Anti-proteolytic activity of Ganoderma lucidum methanol extract against Pseudomonas aeruginosa

Introduction: Protease enzyme is considered one of the most serious virulence factors produced by extended-spectrum β-lactamase-producing and multidrug-resistant Pseudomonas aeruginosa (ESβLMDRPA) clinical isolates. Methodology: The antibacterial activity of methanol extract of Ganoderma lucidum fruiting bodies was tested against a protease-producing ESβLMDRPA clinical isolate, showing its mode of action. Results: The extract showed high antibacterial activity. Its effect on purified protease indicated a reversible non-competitive protease inhibition (kis= 0.45 mg/mL). Conclusions: The G. lucidum extract could be a promising anti-proteolytic active against ESβLMDRPA. It may form a primary platform for further phytochemical studies and development of new drugs for therapy of skin burn infections.


Introduction
Recently, multidrug-resistant (MDR) Gramnegative bacteria have become more prevalent and are causing great problems in the treatment of infections [1].Of clinical significance, Pseudomonas aeruginosa has the ability to secrete several virulence factors, which help the bacteria to adhere and invade to their host by damaging the host's immune responses and forming a barrier to antibiotics [2].Proteases are one of the most crucial virulence factors produced by P. aeruginosa.These proteases work together, leading to significant damage to host tissues.
Ganoderma lucidum is an oriental fungus; it has a long history of use for promoting health and longevity in China and other Asian countries.It is a popular medicinal mushroom that has many biologically active components such as phenolics, flavonoids and polysaccharides, giving its activity as antimicrobial, antioxidant, antiviral, cardiovascular and anticancer [3,4].
Not much literature is available about the antibacterial activity of the fruiting body of G. lucidum for the treatment of ESβLMDRPA which produces protease as a virulence weapon.Therefore, the present study aimed to evaluate the efficiency of G. lucidum extract as a novel alternative and a promising method for virulence attenuation of protease-producing ESβLMDRPA.

Detection of protease activity
An ESβLMDRPA clinical isolate of a burn infection, previously recovered and identified by Khalil et al. [5], was tested for its ability to produce protease enzyme as a virulence factor.Vermelho et al. [6] described the protease activity method.

Production of protease from ESβLMDRPA
Production of protease from ESβLMDRPA was carried out in a proteolytic liquid medium (Sigma Chemicals, St. Louis, USA).The supernatant was extracted for crude enzyme preparation [7].

Protease assay
Proteolytic activity was determined using 0.6 % Hammerstein casein solution (Merck, Darmstadt, Germany) as a substrate (50 mMTris-HCl, pH 7).For activation, 1 mL of the extracted enzyme was mixed with 5 mL of the prepared substrate and incubated at 30 °C for 30 minutes.The reaction was stopped by the addition of 0.5 mL 20 % TCA (Pharmacia, Gothenbu, Sweden) and kept for 10 minutes at 25 °C.After 15 minutes of cooling centrifugation (6.000 rpm), the absorbance was measured at 280 nm.One unit of protease activity was defined as the amount of enzyme required to liberate 1 μg tyrosine per milliliter per minute.The experiments were carried out in triplicate and the mean value was expressed as a unit of protease activity [8].

Ammonium sulphate precipitation
ESβLMDRPA cells were separated by centrifugation (6.000 rpm for 10 min).The supernatant was fractionated by precipitation using ammonium sulfate (50 to 70 % saturation).All precipitates were collected and dialyzed against 50 mM Tris-HCl buffer (pH 7) for 24 h using a dialysis bag.The highest ammonium sulphate fraction was applied gel filtration chromatography [9].

Gel filtration chromatography on DEAE-Sephadex A-50
The dialyzed highest ammonium sulphate fraction was applied on a DEAE-Sephadex A-50 column (GE Health care Biosciences, Uppsala, Sweden) with dimensions 1.5 cm diameter and 20 cm length.The column was pre-equilibrated with 50 mM Tris-HCl (pH 8).The sample was eluted with the same buffer.Ten fractions were collected at a flow rate of 1 mL/minute constant intervals.The most active fractions (6-8) were dialyzed against 50 mM Tris-HCl (pH 7) at 4 °C.

Protein assay and electrophoretic analysis
The protein concentrations in different stages of enzyme purification were estimated according to Bradford [10].

Preparation of fungal extract
The fruiting bodies of G. lucidum were collected from around the Delta region of Gharbia governorate.The identification and extraction were done as described by Kamra and Bhatt [11].

Effect of G. lucidum extract on ESβLMDRPA
Antibacterial activity of G. lucidum extract was measured by using the modified agar well diffusion method [12].One hundred microliters of inoculum (10 7 colony-forming units/mL) using standard turbidity (corresponding to 0.5 McFarland tube) was applied on Muller-Hinton agar plate (Sigma Chemicals, St. Louis, USA).For antibacterial screening, G. lucidum extract was dissolved in dimethyl sulfoxide to a final concentration of 0.0, 0.5, 1.0, 1.5, 2.0, 2.5 mg/mL.Regular wells were made in the inoculated agar plates by a sterile cork borer with 0.8 cm diameter.Each well was aseptically filled up with 0.1 mL volume of extract.The plates were incubated at 37 °C for 24 h.The tests were performed in triplicate.

