The Molecular Mechanisms of the Antibacterial Activity of Sumac (Rhus typhina L.) Tannin Against Pseudomonas aeruginosa

Abstract

Treatment of infections caused by Pseudomonas aeruginosa presents a challenge due to its ability to adapt and acquire drug resistance rapidly. Therefore, a key challenge is identifying and investigating new compounds with antibacterial and anti-virulence activity. Tannins, a group of plant polyphenolic compounds, can interact with bacterial cells and their virulence factors. The purpose of this study was to assess the antibacterial potential of using 3,6-bis-O-di-O-galloyl-1,2,4-tri-O-galloyl-β-D-glucose (C₅₅H₄₀O₃₄) isolated from Rhus typhina against P. aeruginosa ATCC BAA-1744. The investigation involved viability analyses using the INT assay, fluorescence analyses of the tannins’ interaction with the cell membrane and membrane proteins of P. aeruginosa, and analysis of changes in the Zeta potential. The results obtained allowed us to conclude that C₅₅H₄₀O₃₄ exhibits antimicrobial activity by inducing changes in the biophysical properties of P. aeruginosa’s cell membrane. The thermodynamic parameters indicated that C₅₅H₄₀O₃₄ binds to bacterial membrane proteins through hydrophobic interactions. These interactions with proteins may impact their structure and disrupt their functions, such as disturbing or inhibiting the efflux pumps, which are part of P. aeruginosa’s resistance mechanisms. Therefore, C₅₅H₄₀O₃₄ may be a new, natural agent and could potentially be used against P. aeruginosa.

1. Introduction

Pseudomonas aeruginosa (P. aeruginosa) belongs to Gram-negative bacteria. This opportunistic, rod-shaped pathogen can grow in different aerobic environments and demonstrates wide-spectrum tolerance in diverse environmental conditions [1]. P. aeruginosa is responsible for many infections, e.g., ear, bloodstream, urinary tract, respiratory tract, and skin and tissue infections [1], as well as nosocomial infections, pneumonia, and chronic obstructive pulmonary disease [2]. In 2017, the World Health Organization (WHO) placed P. aeruginosa on the list of “critical pathogens” that required new antibiotics [1,3].

Despite the existence of some antibiotics against P. aeruginosa, this pathogen is still challenging to treat due to the broad and different mechanisms of antibiotic resistance in this bacteria, resulting in the development of multidrug resistance (MDR) [1,2,4].

P. aeruginosa’s MDR results from many different mechanisms developed by this pathogen. Some crucial mechanisms include porins, efflux pump systems, and different classes of β-lactamase- and penicillin-binding proteins, as well as lower membrane permeability [2,4,5]. The MDR mechanisms mentioned above can generally be classified as innate immunity. On the other hand, P. aeruginosa can also develop adaptive immunity connected with biofilm formation, quorum sensing, and plasmid transfer of genes responsible for antibiotic resistance [6].

It has been described that P. aeruginosa possesses resistance to such antibiotics as carbapenem, fluoroquinolones, and third-generation cephalosporins [7], as well as aminoglycosides and β-lactams [6].

The strong pathogenicity of P. aeruginosa is related to its high resistivity and many virulence factors. One of the most important is LPS (lipopolysaccharide), which influences antibiotic tolerance and biofilm formation [8]. The second one is the secretion system, formed of six types (T1SS–T6SS) and responsible, i.a., for attachment to host cells, swarming motility, and biofilm formation by the type IV pili and flagella [9]. P. aeruginosa produces and secretes many factors, like exopolysaccharide, siderophores, proteases (e.g., alkaline proteases and elastases A and B), and toxins—for example, exolysin, ExoT, ExoU, Exotoxin A, phospholipase C, Lipase A, leucocidin or pyocyanin [9].

Due to P. aeruginosa’s pathogenicity, connected to its MDR, developing new types of antibiotics is still strongly necessary. For example, Cluck et al. described a new generation of cephalosporin [10]. This method, even though it is essential, has a serious limitation. Developing new antibiotics takes a long time, and bacteria will ultimately develop resistance to new drugs. An alternate method can be searching for and investigating other compounds that demonstrate antibacterial potential and to which bacteria will not develop resistivity. Plant polyphenols are compounds that exhibit strong antibacterial activity. Plants naturally synthesize this large group of different molecules that possess a broad spectrum of antibacterial activity against Gram-positive and Gram-negative strains.

