Inhibitory Activity of Chitin, (2-Acetamido-2-Deoxy-Hexopyranose) against Penicillin-Binding Proteins of Staphylococcus aureus

Abstract

Antibacterial compounds from aquatic sources have the potential to contribute significantly to the treatment of the ever-increasing drug-resistant infectious diseases. The chitin 2-acetamido-2-deoxy-hexopyranose from the freshwater prawn Caridina gracilirostris showed antibacterial activity against Staphylococcus aureus. Two different types of chitin films, (1) chitin and starch, and (2) chitin, starch, and ascorbic acid were tested against S. aureus using in silico molecular docking. The inhibitory action of 2-acetamido-2-deoxy-hexopyranose was recorded against the penicillin-binding protein (PBP2a) of S. aureus. The chitin films exhibited their potential as an effective antibacterial agent through binding energy and ligand efficiency. Further, the inhibitory constant also indicated the potent antibacterial nature of the chitin films. The hydrogen bond interaction of chitin with PBP2a was with serine 49 and threonine 413, at 2.2 Å and 3.4 Å, respectively. The druggability of 2-acetamido-2-deoxy-hexopyranose showed good oral bioavailability, and pharmacokinetics properties were within a normal range. The chitin did not undergo any metabolism and appeared to be of a nontoxic nature. The 2-acetamido-2-deoxy-hexopyranose could be suggested as a novel drug against S. aureus.

1. Introduction

Antibiotic resistance is a significant public health concern [1]. The evolution of resistant strains has become a significant challenge to combat infections caused by bacterial pathogens that were previously treatable using antibiotic therapy [2]. Among the antibiotic-resistant bacteria, Stapylococcus aureus is the principal nosocomial and skin colonizer for humans and primordial cause of hospital- and community-acquired infections [3,4]. The World Health Organization (WHO) denoted S. aureus as one of the highest prioritized bacteria due to the prevalence of recalcitrant isolates and the fact that this pathogen is capable of colonizing a dynamic range of host tissues; as well as this, S. aureus is the leading cause of skin and soft tissue infections, as well as invasive tissue diseases such as endocarditis and osteomyelitis [3]. Recently, several conventional antibiotics such as vancomycin, clindamycin, levofloxacin, betalactamics, quinolones, and glycopeptides used [4,5,6] to combat this bacteria have failed and are considered unsatisfied drugs against S. aureus. Hence, alternative natural therapy is required that can be an effective therapeutic option for the treatment of infectious diseases caused by S. aureus.

The Gram-positive bacteria S. aureus impacts the human population through hospital and community spread and rapidly develops resistance to many antibiotics [1,2]. Survival of bacteria depends on the health of the cell wall. During growth and division, bacteria biosynthesize the cell wall, which is a polymer made up of peptidoglycan as the principle building unit [2]. Worldwide, S. aureus is a significant major public health issue, gaining resistance to major antibacterial therapies. Mostly, microbial sources are easily available, accessible, eco-friendly, and low-cost because plant-based antibiotics have been ignored. The penicillin-binding proteins (PBP2a) of S. aureus are final steps of peptidoglycan biosynthesis of macromolecules (nucleotide precursors, lipid-linked intermediates, and polymerization reactions) that play an important role for synthesis of the cell wall [3]. Although there have been many reports on the inhibition of penicillin-binding protein (PBP2a) on S. aureus, still no satisfactorily effective drug from natural resources is available [4].

Chitin, apart from being a copious polysaccharide richly occurring in crustacean exoskeletons, also forms a vital source of carbon and nitrogen from bio resources of the ocean [5]. Several studies have shown that chitin has been successfully used to fabricate polymeric scaffolds for the purposes of tissue repair and regeneration [6]. Chitin and chitosan are extracted mostly from the exoskeleton of marine shrimps due to their desirable chemical and physical properties for medical and food preservation applications [7]. Nevertheless, chitin and chitosan extracted from different crustacean sources are reported to have variation in their antimicrobial activity [8]. There may be differences in the nature of the exoskeleton of different crustacean species. There is no report on the chitin from the soft-shelled freshwater Caridine prawn Caridina gracilirostris and its biomedical efficacy. The present study reports the potentials of chitin film polymerized from the exoskeleton of C. gracilirostris as a therapeutic agent against pathogenic S. aureus.

