Antimicrobial Compounds from the Mucus of Garden Snail Cornu aspersa

Natural products have long played a major role in medicine and science. The garden snail Cornu aspersa is a rich source of biologically active natural substances which might be an important source for new drugs to treat human disease. Based on our previous studies seven fractions containing compounds with Mw &lt;3 kDa, &lt;10 kDa, &lt;20 kDa, &gt;20 kDa, and between 3-5 kDa, 5-10 kDa, and 10-30 kDa were purified from the mucus of C. aspersa and analyzed by tandem mass spectrometry (MALDI-TOF/TOF). Seventeen novel peptides with potential antibacterial activity have been identified by de novo MS/MS sequencing using tandem mass spectrometry. The different fractions were tested for antibacterial activity against Gram─ (Pseudomonas aureofaciens and Escherichia coli) and Gram+ (Brevibacillus laterosporus) bacterial strains as well anaerobic bacterium Clostridium perfringens. These results revealed that the peptide fractions exhibit a predominant antibacterial activity against B. laterosporus, the fraction with Mw 10 – 30 kDa against E. coli, another peptide fraction &lt;20 kDa against P. aureofaciens, and the protein fraction &gt;20 kDa against the bacterial strain C. perfringens. The discovery of new antimicrobial peptides (AMPs) from natural sources is of great importance for public health due to their effective antimicrobial activities and low resistance rates.


Mucus collection and separation of different fractions
The mucus was collected and purified from C. aspersum snails grown in Bulgarian farms using patented technology without any snail suffering [23]. After ultrafiltration using different membrane filters (10 and 20 kDa), the crude mucus extract was separated into two fractions: a peptide fraction with Mw below 10 kDa and a fraction containing compounds with Mw above 20 kDa. The peptide fraction with Mw below 10 kDa was additionally separated using Amicon® Ultra-15 centrifugal tube filters with 3 and 5 kDa membranes into three fractions. Finally, the following samples have

Molecular mass analysis and de novo sequencing of peptides by mass spectrometry
The isolated peptide fraction with Mw <3 kDa (Sample 1) was lyophilized and analyzed by MALDI-TOF-TOF mass spectrometry on an AutoflexTM III. High Performance MALDI-TOF&TOF/TOF System (Bruker Daltonics) which uses a 200 Hz frequency-tripled Nd-YAG laser operating at a wavelength of 355 nm. Analysis was carried out after mixing 2.0 μl of the sample with 2.0 μl of matrix solution (7 mg/ml of α-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN containing 0.1% TFA), but only 1.0 μl of the mixture was spotted on a stainless steel 192-well target plate. The samples were allowed to dry at room temperature and analysed. A total of 3500 shots were acquired in the MS mode and a collision energy of 4200 was applied. The mixture of angiotensin I, Glu-1-fibrinopeptide B, ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and ACTH was used for calibration of mass spectrometer. The MS/MS spectra were carried out in reflector mode with external calibration using fragments of Glu-fibrino-peptide B. Amino acid sequences of peptides were identified by precursor ion fragmentation using MALDI-MS/MS analysis.

Carbohydrate test
Seven isolated fractions from the mucus were analyzed by the orcinol-sulphuric test to determine the carbohydrate content. About 2 μl of the purified samples were applied to a thin layer plate and air dried. The plate was sprayed with orcinol/H2SO4 and heated for 20 min at 100 o C. The orcinol/H2SO4 solution contained 0.02 g of orcinol, 20% H2SO4, and H2O to a total volume of 10 ml.

SDS-PAGE electrophoresis
Protein fractions with antibacterial activity were analyzed by SDS-PAGE electrophoresis.

Nutrient media and culture conditions
Solid MPA (Meat-Peptone Agar) medium /Nutrient Agar/ was used to investigate the antibacterial activity through cultivation assays. A mesopeptone broth was used to multiply the microorganisms. A nutrient liquid media was used to multiply the microorganisms. Standard microbiological apparatus and glassware were also used following all the rules of good microbiological practice. The test strains used were stored as lyophilized microbial cultures in glucose media and peptone protector. After rehydration in saline the strains were maintained on slop agar in standard tubes.

Studies of antibacterial activities, using different methods
Cultivation methods were applied in mesopeptone (nutrient) agar as follows: Two types of inoculation were used to test the microorganisms. The results are presented as mm 2 /mgP/µ Mol.

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Тhe impact of active fractions isolated from the mucus of garden snail C. aspersum on the cell structure of bacteria was examined by scanning electron microscopy (SEM). Samples were prepared by treatment with ethyl alcohol in a series of increasing concentrations. All data are arithmetic averages of three independent repeats.

