobial activity of bat cathelicidins against diverse bacteria and fungi associated with human health and food pathogens.
A previous study briefly reported the bactericidal effect of a bat cathelicidin, Ml-LN-35, which is identical to ML-CATH, against E. coli and B. cereus [50]. The results were consistent with the results in this study with MICs ranging from 0.5 to 0.8 µM (Table 2). In contrast, antibacterial activity against S. aureus ATCC 6538 was not detected in the previous study, while the peptide showed highly potent anti-staphylococcal activity (1 µg/mL or 0.3 µM) as shown in Table 2. This inconsistency might be attributed to different experimental condition including media composition and peptide synthesis with some modification such as acetylation and amidation in the N-and C-terminal end, respectively, which can affect the nature and structure of AMPs and influence biological activity and stability of the molecules in turn [51][52][53].
In our functional analysis of bat cathelicidins, all three mature peptide region-derived peptides showed antibacterial activity against diverse bacterial strains. However, the truncated peptides corresponding to the predicted antimicrobial activity core region as a minimal unit for biological activity did not show substantial antibacterial activity, although the two different forms of peptides shared common characteristics in hydrophobicity and positive net charge (Table 1). This is somewhat different from the results of our previous studies to characterize cathelicidins of non-bat species [25,47,54].
The alteration of primary sequences and/or physicochemical properties of α-helical cationic AMPs, especially cathelicidins, could affect the formation of amphipathic α-helical structures, hydrophobicity, the density and distribution of charge locally or overall, and minimal length, influencing biological activities, including antimicrobial and mammalian cytotoxicity [55][56][57][58]. For example, a previous study showed that IG-13, corresponding to the minimal active domain of LL-37, a well-known human cathelicidin, was approximately 30-fold less active than LL-37 [58]. The smallest fragment, LL-19, did not form a clear α-helix in the buffer, although the peptide region originated from the α-helical region [55]. In addition, the hydrophobic tail length of the peptides could affect their ability to insert and penetrate the bacterial membrane [57]. Therefore, the loss of antimicrobial activity in the predicted antimicrobial activity core region peptides 18 to 25 amino acids in size of bat cathelicidins could be influenced by peptide length. Further studies are necessary to determine the amino acids critical for maintaining the antimicrobial activity of nascent mature peptides.
Among the mature peptide regions of the three cathelicidins, the specificity and strength of the antibacterial activities of ML-CATH and PD-CATH were highly similar ( Table 2). This could be attributed to the similarities in peptide length, hydrophobicity, and net charge between the two peptides despite their sequence differences (Table 1; Figure S1).
In addition to the extensive functional studies on LL-37 and CRAMP, recent interests in AMPs as potent antibiotic agents lead to the identification and characterization of novel cathelicidins from diverse species including Python bivittatus (Burmese python), Heterocephalus glaber (naked mole rat), Monodelphis domestica (gray short-tailed opossum), Lates calcarifer (Asian sea bass) and Sarcophilus harrisii (Tasmanian devil) [25,47,54,59,60]. These studies suggested that cathelicidins are promising candidates to develop antimicrobial therapeutics considering potent broad-spectrum bactericidal activity, minimal cytotoxicity to mammalian cells, and inducibility of lower antibiotic resistance compared to classical antibiotics. Interestingly, bat cathelicidins, ML-CATH and PD-CATH, showed much broader and stronger antibacterial activities than all the peptides described above (Table 2). Furthermore, ML-CATH and PD-CATH showed milder toxicity compared to ModoCath1 which is one of the cathelicidins showing broad spectrum with high potency. The viability of human keratinocyte was affected at a five-fold higher concentration (160 µg/mL) in PD-CATH comparing to ModoCath1 (32 µg/mL) ( Table 4). These characteristics could make ML-CATH and PD-CATH more suitable for pharmaceutical applications than other cathelicidins. However, further studies are still required to overcome intrinsic disadvantages of AMPs including high production cost, low stability in vivo, and deficiency of proper delivery system. An effective production system of recombinant cathelicidins using an engineered green fluorescent protein (GFP) as a tag protein was attempted for large-scale production of AMPs [61]. The conjugation of AMPs with nanoparticles has been suggested to overcome poor stability of AMPs in biological fluids and proteolytic degradation and to control pharmacokinetics [62][63][64].
In mammals, the copy number of cathelicidin genes varies among species, and a single functional cathelicidin gene is present in dogs, mice, and humans, whereas more than one functional gene is found in pigs, goats, sheep, cattle, and whales [65]. Intraspecies copy number variation has also been reported in pigs [48]. It is interesting to note that the antimicrobial potency of ML-CATH and PD-CATH, obtained from multiple cathelicidin gene-carrying bat species, was higher and more broadly effective against diverse bacterial strains than HA-CATH, obtained from species carrying only a single cathelicidin gene. The expansion of cathelicidin genes in certain bat species may be due to the functional importance of the molecule for their survival. It may also enhance the antimicrobial potency of paralogous genes in parallel.
The variations in the number of cathelicidin genes among different bat species can be attributed to the large phylogenetic diversity of bats in the order Chiroptera [66]. Moreover, in our analysis of the seven bat species, all annotated cathelicidin genes were mapped to the CDC25A and NME6 interval, the evolutionarily conserved cathelicidin residing region in mammals [48], except M. lucifugus with less complete genome information, suggesting the evolutionary conservation of synteny in bats, similar to other mammals. Therefore, the copy number of cathelicidins in bats could vary from a single copy to multiple copies depending on the species, which is consistent with a previous analysis [43]. However, further refinement of the current bat genome assemblies and annotations is needed to determine the copy number of cathelicidin family genes more accurately.
Endogenous AMPs also showed antiviral activity against diverse viruses, including the human influenza virus, human immunodeficiency virus, rabies virus, West Nile virus (WNV), and herpes simplex virus (HSV) [25,[67][68][69][70]. Among them, gramicidin S and melittin from Bacillus brevis and bee venom, respectively, showed noteworthy anti-SARS-CoV-2 effects with viral particle reduction compared to remdesivir, a repurposed drug used to treat the infection [71]. Antiviral activities against HSV and WNV were also reported for other cathelicidins, such as LL-37, Pb-CATH4, Hg-CATH, and ModoCath5 [25,70,72]. Because of the therapeutic potential of these peptides, it will be interesting to evaluate the antiviral activity of bat cathelicidins, which also showed negligible cytotoxicity to mammalian cells in this study. The antibacterial and antifungal activities of HA-CATH, ML-CATH, and PD-CATH observed in this study can serve as references for the potency and antimicrobial spectrum analyses of bat cathelicidins.