2. Material and Methods

2.1. Sampling and Systematics of Freshwater Prawn

For the present study, freshwater prawns (C. gracilirostris) were collected from Adyar creek, Chennai (13.0128° N, 80.2285° E), and on the basis of the taxonomic key characters described by Richard and Chandran [9], they were identified at the species level.

2.2. Extraction of Chitin

Extraction and depolymerization of chitin from prawns was carried out using chemical methods. Sodium hydroxide (2N) and hydrochloric acid (2N) solutions were used for deproteination and demineralization, respectively, while acetone was used for decolorization [10].

2.2.1. Deproteinization

The dried powdered prawns were treated with 2 Normality (N) of sodium hydroxide (NaOH) solution (solid/liquid ratio of 100:1000 (g/mL)) and stirred using a magnetic stirrer with a hot plate at 60 °C for 2 h to remove the remaining proteins and other organic materials. After the reaction, the sample was washed repeatedly with distilled water and then dried in a vacuum oven at 60 °C.

2.2.2. Decoloration

Deproteinized shells were treated with acetone at room temperature for 24 h to remove pigments. The washed shells were filtered and dried in a vacuum oven at 60 °C.

2.2.3. Demineralization

The deproteinized and decolorized material was treated with 2 N of HCl solution (solid/liquid ratio of 1:15 (g/mL)) for 24 h at 60 °C using a magnetic stirrer. Then, the powders were washed with distilled water to remove the minerals and other water-soluble impurities. The obtained chitin from prawn shells was characterized to determine the purity of the chitin.

2.3. Characterization of Chitin

2.3.1. Determination of Yield, Sulfated Ash, and Insoluble Content in Chitin

The chitin yield was calculated by deducting the weight of chitin weight from dried shrimp powder [11]. The sulfated ash content was determined using the method of De Queiroz et al. [12], and using 1% dilute glacial acetic acid, chitin solubility was determined [12]. Three trials were conducted for extracting chitin using chemical methods.

2.3.2. Characterization of Physicochemical Properties

The physicochemical properties of chitin were analyzed using different instrumental techniques such as Fourier transform infrared spectroscopy analysis (Perkin Elmer FTIR model 2000 spectrophotometer), scanning electron microscopy (Quanta 200 FEG ESEM), and energy-dispersive X-ray analysis.

2.4. Preparation of Chitin Composite Film

The chitin (2 mg/mL) and starch (2 mg/mL) blended biopolymer films were processed using glycerol as a plasticizer (100 µL/mL). The homogenized mixtures were transferred to sterile Petri dishes and immobilized using ascorbic acid (1 mg/mL). The control film was prepared without ascorbic acid. The films were dried for 72 h at 25 °C under relative humidity of 50% and then removed manually. The films were stored at 4 °C in sealed Petri dishes prior to further study [13].

2.5. Antimicrobial Activity of Chitin Film

For determining antibacterial activity of chitin films, the bacterial cultures (clinical isolates) were obtained from Meenakshi Hospital, Thanjavur [14]. The overnight grown broth culture of S. aureus was disseminated on nutrient agar (Himedia) plates. Square pieces of control and experimental films were inoculated on plates, and the same was incubated at 37 °C for 24 h. Subsequently, antibacterial activity of the chitin films was evaluated by observing the clear inhibition zone around the films.

2.6. In Silico Inhibitory Mechanisms of Penicillin-Binding Protein (PBP2a) in S. aureus

In silico inhibitory mechanisms of molecular docking analysis was performed using the molecular docking software AutoDock 4.2.6. For ligand preparation, the compound (2-acetamido-2-deoxy-hexopyranose) was download from the PubChem database and converted to a pdp file using open babel 2.4, and following this, the structure was optimized using Argus Lab [15,16]. For the preparation of the protein, the three-dimensional structure of PBD2s was downloaded from the RCSB protein Data Bank, and the binding site was optimized. In addition, the pharmacokinetic assessment (ADMET) and druggability of the compound were conducted using pkCSM and Medchem designer 5.0 software, respectively.