The antibacterial potential of polyphenols occurs at different levels, i.e., at the level of the bacteria cell (including damage of the bacterial membrane and inhibition of metabolism and toxin secretion, as well as a decrease in biofilm formation), the toxin level, and the host cell level [11].

Plant polyphenols can also act against P. aeruginosa. For example, betulin and betulinic acid have antiseptic activity, inhibit the formation of P. aeruginosa biofilms, and quorum sensing controlled virulence factors [12]. Vandeputte described that catechin from Combretum albiflorum (Tul.) Jongkind (Combretaceae) bark extract reduced the biofilm activity of P. aeruginosa by around 30% [13], while Yi [14] noted that tea polyphenols induced morphological changes in P. aeruginosa cells, as well as damaging the cell membrane of this pathogen.

A very interesting sub-group of polyphenols with strong antibacterial activity is tannins. These secondary plant metabolites strongly interact with lipids, proteins, and polysaccharides, as well as possess antibacterial and antiviral activity [11,15,16]. Kaczmarek described how tannic acid possesses broad antibacterial activity against such bacteria as Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, Enterococcus faecalis, P. aeruginosa, Yersinia enterolityca, Bacillus cereus, and Listeria innocua [17], whereas Štumpf described how chestnut extract, as well as such pure tannins as vescalin, castalin, castalagin, vescalagin, and gallic acid, demonstrated antibacterial activity against S. aureus [18].

Also, we have previously demonstrated that tannins have strong antibacterial activity. For example, two hydrolysable tannins, i.e., 1,2,3,4,5-penta-O-galloyl-β-D-glucose (PGG) and 1,2,-di-O-galloyl-4,6-valoneoyl-β-D-glucose, inhibited the growth of S. aureus (the 8325-4 strain). Additionally, PGG increased the antibacterial activity of such antibiotics as oxacillin, penicillin, and methicillin. Both tannins changed the lipid order parameter and decreased the rigidity of the bacterial membrane. Additionally, these compounds strongly interacted with S. aureus membrane proteins and changed the size and chemical composition of staphylococcal membrane vesicles [19].

We have also demonstrated that PGG, 1,2-di-O-galloyl-4,6-valoneoyl-β-D-glucose, and 2-O-bis-digalloyl-4,6-valoneoyl-β-D-glucose have antibacterial potential for use against Streptococcus mutans (S. mutans), responsible for the development of dental caries [16]. The antibacterial activity of tannins is connected not only with their bactericidal or bacteriostatic activity. PGG and 1,2-di-O-galloyl-4,6-valoneoyl-β-D-glucose inhibited α-hemolysin channel formation as well as its conductivity. Also, PGG halted α-hemolysin release/secretion from S.aureus, which can be connected both with PGG interaction with bacterial membranes as well as with the influence on hla gene expression responsible for the biosynthesis of α-hemolysin [20].

As we described above, this pathogen was placed by the WHO on the list of “critical pathogens,” and the development or discovery of new anti-pseudomonas compounds is very urgent. Due to the high and well-documented antibacterial activity of tannins, the main goal of our studies is to investigate and explain the molecular mechanisms of 3,6-bis-O-di-O-galloyl-1,2,4-tri-O-galloyl-β-D-glucose interaction, a tannin that is the main compound of Rhus typhina L. (R. typhina) extract along with P. aeruginosa. Therefore the analyses at the bacteria protein and membrane level were carried out, and the biophysical and physicochemical parameters characterizing such interactions have been calculated.

2. Materials and Methods

2.1. Chemicals

Hydrolysable tannin—3,6-bis-O-di-O-galloyl-1,2,4-tri-O-galloyl-β-D-glucose (C₅₅H₄₀O₃₄) was isolated from R. typhina L. (which, according to invasive.org [21], belongs to the genus Rhus, family Anacardiaceae, order Sapindales, and class Magnoliopsida) and characterized as we described in our previous work [11,22]. INT (iodonitrotetrazolium chloride), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene (DPH), DMSO and phosphate-buffered saline (PBS) were obtained from Merck (KGaA, Darmstadt, Germany). Di-4-ANEPPDHQ was from Thermo Fisher Scientific (Walthman, MA, USA). MH broth and MH agar were from Oxoid (Basingstoke, UK). All other reagents were purchased from POCH (Gliwice, Poland).

