Cannabidiol Interactions with Outer Membrane Proteins in Salmonella Typhimurium LT2

doi:10.21203/rs.3.rs-4858257/v1

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Cannabidiol Interactions with Outer Membrane Proteins in Salmonella Typhimurium LT2

https://doi.org/10.21203/rs.3.rs-4858257/v1

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Cannabidiol (CBD), the non-psychoactive component of the hemp plant has tremendous potential as a novel antimicrobial agent. This study aimed at understanding the interactions between CBD and the outer membrane proteins (OMPs) of Salmonella Typhimurium LT2. Employing in silico techniques, we analyzed the binding affinities, interaction dynamics, and drug-likeness of CBD with key OMPs such as OmpA, OmpC, OmpD, OmpF, OmpX, and NompC. The molecular docking results showed that CBD exhibits varying binding affinities across the OMPs, with OmpX and NompC showing the highest binding affinity of -6.6 kcal/mol and − 6.4 kcal/mol respectively, indicating strong and stable interactions. The results also revealed several key interactions such as hydrogen bonds, Pi-stacking, and hydrophobic interactions, playing crucial roles in the stability and specificity of these protein-ligand complexes. Notable interactions were identified in OmpA with a binding affinity of -5.9 kcal/mol involving hydrogen bonds at 3.2 Å and Pi-Sigma interactions at 3.4 Å. We included phylogenetic analysis of fifty different strains of Salmonella Typhimurium, and we observed high conservation levels among the OMPs, with a sequence similarity threshold of 90%. This high conservation underscores the potential of CBD to act as a broad-spectrum antimicrobial agent. Furthermore, our comparative structural analysis revealed both conserved and variable regions within the OMPs, highlighting the significance of targeting these regions to mitigate resistance development. Using KEGG Pathway analysis, we analyzed OmpC and OmpF, given their roles in nutrient transport and permeability. The disruption of these pathways by CBD binding could impair the bacteria’s ability to manage environmental stresses and evade host immune responses. Beta-lactam resistance pathway analysis was also considered, we observed that CBD could potentially disrupt resistance mechanisms by binding to OMPs, enhancing the efficacy of existing antimicrobial treatments. In conclusion, our findings suggest that CBD, through its interaction with critical OMPs, has the potential to serve as a potent antimicrobial agent against Salmonella Typhimurium LT2. These findings lay the foundation for further studies of CBD as a novel therapeutic agent in combating bacterial infections and addressing the global challenge of antibiotic resistance.

General Microbiology

Outer membrane proteins

Gram-negative bacteria

Antibiotic resistance

Cannabidiol

Phylogenetics

In silico

Salmonella Typhimurium is a recalcitrant pathogen that remains the leading cause of foodborne infections worldwide. Salmonella outer membrane proteins (OMPs) significantly affect its pathogenicity. Omps is primarily involved in a diverse role in structural integrity, nutrient uptake, and immune system interactions (Futoma-Kołoch et al., 2019). These proteins facilitate the bacterium's survival in harsh environments and invasion of host immune defenses. It is incumbent to understand the dynamic roles of these Omps; hence, understanding their function is crucial in the face of the ever-rising incidence of antibiotic resistance (Chen et al., 2020). Omp's are pertinent to the survival and virulence of Salmonella enterica, and they play an enormous role in nutrient uptake and transport, an essential process for the growth and survival of the bacteria within its host. Omps such as OmpC and OmpF are proteins that form channels and allow the passive diffusion of minute hydrophobic molecules, such as essential nutrients, across the plasmalemma. This enhances the bacterium's ability to thrive in nutrient-limited environments such as the host gut (Chamachi 2021).