Purification and characterization of different fractions from mucus
The purified crude mucus extract was separated into various fractions by ultrafiltration under pressure with membrane filters of different pore sizes of 1, 10, and 20 kDa and centrifugal tube filters  Using the same method, the amino acid sequences of 17 novel peptides in Sample 1 with molecular masses between 1000-2800 Da were identified ( Table 1). The isoelectric points (pI) and grand average of hydropathicity (GRAVY) of the peptides were predicted by the ExPASy MW/pI tool program and ExPASy ProtParam tool (Table 1). Table 1. Amino acid sequences of peptides from the mucus of garden snail C. aspersum, identified by de novo sequencing on MALDI-MS/MS.

A glycosylation screening
A screening of the fractions from mucus performed by orcinol/H2SO4 assay identified several glycosylated fractions. As is shown in Figure 2 all fractions except the control fraction water

Antibacterial activity of different fractions from the mucus of garden snail C. aspersum
It is known that the Gly and Pro content in peptides plays a crucial role in the activity against   Figure 3B). Furthermore, only one tested fraction, with high carbohydrate content (Sample 7 with Mw >20 kDa) ( Figure 3B The large sterile zones formed due to a strong inhibition effect of Fraction 7 against Clostridium perfringens NBIMCC 8615 is illustrated with three repetitions in Figure 4A due to the deep inoculation of the bacteria in comparison to the control. For comparison, the antibacterial effect of AMCs (Sample 5) against Escherichia coli NBIMCC 8785 at surface inoculation of the bacteria is illustrated in Figure 4B. It clearly shows that fraction 7 of AMCs displays an antibacterial effect against Clostridium perfringens NBIMCC 8615. This allows to speculate that this fraction is may become a candidate for medical treatment of anaerobic infections caused by clostridia.
These two fractions with antibacterial activity were subject to SDS-PAGE in order to determine the approximate size of the antimicrobial substances. The electrophoresis (  To shed light on the mechanism of antibacterial action, we studied the effect of the active fraction with Mw 10-30 kDa on the cell structure of the Gram-bacterial strain E. coli NBIMCC 8785 by scanning electron microscopy (SEM). As is shown Figure 5, the results obtained by SEM from control -18-hour culture of E. coli in nutrient broth ( Figure 5A) and the damaged membranes of cell of 18 hour culture of E. coli in nutrient broth after 1 hour action of peptide fraction 5 with at magnification 10000X ( Figure 5B) and 30000X ( Figure 5C).