2.2. Bacterial Strain and Growth Conditions

P. aeruginosa ATCC BAA-1744 strain (Microbiologics, MN, USA) was used for studies. Bacteria were incubated overnight at 37 °C in Mueller–Hinton (MH) broth with shaking at 200 rpm. Next, the supernatant (MH broth) was removed by centrifugation at 2300× g for 15 min, bacterial cells were diluted in PBS, and such obtained suspensions were standardized at λ = 600 nm to receive an optical density OD = 0.1 (to measure the interaction with bacterial membrane proteins and Zeta potential analyses) or OD = 0.01 (for membrane analyses).

2.3. Determination of P. aeruginosa Viability Using INT Assay

Bacterial viability was determined using p-iodonitrotetrazolium chloride (INT) by monitoring the growth of the P. aeruginosa bacteria using the microdilution method. The previous studies established the minimum inhibitory concentration (MIC) of C₅₅H₄₀O₃₄ against the P. aeruginosa ATCC BAA-1744 strain [23].

C₅₅H₄₀O₃₄ was dissolved in water at a concentration of 1000 µM. The samples were then serially diluted twice in MH broth from 500 to 15.625 µM in a 96-well plate with a final volume of 100 µL. Next, 100 µL of the bacterial suspension (1 × 10⁶ CFU/mL) was added to each well. The final concentrations of C₅₅H₄₀O₃₄ in the wells ranged from the MIC value (250 µM, according to Sekowski et al. 2023 [23]) to 1/32 of the MIC (7.8125 µM). Samples were incubated for 24 h at 37 °C. Then, 50 µL of INT dye (0.4 mg/mL) was added and incubated for 15 min at 37 °C. Next, 50 µL of DMSO was added to each well. Absorbance values were measured on a SpectraMax M2 (Molecular Device, San Jose, CA, USA) at λ = 490 nm after 30 min of incubation.

2.4. Measurements of P. aeruginosa Membrane Fluidity

The bacterial suspension (OD₆₀₀ = 0.01) was labeled with TMA-DPH or DPH at a final concentration of 1 μM (15 min, 37 °C). Next, the samples were incubated with C₅₅H₄₀O₃₄ (10 min, 37 °C) in the concentration range of 2–10 μM, and the fluorescence anisotropy signal was measured using the excitation and emission wavelengths λ_exc = 340 nm, λ_em = 430 nm for TMA-DPH and λ_exc = 348 nm, λ_em = 426 nm for DPH, respectively. Bacteria without studied compounds were taken as control. The investigations were conducted using a PerkinElmer LS-55 spectrofluorometer (PerkinElmer, Buckinghamshire, UK). Changes in the fluidity of P. aeruginosa membranes induced by tannins were assessed based on the fluorescence anisotropy values of the samples (r). The anisotropy values were calculated using the Jablonski equation [11]:

$$\mathrm{r}={\displaystyle \frac{{\mathrm{I}}_{\mathrm{V}\mathrm{V}}-\mathrm{G}{\mathrm{I}}_{\mathrm{V}\mathrm{H}}}{{\mathrm{I}}_{\mathrm{V}\mathrm{V}}+2\mathrm{G}{\mathrm{I}}_{\mathrm{V}\mathrm{H}}}}$$

(1)

where I_VV and I_VH are the vertical and horizontal fluorescence intensities, respectively, for the vertical polarization of the excitation light beam. Before analysis, the G factor (grating correction factor), which corrects the polarizing effects of the monochromator, was registered.

The obtained values were used to calculate the order parameter using the below Equation (2) [11]:

$$\mathrm{S}={\displaystyle \frac{\sqrt{\left[1-2\left(\frac{\mathbf{r}}{{\mathbf{r}}_0}\right)+5{\left(\frac{\mathbf{r}}{{\mathbf{r}}_0}\right)}^2\right]}-1+\frac{\mathbf{r}}{{\mathbf{r}}_0}}{2\left(\frac{\mathbf{r}}{{\mathbf{r}}_0}\right)}}$$

(2)

where r₀ is the fluorescence anisotropy of probes in the absence of any rotational motion of probes.