Furthermore, OMPs play a crucial role in bacterial structural integrity. Salmonella Typhimurium and other Gram-negative bacteria have an outer membrane consisting of an asymmetric bilayer with phospholipids on the inner leaflet and lipopolysaccharides on the outer leaflet, creating a formidable barrier against hostile environment and antimicrobial agents (Yang et al., 2020). On the other hand, OmpA and OmpD are anchor proteins that support the outer membrane with peptidoglycan layers, providing structural support and ensuring the bacteria's survival under mechanical and chemical stress (McClean, 2012). Additionally, OMPs are crucial in association with the host immune system. An immune response is triggered when the host recognizes these proteins (Roy et al., 2022). Nevertheless, some OMPs, such as OmpX, can evade immune detection and reduce the visibility of the bacteria to the host immune response (Tsaplina, 2024). At the same time, OmpA can interact with the host cell receptors to enhance invasion and cause infection (Confer, and Ayalew, 2013). Bacteria require these interactions to establish a niche within their host and evade immune clearance (Nie et al., 2020).

In our quest to explore CBD's potential as a novel antimicrobial agent, we must examine the interactions between Salmonella Typhimurium OMPs and CBD. This will provide a valuable insight into the mechanisms fostering CBDs antimicrobial potential. We hypothesize that CBD can interact with and disrupt the function of Salmonella Typhimurium, thereby mutilating its ability to accommodate and transport nutrients, maintain structural integrity, and evade host immunity. To achieve this, we used computational studies to determine the binding affinities of CBD to various Salmonella Typhimurium OMPs such as OmpA, OmpC, OmpD, OmpF, OmpX and NompC. We identified and characterize the binding sites and residues influencing the interactions between CBD and the OMPs. We also employed root mean squares deviation analysis to assess the structural stability and conformational alterations following CBD and OMP binding (Gesualdo et al 2021). Finally, we explored the genetic diversity and evolutionary relationships of the OMPS among varied Salmonella enterica strains using phylogenetic analysis.

Thus, providing deeper understanding of CBD interactions with Salmonella OMPs. This knowledge may help unearth new realms in antimicrobial therapy, potentially providing effective treatments against bacterial infections and reducing the rampant incidence of antibiotic resistance.

Protein Selection and Preparation

We initiated our exploration by selecting and preparing various outer membrane proteins that play a crucial role in the pathophysiology and pathogenicity of Salmonella Typhimurium LT2. Thus, the proteins of interest for this study included OmpA, OmpC, OmpD, OmpF, OmpX, and NompC. These OMPs were chosen based on their significant contribution to the bacterial cell envelope's structural integrity and other essential functions and significance in antibiotic resistance. We used UniProtKB (https://www.uniprot.org/) and NCBI databases (https://www.ncbi.nlm.nih.gov/) to retrieve the sequences of these OMPs. UniProtKB and NCBI are comprehensive public resources for protein and nucleotide sequences. A Protein Data Bank (PDB) was used to retrieve the 3D (three-dimensional) structures of these OMPs. In cases where we could not retrieve the 3D structures from PBD, we employed Swiss-Model (https://swissmodel.expasy.org/) (a web-based tool for structure homology modeling) for homology modeling. AutoDock tools were used to prepare the structures for docking after retrieving them. The preparation involves several significant steps to ensure the proteins are optimal for binding studies. Thus, we assigned protonation states to the amino acids by adding polar hydrogens, removing all water molecules, and adding Kolman charges. The structures were optimized to ensure accuracy in the subsequent docking studies.

Ligand Preparation

PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/644019#section=2D-Structure) (a free chemistry database managed by NCBI) was used to retrieve our compound of interest (CBD). We optimized CBDs' structure and prepared it to dock using the Pymol Visualization tool. This ensures that CBD was converted to PBD format, and the right protonation state was maintained to minimize its energy. A crucial step for obtaining accurate and reliable docking results.

Molecular Docking and Binding Affinity Calculations.

AutoDock and AutoDock Vina were used to predict the interactions/binding of CBD to the selected OMPs. Thus, the grid parameters were set around the active sites of the proteins to maximize the docking search space. Discovery Studio (https://discover.3ds.com/discovery-studio-visualizer-download) was used to predict the optimal binding site for each protein, and the binding site parameters were used to set up the grid parameters in AutoDock. The ligand (CBD) was then docked into the interaction site for each OMP. Multiple poses were generated to sample the conformational space comprehensively, and the best pose was selected based on the binding score and root mean square deviation (RMSD) values (both lower and upper bound) and visual inspection of the binding. This ensures that the most accurate representation of the binding mechanisms is achieved. We calculated the binding affinities of CBD to each protein using AutoDock Vina and AutoDock mgl tools. These provide us with quantitative data on the relative strength of each protein interaction with CBD. We interpreted each result to ascertain the best or strongest affinities and critical residues involved in the interaction.