3. Results and Discussion

The present study is the first attempt made to extract chitin from C. gracilirostris using a chemical method. The chitin yield of C. gracilirostris was 3.70 ± 0.37 g from 100 g of prawn powder (Figure 1 and Figure 2). Although the amount of yield of chitin from C. gracilirostris was less than that reported from zooplankton such as copepods (8.58 g) and cladocerans (12.22 g) [17], it was higher than that of Fenneropenaeus indicus (2.77 g) and Fenneropenaeus semisulcatus (3.21 g) [18]. Due to its complex structure, the solubility of chitin is one of the limiting factors that restricts its utilization in pharmacology, and many efforts are made to establish enhanced chitin solubility through modification of chemical composition. Higher solubility might render the wider application of chitin in many fields. Further, there is growing interest in producing chitin [19]. In the recent past, considerable interest is generated to extraction of chitin from different sources and its application in the medical field due to its advantageous and beneficial biological properties [20]. In the present study, the percentage of insoluble content of chitin in the chemically extracted method was 1.166% ± 0.154%. In general, an efficient demineralization process in chitin extraction is denoted by the resultant ash content of chitin and less than 1% ash content reported to be of good quality chitin [21]. The present study recorded less than 1% ash content (0.33 ± 0.04%). This ash content implies that there is effective demineralization of the chitin from C. gracilirostris powder with 2N HCl in the chemical method.

FTIR analysis is an analytical technique used to identify polymeric materials. In the present study, the FTIR spectrum of chitin from prawn shell showed absorbance peaks at 3500–3200 cm⁻¹, 1700–1500 cm⁻¹, 1110 cm⁻¹, 950–800 cm⁻¹, and 600–500 cm⁻¹, which were characteristic bands of chitin and were attributed to O–H (stretching) and N–H (bending), v N–H (amide-I), C–O–C, saccharides, and O–H stretching, respectively (Figure 3). The chitin from Brachytrupes portentosus showed peaks at 3433 cm⁻¹, 3257–3103 cm⁻¹, 1653 cm⁻¹, 1622 cm⁻¹, 1554 cm⁻¹, and 1311 cm⁻¹, indicating similarity to the present finding. Further, chitin extracted from other sources such as Bombyx mori [22], Cicada sloughs [23], and Orthoptera sp. [24] showed similar absorbance peaks. The chitin obtained from the shrimp Parapenaeus longirostris showed absorbance peaks at 3500–3200 cm⁻¹, 3100–2900 cm⁻¹, 1659–1624 cm⁻¹, 1554 cm⁻¹, 1315 cm⁻¹, 1200–1110 cm⁻¹, 900–800 cm⁻¹, and 600–500 cm⁻¹, which were attributed to the –NH2, –OH, amide I, C=O, N–H, C–O–C, saccharides, and O–H stretching, respectively, also showing close similarity with the present study [25]. Povea et al. [26] recorded chitin absorption ranges of 3450 cm⁻¹, 1870–2880 cm⁻¹, 1655 cm⁻¹, 1580 cm⁻¹, and 1320 cm⁻¹, which were attributed to O–H stretching, CH-stretching, amide I, –NH2 bending, and amide III, respectively. The absorption bands at 1160 cm⁻¹ (C–O–C bridge), 1082 and 1032 cm⁻¹ (skeletal vibrations involving the C–O stretching), 896 and 894 cm⁻¹ (saccharide rings), and 600–500 cm⁻¹ (alkyl halides) denote groups in parentheses. Similarly, the chitin characterized from shrimp exoskeletons appeared as a band of –OH, –NH2, and –CO groups, which indicated the frequency wavelengths at 670, 700, and 1000 cm⁻¹, respectively, indicating the N-acetylation [27,28]. On the basis of the literature, it is inferred that the long-chain polymer of chitin with strong interactions between sugar chains and solvent revealed by the widening of bands could be attributed to –NH₂, –OH (3390–3418 cm⁻¹), C=O (1716–1724 cm⁻¹), amide I (1626–1633 cm⁻¹), amide II (1520–1531 cm⁻¹), C₃–OH (double peaks, 1178–1189 cm⁻¹ and 1148–1153 cm⁻¹), C₆–OH (1073–1074 cm⁻¹), 897 cm⁻¹ (C–O–C bridge as well as glycosidic linkage), 700–610 cm⁻¹ (–C≡C–H:C–H), and 690–515 cm⁻¹ (C–Br stretch) [25,29,30,31,32,33,34]. A close resemblance is suggested between commercially available chitin and chemical composition and bonding types of the chitin revealed in the FTIR results of the present study.