2.5. Investigation of Functional Membrane Microdomains of P. aeruginosa

Bacterial suspension (OD₆₀₀ = 0.01) was labeled with di-4-ANEPPDHQ probe at a concentration of 5 µM and incubated for 15 min at 37 °C. Next, the bacteria were incubated with C₅₅H₄₀O₃₄ (2–10 µM) for 10 min at 37 °C. Bacteria without tannins were taken as control. Fluorescence intensities di-4-ANEPPDHQ probe registered at an excitation wavelength of λ = 475 nm and an emission wavelength of λ₁ = 560 nm and λ₂ = 620 nm.

2.6. Fluorescence Analysis of Tannins Interactions with P. aeruginosa Membrane Proteins

The bacterial suspension (OD₆₀₀ = 0.1) was incubated with C₅₅H₄₀O₃₄ in the 2–10 µM concentration range for 10 min at 37 °C. Bacteria without tannins were taken as control. The fluorescence intensities of P. aeruginosa membrane proteins were determined using a Perkin-Elmer LS-55B spectrofluorometer. The readings were taken at wavelengths λ_exc = 295 nm and λ_em = 350 nm, and experiments were performed at three temperatures: 296 K (23 °C), 303 K (30 °C), and 310 K (37 °C).

Fluorescence quenching was characterized using the Stern–Volmer Equation (3) [24], and the Stern–Volmer plots were employed for its graphical representation.

$${\displaystyle \frac{{\mathrm{F}}_0}{\mathrm{F}}}={\mathrm{K}}_{\mathrm{s}\mathrm{v}}\left[\mathrm{Q}\right]+1$$

(3)

where F₀ and F—fluorescence without and with the presence of quencher; K_sv—Stern–Volmer constant; [Q]—quencher concentration

For the detection of which mechanism (static or dynamic) is responsible for both the fluorescence quenching and tannin–protein molecule interactions, the quenching constant (k_q) [24] was calculated using the following Equation (4):

$${\mathrm{k}}_{\mathrm{q}}={\displaystyle \frac{{\mathrm{K}}_{\mathrm{S}\mathrm{V}}}{{\mathrm{\tau }}_0}}$$

(4)

where k_q—quenching constant; K_SV—Stern–Volmer constant; τ₀—fluorescence life-time of fluorophore molecules (5 × 10⁻⁹ s).

To determine if the tannin–P. aeruginosa interaction is reversible, the binding constant (log K_b) was calculated based on the double logarithmic plots and double logarithmic Equation (5) [25].

$$\mathrm{l}\mathrm{o}\mathrm{g}\left[{\displaystyle \frac{({\mathrm{F}}_0-\mathrm{F})}{\mathrm{F}}}\right]=\mathrm{l}\mathrm{o}\mathrm{g}{\mathrm{K}}_{\mathrm{b}}+\mathrm{n} \mathrm{l}\mathrm{o}\mathrm{g}\left[\mathrm{Q}\right]$$

(5)

where F₀ and F—fluorescence without and with the presence of quencher; K_b—binding constant; n—number of binding sites; [Q]—quencher concentration

In order to detect which bond types are responsible for the interactions of C₅₅H₄₀O₃₄ with P. aeruginosa membrane proteins, the thermodynamic parameters of the reaction were determined using the following Equations (6) and (7) [26]:

$$\mathrm{l}\mathrm{n} {\mathrm{K}}_{\mathrm{b}}={\displaystyle \frac{∆\mathrm{H}}{\mathrm{R}\mathrm{T}}}+{\displaystyle \frac{∆\mathrm{S}}{\mathrm{R}}}$$

(6)

ΔG = ΔH − TΔS

(7)

where ΔH—enthalpy changes, ΔS—entropy changes, ΔG—free energy changes, T—temperature at Kelvin scale, R—gas constant, and lnK_b—natural logarithm of binding constant.

2.7. Analysis of ϛ-Potential

The bacterial suspension (OD₆₀₀ = 0.1) was incubated with C₅₅H₄₀O₃₄ in the 2–10 μM concentration range for 15 min at 25 °C. Changes in Zeta potential and cell size of P. aeruginosa in the presence of the test compound were measured by electrophoresis (Zeta potential) and dynamic light scattering DLS (cell size) using a Zetasiser ULTRA (Malvern, Worcestershire, UK).