Interaction Analysis

Discovery Studio was also used to visualize the protein-ligand complex of the best binding affinities (those with the best binding pose, usually pose 1) to generate 2D and 3D interactive diagrams. These visualizations enabled us to identify critical binding residues that played a significant role in the CBD-OMP’s interactions. The visualization of these interactions also helped us to ascertain the stability and conformational alteration of the complexes.

Phylogenetic Analysis

Fifty different strains of Salmonella enterica, including Typhimurium LT2, were selected and explored the evolutionary relationship and genetic conservation among the OMPs using phylogenetic analysis. Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo) was used to align the sequences of the different strains, and phylogenetic trees were built to represent the evolutionary landmark of the strains. Jalview (https://www.jalview.org/download/) was used to visualize the multiple sequence similarities among the proteins. Thus, after retrieving the protein sequences from PDB, we loaded them onto Jalview to create a visual representation of the conserved and variable regions. In cases where protein sequences were unavailable in the protein databank, we retrieved the DNA sequences from NCBI databases. Then we converted the nucleotide sequences into the corresponding peptide sequences using Expasy Translate Tool (https://web.expasy.org/translate/).

Comparative Structural Analysis

For the comparative structural analysis of the OMPs, Swiss-Model, and pairwise protein structure alignment tools were used. We aligned the structures and identified the conserved and divergent regions. The Swiss-Model generated 3D structures of proteins whose crystal structures were unavailable in PBD. Hence, we obtained the nucleotide bases from UniprotKB and NCBI databases. The generated nucleotide bases were loaded onto the Swiss-Model page, and the best and most probable 3D conformations based on homologous protein structures were predicted. This ensured we used only accurate structural representations of the proteins for further analysis. Following the prediction of the protein structures, we used the pairwise protein structure alignment tool to compare the various OMPs. This allowed us to superimpose the 3D models of the varied OMPs. OmpD was chosen as the reference protein because it is well characterized, and all the other OMPs were aligned against OmpD. These alignments reveal regions of divergence and convergence.

Pathway Mapping

For the pathway analysis, we narrowed down to only OmpC and OmpF because only these two have their pathways retrievable form the KEGG database (https://www.genome.jp/kegg/). Using the protein sequences retrieved from UniProtKB and NCBI databases, the sequences of the two proteins were uploaded to the BlastKOALA search box in the KEGG homepage to generate the annotated data. Using the KO values, we accessed the KEGG pathways such as the two-component systems and the beta-lactam resistance. After the generation of the various pathways, we trimmed down the two-component system pathway to cover only relevant pathways in Salmonella Typhimurium LT2 pathogenesis.

Data Analysis and Interpretation

All data from docking studies, binding affinity calculations, interaction analyses, phylogenetic studies, and pathway mapping were compiled and thoroughly analyzed. We used MS Excel (2016 version) and R 4.4.1 for all quantitative data graphs generated. The results were interpreted to evaluate CBD's potential as a broad-spectrum antimicrobial agent against Salmonella enterica.

Figure 1 presents the 2D chemical structure of Cannabidiol (CBD), obtained from PubChem. The figure details the molecular structure and various resolution parameters, highlighting the chemical configuration of CBD that facilitates its interaction with outer membrane proteins. The CBD molecule, characterized by its hydroxyl groups and aromatic rings, is shown in multiple resolutions to emphasize its structural details, crucial for understanding its binding potential with bacterial proteins.

Figure 1a: 2D structure of CBD retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/644019).