In Silico Identification of Cathelicidin-like Sequences in the Genome of Seven Bat Species
Four hundred sequences belonging to five major AMP families of vertebrates, alphadefensins, beta-defensins, cathelicidin, hepcidin, and LEAP-2, were obtained similarly to the previous studies [25,47]. Briefly, the sequences of 2477 nonredundant AMPs were downloaded from UniProtKB/Swiss-Prot (http://www.uniprot.org/uniprot/, accessed on 16 March 2022; [73]) using the query "antimicrobial peptide AND reviewed: yes". After removing sequences originated from nonvertebrates, the sequences corresponding to five major AMP families described above were collected (Table S7) [25]. The sequences were blasted using the NCBI blastp mode

Peptide Synthesis
Peptides corresponding to the predicted active core and mature peptide regions of cathelicidins were generated via solid-phase peptide synthesis, purified via high-performance liquid chromatography (HPLC), and confirmed by mass spectroscopy using a commercial service (GenScript, Piscataway Township, NJ, USA). Peptide names were given according to the acronym of binomial nomenclature of the species followed by "CATH" indicating cathelicidin. The peptide names and sequences for each species are ∆HA-CATH (N-LLRRGGRKIGQGLERIGQRIQGF-C) and HA-CATH (N-ILGRLRDLLRRGGRKIGQGLERI GQRIQGFFSNREPMEES-C) for H. armiger, ∆ML-CATH (N-GIFILKHRRPIGRGIEIT-C) and ML-CATH (N-LNPLIKAGIFILKHRRPIGRGIEITGRGIKKFFSK-C) for M. lucifugus, and ∆PD-CATH (N-IAGRIAGKLIGDAINRHRERNRQRR-C) and PD-CATH (N-ILGPALRIGGR IAGRIAGKLIGDAINRHRERNRQRRG-C) for P. discolor. The synthesized peptides were dissolved in distilled water at a concentration of 4 mg/mL.