2.8. Statistical Analysis

At least three independent trials were conducted for each of the experiments. The data are presented as the mean values and their mean standard deviation (X ± SD). Statistical analysis was performed using Student’s t-test (p < 0.05) using GraphPad software QuickCalc (https://www.graphpad.com/quickcalcs/ accessed on 12 November 2024). Graphs and figures were created using Origin 8.5.1 software (Northampton, MA, USA).

3. Results and Discussion

3.1. Bacterial Cell Viability of P. aeruginosa Under the Influence of C₅₅H₄₀O₃₄

It is well known that tannins have antibacterial activity. This potential can be connected with the decrease in bacterial viability. Therefore, the experiments were carried out using INT test to estimate the impact of C₅₅H₄₀O₃₄ on the viability of P. aeruginosa. This colorimetric test is based on the ability of bacteria to change in INT color from yellow to pink product, whose absorbance is analyzed. As we determined previously, the minimum inhibitory concentration (MIC) of C₅₅H₄₀O₃₄ against the P. aeruginosa was 250 µM [23]. Therefore, we used 250 µM concentration (as 1 MIC) and lower ones in these studies. Studies of P. aeruginosa viability demonstrated that at a concentration of 250 µM (equal 1MIC), the compound reduced bacterial cell viability by 70% in comparison to control (taken as 100%) (Figure 1A). In order to calculate IC₅₀ value results, they are presented as logistic functions (Figure 1B), and the designated IC₅₀ value was 78.89 ± 4.44 µM.

The literature reports the antimicrobial activity of extracts from Rhus sp. against P. aeruginosa. Borchardt et al. [27] demonstrated that R. typhina fruit extract inhibited the growth of P. aeruginosa. This result was also confirmed by Vandal et al. [28], using a 50% ethanolic extract from the fruit of the same species. Alsamri et al. [29] also described the antimicrobial activity of the aqueous and ethanolic extract of Rhus coriaria on three strains of P. aeruginosa isolated from human epithelial cells. These researchers used extracts containing multiple biologically active compounds. The results obtained in the present study are consistent with the literature data, but at the same time, indicate the strong antibacterial properties of one type of extract molecules, i.e., the tested 3,6-bis-O-di-O-galloil-1,2,4-tri-O-galloil-β-D-glucose (C₅₅H₄₀O₃₄). It should be emphasized that the antibacterial activity used in our studies is very strong. Trentin’s team [30] investigated the effect of tannin from a tree of the Anacardiaceae family (Myracrodruon urundeuva) on P. aeruginosa and showed that the MIC was 4 mg/mL. In our studies, the MIC was 250µM (equal 0.3 mg/mL).

3.2. Changes in Lipid Order Parameter in P. aeruginosa Cell Membrane

Tannins can influence bacterial activity through the interaction with their cell membrane, which occurrs by interaction with polar parts of membrane or by incorporation deeply, into hydrophobic fatty-acid tails of phospholipids. Such a process results in changes in membrane physicochemical properties [25]. Therefore, the effects of C₅₅H₄₀O₃₄ on bacterial cell membrane structure were investigated using fluorescence techniques. Two labels, TMA-DPH and DPH, located in different parts of the lipid bilayer were used to analyze changes in P. aeruginosa membrane fluidity. TMA-DPH fluorescent marker is located in the membrane’s outer (hydrophilic) part, while DPH binds to the inner phospholipid’s region (hydrophobic). Firstly, the fluorescence anisotropy changes in P. aeruginosa without and in the presence of studied compound were monitored. It was noted that the bacterial membrane became more fluid with increasing concentration of C₅₅H₄₀O₃₄. Changes were observed in both the hydrophilic and hydrophobic parts. Based on the fluorescence anisotropy, the order parameter ‘S’ was calculated according to Equation (2), presented as the ratio (S/S₀) and demonstrated below (Figure 2).