Discussion
The discovery of new antimicrobial compounds (AMCs) from natural sources is of great importance for public health, since these molecules are pharmacological candidates due to their effective antimicrobial activity and low resistance rates. The resistance against them is not prevalent although AMPs have been exposed to microbes for millions of years [27]. Besides, mutations in the microbes during the course of evolution led to the diversification of AMPs [28] In our previous work we have determined the primary structure and their antimicrobial activity of only 9 peptides produced by the mucus of the garden snail H. aspersa, in particular against Gram ─ Pseudomonas aureofaciens AP9 and Gram + Brevibacillus laterosporus BT271 bacteria [24]. We hypothesized that other peptides from the mucus would also be effective against bacterium Clostridium perfringens NBIMCC 8615.
In certain primary structures (Table 1) membranes. Generally, these proteins could attain diverse conformations such as α-helices, β-sheets, mixed conformations, loops, and extended structures [28,29]. Analyses using the ExPASy ProtParam tool indicate that the mucus fraction with Mw<3kDa contains both cationic and anionic AMPs, but is dominated by cationic AMPs. Most of the peptides, identified in fraction with Mw<3kDa, are characterized by an amphipathic structure and display generally hydrophobic surfaces (Table 1).
This fact is considered as a prerequisite for the disruption of biological membranes and direct cell lysis [29]. It is known that cationic AMPs kill microbes via mechanisms that predominantly involve interactions between the peptide's positively charged residues and anionic components of target cell membranes. These interactions can then lead to a range of effects including membrane permeabilization, depolarization, leakage or lysis resulting in cell death [30]. Generally, cationic antimicrobial peptides act on the membrane of microorganisms by an electrostatic difference, but there are AMPs that act internally and can interact on ribosomes, internal proteins, or nucleic acids.
Some of the identified peptides in Table 1 have a primary structure similar to glycine-rich linear antimicrobial peptides, as Ctenidin1-3 with activity against the Gram-negative bacterium E. coli, isolated and characterized from hemocytes of the spider Cupiennius salei [31]. Also acanthoscurrins isolated from the hemocytes of the spider Acanthoscurria gomesiana act against Gram-negative bacteria, Escherichia coli, and the yeast Candida albicans [32], are characterized as cationic peptides with high glycine content. Using alignment of amino acid sequences presented in Table 1 with CAMPSing (http://www.campsign.bicnirrh.res.in/blast.php), the peptides №s 5 and 6 showed ~72% identity with Ctenidin 1 and Ctenidin 3, and peptides №s 9, 10, 13, and 17 have about 73-76% identity with two isoforms of acanthoscurrin.
Some of the identified peptides shown in Table 1  last decade. Previous research also identified AAMPs in mucus fraction with Mw below 10 kDa, isolated from garden snail H. aspersa [23,24]. Usually AAMPs show antibacterial activity, but a number of them are multifunctional, variously showing antifungal, anticancer, a neuropeptide activity, and the probability to become potential conventional antibiotics [29]. Antimicrobial mechanisms proposed for these peptides include toroidal pore formation and membrane interaction according to the Shai-Huang-Matsazuki model along with pH-dependent amyloidogenesis and membranolysis via tilted peptide formation [33]. AAMPs generally adopt amphiphilic structures.
For a number of these peptides, post-translational modifications are essential for antimicrobial activity. Membrane interaction appears a key to the antimicrobial function of AAMPs. The architectures of AAMPs vary from alpha-helical peptides from some amphibians to cyclic cysteine knot structures observed in some plant proteins. But in many cases, the mechanisms underlying the antimicrobial action of these peptides are unclear or have not been elucidated [30].
The detected peptides (shown in  [35,36]. Due to our results that the identified sequences, 12 peptides of the identified sequences contain 1 to 3 Pro amino acid residues in the polypeptide chain. In nine peptides Pro residues are incorporated in the C-terminal region of the polypeptide chain, but only in 5 peptides Pro is located in the N-terminal region. Two peptides contain proline residues both in the N-terminal and C-terminal polypeptide chain. Moreover, one proline residue was found in the center of the polypeptide chain for two peptides (№s 8, 15). the appearance of these peptides in the mucus. Furthermore, the majority of known AMPs originate from processing of larger inactive proteins, and some studies suggest that biologically active proteins, such as hemocyanins [37] or hemoglobins [38] can be processed to produce bioactive aspersa which appear to have activity against Ps. aeruginosa [21].
Results from comparative analysis of the antibacterial activity of fractions against C. perfringens NBIMCC 8615 (Gram ─ ) and other tested bacterial strains by surface and deep inoculation of the bacteria reveal that a protein fraction with Mw >20 kDa (one of the two fractions with the highest carbohydrate content) is the most effective against the bacterial strain C. perringens at deep anaerobic cultivation. Probably the proteins determined at ~37 kDa, ~ 42 kDa, 45-50 kDa, ~65kDa, 80-90 kDa and between 150-250 kDa (electrophoresis, Figure 4C) are responsible for the anticlostidial effect.
Some of the found proteins may be related to example to a new protein named Aspernin with a molecular weight 37.4 kDa and protein with a molecular weight ~50 kDa, determined previously in fractions from the mucus of H. aspersa with anti-pseudomonal properties [21], as well as protein with Mw 50.81 kDa from A. fulica mucus [39]. Furthermore, the protein determined between 80-90 kDa probably corresponds of protein with MW of 83.67 kDa (achacin) isolated from A. fulica mucus, actives against Streptococcus mutans and Actinobacillus actinomycetemcomitans [39]. Our new data are in agreement on the antimicrobial properties of the mucus from H. aspersa, and A. fulica [14,21,39].
The combination of two vectors of action of protein fraction >20 kDa against clostridial infections, an antibacterial and a regenerative effect, will be the basis for the development of synergistic therapeutic agents.
Our results show that the antibacterial activity of fraction with Mw 10-30 kDa (Fraction 5 with Mw 10-30 kDa) induces serious damaging of the bacterial membrane changing of the shape, activity and metabolism of the bacteria strain Escherichia coli NBIMCC 8785 (Figures 5 A, B, C). These results will be extended by investigations on the mechanism of the antibacterial effect against Clostridium perfringens.

Conclusions
In conclusion, the presents work gives an example on isolation and structural identification of new biomolecules from a complex mixture of H. aspersa mucus using advanced sophisticated instrumentation. Du to different biotests, several of the mucus fractions have considerable antimicrobial activity which could probably add to the arsenal of antibiotics as candidates with low resistance rates.