Evaluation of Antibacterial Activity
The antibacterial activities of the chemically synthesized peptides were evaluated against a panel of bacteria consisting of 6 Gram-negative strains, including E. coli Lakes, NJ, USA) or brain heart infusion broth (BHIB; BD Bioscience) at 37 • C for 6 h with shaking at 220 rpm. E. faecalis and streptococci were cultured in BHIB because of their slow growth, whereas all other bacteria were cultured in LB medium. The cells were washed by sterile saline (0.9% NaCl) twice and adjusted in a single well of a 96-well plate at a cell density of 1.5 × 10 5 CFU/well in accordance with 0.5 McFarland standard. Subsequently, 180 µL/well of fresh Mueller-Hinton broth (MHB; BD Bioscience), except for E. faecalis and streptococci using BHIB was added to the plate using BHIB. Final concentrations of 1-40 µg/mL (0.3-19.3 µM) of each peptide and reference antibiotic were added to each well. The plates were then incubated at 37 • C for 6 h. Subsequently, 10 µL of the coloring reagent was added, and the mixture was incubated at 37 • C for 2 h. The absorbance of each well was measured at 450 nm using a microplate spectrophotometer (Bio-Rad, xMark spectrophotometer, Hercules, CA, USA). MIC values were determined when the difference in the absorbance values between treatments and blanks (media and coloring reagent only) was less than 0.05. Experiments were performed in triplicate.

Evaluation of Antifungal Activity
The antifungal activities of the peptides were evaluated against two yeast fungi, C. albicans KCTC 7270 and C. cuniculi KCTC 17232, and a mold fungus, M. hiemalis KCTC 26779. C. albicans and C. cuniculi were cultured in yeast mold broth (YMB; BD Science) agar at 35 • C and 25 • C, respectively. Cells were prepared in YMB for C. albicans and RPMI 1640 medium (with l-glutamine, without phenol red and sodium bicarbonate, Sigma Aldrich, Saint Louis, MO, USA) and adjusted to pH 7.0 with 0.165 M 3-(N-morpholino) propanesulfonic acid (MOPS) (iNtRON Biotechnology, Seoul, Korea), as previously described [80] for C. cuniculi. According to the 0.5 McFarland standard, cells at 1.5 × 10 5 CFU/well were seeded into a 96-well plate containing 180 µL of fresh medium. Subsequently, peptides at final concentrations of 5-45 ug/mL (1.2-19.3 µM) peptides were added to each well. Ciclopirox (≥98% (HPLC), Sigma Aldrich) was used as a reference for the antifungal activity. The antifungal assay was conducted in the same manner as the antibacterial assay described above, according to the manufacturer's protocol. Briefly, C. albicans and C. cuniculi were incubated at 35 • C for 6 h and 25 • C for 47 h, respectively, and 10 µL of the coloring reagent was added and further incubated for 18 h and 25 h. The MIC values were determined in the same way as for the antibacterial assay.
To evaluate the antifungal activity of peptides against M. hiemalis, the metabolic activity of hyphae was measured using the same colorimetric assay as described above, with slight modifications to a previous study [81]. Briefly, M. hiemalis was streaked onto malt extract broth (BD Biosciences) agar in a Petri dish and cultured at 25 • C for seven days. Then, conidia were collected from the dish by adding phosphate buffered saline (PBS) containing 0.05% Tween-80 (Sigma Aldrich) and filtered through sterilized 40 um cell strainer (SPL Life Science, Pocheon, Korea) to remove hyphae. After washing twice with PBS and centrifugation, 5 × 10 4 conidia were counted using a hemocytometer, suspended in 50 µL of RPMI 1640 as described above, and seeded in wells of a 96-well plate. After 16 h of incubation at 25 • C for hyphae formation, the plate was centrifuged at 3000× g and the media were changed. The peptides were then added at final concentrations of 20, 40, and 80 µg/mL. Fluvastatin sodium (70 µM; Sigma-Aldrich) was used as a reference for antifungal activity. For the coloring reaction, 10 µL of the coloring solution (Microbial Viability Assay Kit-WST, Dojindo) was added to each well and incubated for 2 h. Subsequently, the plate was centrifuged, and the supernatant was transferred to a new 96-well plate. The absorbance at 450 nm (for the treated group and control) and 650 nm (for the background) was measured for each well using a microplate reader (xMarkTM spectrophotometer; Bio-Rad). The metabolic activity of the fungal cells was calculated using the following equation. All experiments were performed in triplicate.