The obtained results presents concentration-dependent changes in the ordering parameter (S). The polar region of the membrane experienced strong fluidization for the highest C₅₅H₄₀O₃₄ concentration (10 µM) order parameter lowered from 1 (control) up to 0.59 ± 0.11. It can be the result from the interaction between C₅₅H₄₀O₃₄ and phospholipid polar heads on the lipid bilayer surface. Such effect was previously described for the tannins’-OH group’s interaction with phospholipids’ PO⁻² groups [26]. A lower effect was observed for non-polar membrane parts where, for the highest C₅₅H₄₀O₃₄ concentration, the order parameter decreased from 1 (control) up to 0.86 ± 0.10. There were similar effects for PGG and dGVG interaction with S. aureus (8325-4) [19]. The fluidization of the bacterial membrane may lead to modifications in membrane transport processes and increased permeability, resulting in a loss in cell integrity [30]. For example, resveratrol derivatives [31], catechins [32], and rutin [33] were capable of damaging bacterial cell membranes. Also, Trentin et al. [30] demonstrated that tannins isolated from three South American trees disrupted the membrane structure of P. aeruginosa, leading to a rapid loss of bacterial viability.

3.3. Analysis of P. aeruginosa Membrane Microdomains in the Presence of C₅₅H₄₀O₃₄

Bacterial microdomains are highly active cell membrane areas, rich in sterols (e.g., cholesterol) and sphingolipids. Their function is to segregate or concentrate specific membrane proteins and lipids. The above results clearly demonstrate that C₅₅H₄₀O₃₄ interacts with P. aeruginosa membrane and changes its physicochemical properties. Such interaction can additionally trigger the formation of membrane microdomains. Therefore, it was also investigated if C₅₅H₄₀O₃₄ influences the level of membrane microdomains in P. aeruginosa cells.

It was observed (Figure 3) that C₅₅H₄₀O₃₄ caused stiffening and increased the level of membrane microdomains in P. aeruginosa in a concentration-dependent manner. For the most significant amount of C₅₅H₄₀O₃₄, the microdomain level increased from 1 to 1.60 ± 0.06.

The explanation of microdomain formation in bacteria is complex. There are only a few studies on bacterial microdomains and the most extensively studied Gram-negative bacteria so far include Helicobacter pylori, Campylobacter jejuni, and Borrelia burgdorferi [34,35]. We assume that P. aeruginosa, under stress caused by the antimicrobial action of the C₅₅H₄₀O₃₄, began synthesizing microdomains as a defense mechanism. According to Borisova, tannins can impact membrane integrity by interacting with proteins and lipids or forming membrane pores [36]. Additionally, the compound may affect sterol levels or embedding itself in the membrane, as confirmed by studies using TMA-DPH and DPH markers, contributing to local phospholipid aggregation, acting as a binding molecule, and leading to an increase in microdomain levels compared to the control.

3.4. Fluorescent Analysis of C₅₅H₄₀O₃₄ Binding to Membrane Proteins of P. aeruginosa

The biological activity of tannins is connected with their strong interaction with lipids and proteins, including proteins anchored in the membrane structure. Tannin–protein interactions can be also responsible for the tannins’ antimicrobial activity [37]. Therefore, it was investigated if C₅₅H₄₀O₃₄ interacts with membrane proteins of P. aeruginosa. In studies, the changes in fluorescence intensity descended from tryptophan in proteins were monitored. Tryptophan (Trp) fluorescence is highly sensitive to changes in their microenvironment. It allows for analyzing structural changes in the protein molecules [19].

As can be observed in Figure 4, C₅₅H₄₀O₃₄ was responsible for a concentration-dependent decrease in Trp fluorescence intensity at all three temperatures. Based on the obtained results, it was concluded that the studied tannin has a strong affinity to the membrane proteins of P. aeruginosa. C₅₅H₄₀O₃₄ interaction with proteins can trigger the changes in the protein’s secondary and tertiary structure, as well as, disturb protein functions, which, in the case of membrane proteins like efflux pumps (one from P. aeruginosa virulence factor responsible for MDR) can partially or entirely inhibit their activity leading to “turn off” one of the MDR crucial factors. Similar observations were described for other polyphenols whose activity led to the inactivation of several virulence factors in S. aureus [14,38].

The above results showed that C₅₅H₄₀O₃₄ interacts with P. aeruginosa membrane proteins. Some physicochemical parameters were calculated using Stern–Volmer plots to describe these interactions and better explain their mechanisms (Figure 5A,C,E). It allowed us to obtain the K_SV constants, which informed us about the quencher’s affinity to the quenched molecules. Received Stern–Volmer constants (

Table 1. Binding parameters of the C₅₅H₄₀O₃₄–protein interaction of P. aeruginosa membranes for C₅₅H₄₀O₃₄.