SEM micrographs providing the topography and ultrastructure of the surface of the chitin prepared from chemical methods are presented in Figure 4. Chemically extracted chitin exhibited rough and thick surface morphology under scanning electron microscopic examination at 10 µm, 2 µm, and 1 µm× magnification. These images indicate chitin being a biosynthetic material and its existence as a sponge form or nanofibers could have a role as scaffolding films or sheets in the treatment of wounds and ulcers. SEM studies of Sajomsang et al. [23] in Cicada sloughs and Liu et al. [34] in Holotrichia parallela showed a comparable ultra-structure of chitin to the present report. The energy-dispersive X-ray spectroscopy provided accurate information regarding chemical composition of subcellular structures that could be compared with their high-resolution images [35]. The EDX results of the present study from the chemically extracted chitin sample shows the existence of elements Ca and Na in the prawn [35]. The presence of the Si element was recorded in the prawn shells (Figure 5). This study suggests that the EDX spectra for chitin showed the presence of elements in the chitin from C. gracilirostris without any impurity peaks (

Table 1. Energy-dispersive X-ray spectroscopy of chitin.

Elements	Weight (%)
Carbon (C)	20.9
Nitrogen (N)	3.8
Oxygen (O)	42.2
Sodium (Na)	32.1
Silicon (Si)	0.9

).

The chitin films produced antimicrobial activity against the clinical pathogen S. aureus. Of the two different chitin films tested for antimicrobial activity, chitin-, starch-, and ascorbic-acid-mediated film showed higher inhibition activity (23 mm) than chitin and starch-mediated film (9 mm) against S. aureus (Figure 6). Velásquez et al. [36] reported that the electrostatic interaction between positively charged chitin with the negatively charged cellular membranes of bacteria significantly alters the barrier properties of the membranes, and the authors further reported that the chelating capacity of the chitin can also affect microbial growth. Similarly, our results indicate the phospholipids present in the cellular membranes of Gram-positive bacteria/S. aureus interact with the NH- groups of the chitin, causing the bacteria to lose cellular material. The chelating capacity of the chitin could effectively inhibit growth of S. aureus. The use of organic acids with low carbon number such as acetic acid as a chitin solvent improves the antimicrobial activity of chitin [36]. Consequently, there is a possibility of cell membranes of the bacteria being broken by the action of chitin film, promoting the entry of natural antioxidants into the cells. The antioxidants (ascorbic acid) have antimicrobial properties and can therefore increase the antibacterial properties of chitin film [37]. The clear zone around the chitin film with ascorbic acid denoted potential inhibitor properties against S. aureus.

Molecular docking identifies potential small molecules in drug discovery and active research undertaken on these molecules due to their performance in antibiotics and resistance. Due to the involvement of PBPs in the cell wall synthesis of bacteria, they form ideal targets for drugs [4]. The present study showed effective antimicrobial activity of chitin films against S. aureus. The antibacterial mechanism of chitin film using the in silico model indicated effectiveness of its activity with a binding energy of −5.01044 kcal/mol and ligand efficiency of 0.38 (Figure 7). Further, firm-binding affinity of these compounds is also affirmed by possessing an inhibitory constant (ki) value of 239.81 nM (

Table 2. Molecular interaction of 2-acetamido-2-deoxy-hexopyranose with the binding pocket of penicillin-binding protein.

Binding Energy (kcal/mol)	Ligand Efficiency (kcal/mol)	Inhibitory Constant (Ki)	Hydrogen Bond Interaction
−5.01044	−0.38	239.81	SER 49 THR 413

). The interaction mode of chitin to PDB ID: 2Y4A with hydrogen bond interaction (SER 49 and THR 413, with 2.2 Å and 3.4 Å) is presented in Figure 8. These results validated the observed antimicrobial activity of the chitin and hence could be a promising antimicrobial inhibitor against S. aureus.

The important functional properties of drugs for action in the human body are defined by Lipinski’s druggability rule, and these properties include absorption, distribution, metabolism, excretion, and toxicity [38]. In the present study using different standalone (MedChem Designer 5.0) and online tools (pkCSM), 2-acetamido-2-deoxy-hexopyranose was assessed for its druggability, and druggability prediction showed good oral bioavailability such as distribution coefficient, Moriguchi estimation of log, nitrogen- and oxygen-based hydrogen bond acceptors, and hydrogen bond donor protons, with molecular weights of 0.953, −2.294, 221, 7, and 221.211g/mol, respectively, and all five druggability parameters are within the permitted limits, showing good bioavailability of the drug for oral administration (

Table 3. Drug ability of 2-acetamido-2-deoxy-hexopyranose.