Mode of inhibition of G. lucidum extract on protease enzyme
The evaluation of G. lucidum extract as a protease inhibitor against ESβLMDRPA was conducted in liquid protease-dependent medium (Sigma Chemicals, St. Louis, USA), as described by Ferrasson et al. [7].Briefly, to each test tube containing 5 mL fractions of medium, about 50 µL of different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 mg/mL) of G. lucidum extract were separately added.Then, the tubes were inoculated with 50 µL of ESβLMDRPA (10 7 colony-forming units/mL) and incubated at 37 °C for three days (the optimum time for protease production).The control tube was free from G. lucidum extract.Quantitative assay of protease activity was measured as described by Khembhavi et al. [8].
The mode of inhibitory action of G. lucidum extract against ESβLMDRPA was tested by using the purified protease dissolved in 50 mM Tris-HCl buffer (pH 7).Quantitative assay of protease activity was determined by mixing different concentrations of G. lucidum extract (0.5, 1.0, 1.5, 2.0, 2.5 mg/mL) and casein substrate (5, 10, 15, 20, 25%) [8].Results were represented graphically by a reciprocal plot between enzyme activity and substrate concentration for each G. lucidum extract concentration [13].

Statistical analysis
In analyzing the data of ESβLMDRPA protease enzyme production and the effect of G. lucidum extract on enzyme activity through analysis of variance (ANOVA), one-way tests by SPSS version 17 were used to evaluate the variation in concentrations of G. lucidum extract on bacterial growth and its enzyme activity.

Results
The ESβLMDRPA protease enzyme was found to possess high proteolytic activity, which appeared as clear areas (Figure 1A), indicating the hydrolysis of the bovine serum albumin (BSA) compared to the control (blue color of BSA) (Figure 1B).The highest significant rate of protease production (27 units/mL) was quantitatively reported in the culture filtrate of ESβLMDRPA on the third day of growth (p < 0.001).In protease purification, the highest active fraction from 70 % ammonium sulphate of culture broth was adsorbed on the DEAE-Sephadex A-50 matrix (GE Health care Biosciences, Uppsala, Sweden).The bound protease was eluted with 10 mM Tris-HCl buffer (pH 7). Figure 2 shows that fractions 6-8 were the most active fractions of ESβLMDRPA protease activity, which confirmed by the appearance of a single protein band at molecular weight 40 kDa during the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Figure 3).After purification, protease enzyme indicated specific activity of 84.6 units/mg from 9 units/mg with a purification fold of nearly 9.4 times that of the crude enzyme (Table 1).
A trial was conducted to find a correlation between protease activity and high inhibition effect of G. lucidum extract against ESβLMDRPA as an important offensive force for the bacteria to establish the infection.The minimum inhibitory concentration (MIC) of G. lucidum extract against ESβLMDRPA was recorded as 2 mg/mL.The highest concentration of G. lucidum extract was accompanied by the lowest activity in protease, which recorded as decreasing from 27 to 3   units/mL at MIC concentration 2 mg/mL (Table 2).In addition, statistical analysis revealed that the variation in the effect of different concentrations of G. lucidum on protease activity was highly significant (p < 0.001).
The highest antiprotease activity of G. lucidum extract could explain its ability to manage infection.As shown in Figure 4A, G. lucidum extract was a reversible, noncompetitive protease inhibitor.Its activity was decreased by increasing substrate concentrations, as confirmed by Km (Michaelis constant) which achieved at 9.7 units/mL.This high Km shows the low affinity between enzyme and substrate.Plotting the slope of the double reciprocal plot of G. lucidum extract inverse against the inverse of protease initial activity indicated an inhibition constant (kis) of 0.45 mg/mL due to the effect of G. lucidum extract on an enzyme reaction slope (Figure 4B).

Discussion
In the present work, the major protease yield of P. aeruginosa with ammonium sulphate is supported by the results of Kumar et al. [14] for Bacillus thuringensis with ammonium sulphate (65% saturation).
SDS-PAGE analytical studies of protease revealed that it possesses a single protein band with molecular weight of 40 kDa.This agrees with P. aeruginosa as described by Gupta et al. [15].
In a trial to explain the ability of G. lucidum extract to manage infection, it was observed that G. lucidum extract has a strong antiprotease activity.Our study determined that the kinetic properties of G. lucidum extract act as reversible non-competitive inhibitor for protease enzyme, in accordance with Arulpandi and Sangeetha [16], who reported reversible non-competitive inhibition of protease by using Cassia fistula leaf extract.

Conclusions
This novel study of G. lucidum as a protease inhibitor suggests that G. lucidum extract is a promising new active source against ESβLMDRPA, which could be a major causative agent of burn infections.

Figure 3 .
Figure 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for different stages of protease purification.ASP: ammonium sulphate precipitation

Table 2 .
Inhibitory effect of G. lucidum on ESβLMDRPA.Each value is the mean of three replicates ± SD; Values with the same letter in the same column are insignificant at p ≤ 0.05.