Binding Parameters	C55H40O34
	Temperature [K]
	296	303	310
KSV [M−1 × 105]	1.66 ± 0.10	1.65 ± 0.10	1.76 ± 0.17
kq [M−1 s−1 × 1013]	3.32 ± 0.20	3.30 ± 0.20	3.53 ± 0.34
Log Kb	5.28 ± 0.21	5.46 ± 0.23	5.56 ± 0.47

) indicate a strong affinity of C₅₅H₄₀O₃₄ to the membrane proteins of P. aeruginosa.

To determine whether C₅₅H₄₀O₃₄ causes static quenching (result in tannin–protein complex formation) or dynamic quenching (tannin–protein collisional encounters), the quenching constant (k_q) was calculated using Equation (4). The k_q values for all studied temperatures (see Table 1) were larger than the maximum collision quenching constants (2 × 10¹⁰ M⁻¹ s⁻¹), which suggests that the fluorescence quenching occurs through a static mechanism. It allowed us to conclude that used C₅₅H₄₀O₃₄ tannin formed complexes with the membrane proteins of P. aeruginosa. Our results are in good agreement with the other works. For example, Czerkas et al. demonstrated that hydrolysable tannins (PGG, dGVG, and b-dGVG) form complexes with the membrane proteins of S. mutans [16]. Olchowik-Grabarek described that PGG as well as dGVG formed complexes with S. aureus membrane proteins [19] as well as with staphylococcal toxin (alpha-hemolysin) [20].

To find out whether the complex formation between C₅₅H₄₀O₃₄ and P. aeruginosa membrane proteins is reversible, the binding constants (log K_b) were determined (Table 1). The obtained logarithmic values ranged between 5.2 and 5.6, which for non-logarithmic values falls within the range of 10⁴–10⁶. The interaction is reversible when non-logarithmic values are between 1 and 15 × 10⁴ M⁻¹ [39]. The binding constants obtained, after being converted from logarithmic values, exceed the range of 1–15 × 10⁴ M⁻¹, suggesting more robust and more durable binding, which could indicate irreversible binding of C₅₅H₄₀O₃₄ to the membrane proteins of P. aeruginosa.

3.5. Thermodynamic Parameters of C₅₅H₄₀O₃₄-Membrane Protein Interactions in P. aeruginosa

Different ligands and biomacromolecules can interact through hydrogen bonds, hydrophobic interactions, van der Waals forces, and electrostatic interactions [40,41,42]. To define which type of interaction is responsible for C₅₅H₄₀O₃₄ affinity to P. aeruginosa membrane proteins, the thermodynamic parameters (ΔH, ΔS, ΔG) were determined (

Table 2. Thermodynamic parameters of C₅₅H₄₀O₃₄–protein bonds of P. aeruginosa membranes.

Temperature [K]	ΔH [kJ × mol−1]	ΔS [kJ × mol−1 K−1]	ΔG [kJ × mol−1]
296	56.689	0.293	−29.892
303			−31.939
310			−33.987

) using the Van ’t Hoff equation (Equation (6)) and Van ’t Hoff plot was shown in Figure 6. Hydrophobic interactions occur when the value of enthalpy (ΔH) and entropy (ΔS) is positive. On the other hand, if both ΔH and ΔS have a negative value, it suggests the presence of van der Waals interactions and hydrogen bonds. When ΔH < 0, and ΔS > 0, then the electrostatic forces are engaged in the interaction [26].

Calculated thermodynamic parameters allowed us to conclude that the interactions between C₅₅H₄₀O₃₄ and the P. aeruginosa membrane proteins are the results of hydrophobic interactions (ΔH > 0 and ΔS > 0), which are observed when a non-polar aromatic ring in phenolic compounds engages in hydrophobic interactions with hydrophobic amino acid residues in proteins [43]. An example is the study by Zhang et al. [44], which confirmed hydrophobic interactions between curcumin and myosin. Also, Czerkas described that dGVG interaction with S. mutans membrane proteins has a hydrophobic nature [16].