Diff Coeff	MLogP	Mw	M_NO	HBDH
0.953	−2.294	221.211	7	5

Note: Diff coeff: distribution coefficient; MlogP: Moriguchi estimation of log; M_NO: nitrogen- and oxygen-based hydrogen bond acceptors; HBDH: hydrogen bond donor protons; Mw: molecular weight.

).

The result of pharmacokinetics properties (ADME) of the 2-acetamido-2-deoxy-hexopyranose indicated the absorption parameters such as water solubility, CaCO₂ permeability capacity, intestinal absorption, and skin permeability at −1.214 log mol/L, −0.217 log papp in 10⁻⁶ cm/s, 26.824%, and −3.137 log kp, respectively. The distribution parameter of VDss, fraction unbound, BBB permeability, and CNS permeability were −0.126 log L/kg, 0.86 FU, −0.664 BB, and −4.043 PS, respectively. The excretion of the compound showed 0.711 log ml/min/kg in total clearance. There was no recorded involvement of 2-acetamido-2-deoxy-hexopyranose in metabolic activity (

Table 4. In silico ADME properties of 2-acetamido-2-deoxy-hexopyranose.

Properties	Model Name	Predicted Value
Absorption	Water solubility	−1.214 mlog mol/L
	CaCO2 permeability	−0.217 log papp in 10−6 cm/s
	Intestinal absorption(human)	26.824% absorbed
	Skin permeability	−3.137 log kp
	P glycoprotein substrate	No
	P glycoprotein inhibitor I	No
	P glycoprotein inhibitor II	No
Distribution	VDss (human)	−0.126 log L/kg
	Fraction unbound (human)	0.86 FU
	BBB permeability	−0.664 Log(BB)
	CNS permeability	−4.043 PS
Metabolism	CYP2D6 substrate	No
	CYP3A4 substrate	No
	CYP1A2 inhibitor	No
	CYP2C19 inhibitor	No
	CYP2C9 inhibitor	No
	CYP2D6 inhibitor	No
	CYP3A4 inhibitor	No
Excretion	Total clearance	0.711 log ml/min/kg
	Renal OCT2	No

). The toxicity of the compound was tested using different toxicity parameters such as max tolerated dose, oral rat acute toxicity, oral rat chronic toxicity, Tetrahymena pyriformis, and minnow toxicity, and all the results observed were within a normal range. The values recoded for max tolerated dose, oral rat acute toxicity, oral rat chronic toxicity, T. pyriformis toxicity, and minnow toxicity were 2.179 log mg/kg/day, 1.729 mol/kg, 3.565 log mg/kg_bw/day, 0.285 log ug/L, and 5.272 log nM, respectively (

Table 5. In silico Toxicity properties of 2-acetamido-2-deoxy-hexopyranose.

AMES toxicity	No
Max tolerated dose (human)	2.179 log mg/kg/day
hERG I inhibitor	No
hERG II inhibitor	No
Oral rat acute toxicity (LD50)	1.729 mol/kg
Oral rat chronic toxicity (LOAEL)	3.565 log mg/kg_bw/day
Hepatotoxicity	No
Skin sensitization	No
T. pyriformis toxicity	0.285 log ug/L
Minnow toxicity	5.272 log nM

). The pharmacokinetics properties (ADMET) of the 2-acetamido-2-deoxy-hexopyranose indicated the nontoxic nature of this compound and hence that it could be an effective antibacterial agent for developing new novel drug against S. aureus.

4. Conclusions

The chitin (2-acetamido-2-deoxy-hexopyranose) extracted from the exoskeleton of the Caridine prawn is an effective antibacterial agent that inhibits the growth of S. aureus. The chitin-, starch-, and ascorbic-acid-mediated film produced higher antibacterial activity than chitin- and starch-mediated film. In vitro studies suggested its potential antibacterial mechanisms, druggability, and nontoxic nature. Further research on the application of 2-acetamido-2-deoxy-hexopyranose film composite in the wound healing process could lead to identification of its role as an antibacterial compound and in particular against S. aureus infection.