In Vitro Cytotoxicity Assay
HaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone TM , Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone TM ) and 1% penicillin/streptomycin (Pen-Strep; Hyclone TM ) up to 80% confluence at 37°C and 5% CO 2 . Cells were detached from the plate by adding 0.25% trypsin-EDTA solution (Gibco TM ; Carlsbad, CA, USA). In a 96-well plate, 2 × 10 4 cells were seeded per well and cultured for 24 h. Subsequently, the medium was replaced with 100 µL DMEM (Hyclone TM ) containing 10% heat-inactivated FBS. Peptides were added to each well at concentrations of 64 µg/mL and 160 µg/mL. As references for complete cell lysis, 64 µg/mL melittin (Sigma-Aldrich) and Triton X-100 (Sigma-Aldrich) were used. Untreated cells were used as negative controls. After 24 h incubation at 37°C and 5 % CO 2 , 10 µL of coloring solution (Cell Proliferation Reagent WST-1 TM ; Sigma Aldrich) was added to each well according to the manufacturer's instructions. The absorbance at 450 nm (treated group and control) and 650 nm (background) was measured in each well using a microplate reader (xMarkTM spectrophotometer; Bio-Rad). The cell viability was calculated using the following equation. All experiments were performed in triplicate.

Statistical Analysis
Statistical analysis was performed using Student's paired t-test with a two-tailed distribution using RStudio (https://www.R-project.org/, accessed on 13 July 2022).

Conclusions
In summary, species-specific differences in genetic and functional characteristics of cathelicidins from seven bat species were studied. We listed 19 cathelicidin-like sequences from seven bat species and tested the biological activity of three cathelicidins (HA-CATH, ML-CATH, and PD-CATH). Among them, ML-CATH and PD-CATH showed potent antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria. The potency of antibacterial activity of ML-CATH and PD-CATH was comparable to that of other highly active cathelicidins reported in vertebrates. ML-CATH and PD-CATH also showed antifungal activities against C. albicans, C. cuniculi, and M. hiemalis. The cytotoxicity on HaCaT cells showed a positive correlation with the potency of bat cathelicidins, but given the effective bactericidal concentration, the cytotoxicity of bat cathelicidins is within the acceptable range for therapeutic exploitation. ML-CATH and PD-CATH are promising candidates for the development of peptide antibiotics, but further studies including peptide production and in vivo activity are in need.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics11080989/s1, Figure S1: Comparison of the amino acid sequences and properties of the three bat cathelicidins evaluated in this study, Table S1: List of the putative antimicrobial peptide (AMP) genes of Rhinolophus ferrumequinum, Table S2: Results of the National Center for Biotechnology Information (NCBI) tblastn analysis of the genomes of seven bat species using 64 vertebrate cathelicidin sequences as queries, Table S3: Results of the NCBI blastp analysis of the genomes of seven bat species using 64 vertebrate cathelicidin sequences as queries, Table S4: Results of the in silico analysis of annotated putative cathelicidin-like sequences of seven bat species in NCBI, Table S5: In silico prediction of the antimicrobial activity-containing regions of five functional bat cathelicidins, Table S6: Comparison of the AMP family gene numbers of six mammalian species, Table S7: A list of antimicrobial peptides queries for the blastp analysis of antimicrobial peptides.

Conflicts of Interest:
The authors declare that they have competing financial interests. The peptide sequences, ML-CATH and PD-CATH were subject to domestic and foreign patent applications by Konkuk University.