By calculating enthalpy and entropy, it is also possible to determine the free enthalpy, also known as Gibbs free energy (Table 2). Free enthalpy provides information about the binding process that can be spontaneous (for ∆G < 0) or not (∆G > 0) [26]. Since C₅₅H₄₀O₃₄ interactions received ∆G < 0, it can therefore be concluded that interaction occurred spontaneously. Interestingly, the value of this parameter decreases with increasing temperature, which may suggest more favorable thermodynamic conditions for the tannin–protein interaction at higher temperatures.

3.6. Zeta Potential and Bacterial Cell Size Determination

The described results clearly present that C₅₅H₄₀O₃₄ interacts strongly with P. aeruginosa cells. Such interactions can modify bacterial Zeta potential and their cell size. In order to study if C₅₅H₄₀O₃₄ can induce changes at the P. aeruginosa size as well as surface Zeta potential, these two values have been measured, and the results are demonstrated in

Table 3. Zeta potential and particle size changes in P. aeruginosa membrane proteins in the presence of C₅₅H₄₀O₃₄. Data represented as mean ± SD, n = 6.

Concentration [µM]	0	2	4	6	8	10
Zeta potential [mV]	−14.75 ± 1.26	−16.36 ± 1.55	−16.44 ± 1.61	−17.31 ± 1.57	−16.58 ± 1.42	−16.62 ± 1.56
Cell size [nm]	1192.30 ± 139.09	1134.85 ± 173.89	1220.99 ± 185.52	1298.66 ± 191.28	1305.81 ± 227.61	1237.64 ± 126.68

. It should be emphasized that the Zeta potential is an important physicochemical parameter that plays a crucial role in the interactions of microorganisms with various ions and molecules. It also provides information on membrane permeability, biofilm formation, and aggregation under the influence of various compounds [45].

The results indicated that C₅₅H₄₀O₃₄ slightly decreased the Zeta potential of the studied bacteria compared to the control bacteria but generally did not induce the changes in P. aeruginosa’s diameter. Small alterations of P. aeruginosa size were observed in the presence of C₅₅H₄₀O₃₄, which are negligible compared to the control. The other studies also observed Zeta potential changes in bacteria cells under the influence of polyphenolic compounds. For instance, [46] demonstrated that ferulic acid, rosmarinic acid, and epigallocatechin gallate affect the Zeta potential, reducing adhesion in P. aeruginosa, Bacillus cereus, and Staphylococcus aureus. Tannins revealed also the ability to change Zeta potential in S. mutans cells [16] as well as in S. aureus cells [11].

4. Conclusions

Tannins belong to plant polyphenols and have strong antibacterial activity. In this study, it was demonstrated that C₅₅H₄₀O₃₄ has strong antibacterial properties against P. aeruginosa. Observed antibacterial activity is the result of influence on the various structural components of the bacteria, including membrane proteins and phospholipids. Specifically, C₅₅H₄₀O₃₄ reduces bacterial cell viability and disturbs the membrane’s hydrophilic and hydrophobic sections, potentially leading to increased permeability and compromised cellular integrity. Additionally, the compound has been shown to interact with membrane proteins through a static mechanism, forming tannin–protein complexes. This interaction can modify the structure and function of the proteins, ultimately contributing to the inhibition of bacterial resistance mechanisms such as efflux pumps. The study highlights that C₅₅H₄₀O₃₄ can form stable complexes with P. aeruginosa membrane proteins via spontaneous hydrophobic interactions between the compound and bacterial proteins. Furthermore, C₅₅H₄₀O₃₄ induces changes in the Zeta potential of P. aeruginosa cells. Our highly innovative studies demonstrate that 3,6-bis-O-di-O-galloyl-1,2,4-tri-O-galloyl-β-D-glucose possesses strong antibacterial activity against P. aeruginosa. According to the obtained results, it can be concluded that C₅₅H₄₀O₃₄ possesses strong antibacterial activity against P. aeruginosa with application potential in treating infections caused by P. aeruginosa, especially in the context of multidrug resistance. Also, it can be assumed that one of the possible modes of C₅₅H₄₀O₃₄ antibacterial action is connected with P. aeruginosa membrane lipids and proteins, but further studies are needed to verify if C₅₅H₄₀O₃₄ activity can occur via other mechanisms, e.g., like inhibition of electron transport, cell wall synthesis or ribosomal function.