The Ramachandran plot for OmpX and OmpD are illustrated in Fig. 1b A and C and highlights the detailed analysis of the phi (Φ) and psi (Ψ) torsion angles of the amino acid residues in two modeled OMPs. The figure showed the favored regions for secondary structures. The green-shaded areas in the plot are regions where β-sheets are likely to form. The data points within these regions indicate that OmpX and OmpD has residues that adopt β-sheet conformations. The less densely shaded areas may suggest regions where α-helices are present, although for OmpX, the emphasis is on β-sheets as compared to OmpD. Figure 1b B and D represents the 3D structures of OmpX and OmpD showing how the proteins are embedded in the outer membranes. Both proteins are characterized by a transmembrane β-barrel structures (a common feature of outer membrane proteins), with Global Model Quality Estimation (GMQE) score of 0.66 and 0.75, and the QMEANDisCo Global scores of ± 0.07 and ± 0.05 for OmpX and OmpD respectively.

Figure 2 illustrates the surface interactions of outer membrane proteins OmpA and OmpC with and without the presence of Cannabidiol (CBD). The structural data for these proteins were retrieved from the RCSB Protein Data Bank and supplemented with models generated using the Swiss-Model repository. The presence of CBD appears to induce conformational changes in the surface topology of both OmpA and OmpC, suggesting potential binding interactions that could alter their function.

Similar to Fig. 2, this Fig. 3 shows the surface interactions of OmpD and OmpF with and without CBD. The structural data were sourced from the RCSB Protein Data Bank, with additional models created via Swiss-Model. The interaction with CBD results in noticeable changes in the surface structure of these proteins, which could impact their permeability and function in the bacterial membrane.

Figure 4 depicts the surface interactions of NompC and OmpX with and without CBD. The protein structures were obtained from the RCSB Protein Data Bank, complemented by models from Swiss-Model. The CBD binding appears to influence the surface morphology of these proteins, indicating possible sites of interaction that could affect their biological activity.

The binding affinities of the different outer membrane proteins (OMPs) from Salmonella Typhimurium LT2 with CBD are presented in Fig. 5. These binding scores represent the best binding affinities (best poses 1) from eight different poses for each OMP in this study. The results showed that OmpX exhibits the highest (strongest) binding affinity at -6.6 kcal/mol. The New outer membrane protein C follows with the strongest binding affinity at -6.4 kcal/mol. OmpF has the lowest binding affinity to CBD with affinity of -5.7 kcal/mol. These results suggest that CBD exhibits varying levels of interaction with different OMPs.

We included docking analysis carried out using AutoDock Vina to compare to docking scores of PyRxd Vina as shown in the histogram below for the OMPs used illustrated in Fig. 7. The comparative analysis reveals that AD has consistent stronger binding affinities as compared to Vina across all the OMPs. For instance, the cluster of OmpA is between − 6.5 to -6.0 Kcal/mol for AD, and ranges between − 5.9 to -4.7 Kcal/mol for Vina. These cluster patterns are seen for the all the other OMPs suggesting stronger binding affinities using AD.

We analyzed the RMSD (Root Mean Square Deviation) of the OMPs interaction (binding) with CBD as shown in Fig. 8. This provides a detailed insight into the stability of the interaction over time. We determined the RMSD values in two different setups (immediate binding representing rmsd l.b, and sustained binding over time representing rmsd u.b) for all the Salmonella Typhimurium LT2 OMPs considered in the study. The results while the best poses (pose 1-those with the best binding affinities) have 0 rmsd values for both the immediate and sustained binding times, the other binding affinities showed varied RMSD values over the observed intervals. RMSD values of 0 suggests that pose 1 (the best binding affinities) are the most stable in the binding interactions. Generally, OmpA showed varied stability in the binding interactions, OmpC have moderate stability with a relative lower RMSD values, OmpD showed relatively higher RMSD values, suggesting a more dynamic association, OmpF exhibits fluctuating RMSD values, suggesting less stability in the binding, OmpX exhibits a trend of increasing RMSD values indicating a gradual lost in stability with time. Similar to OmpD, NompC showed relatively higher RMSD values, suggesting a dynamic and less stable associations with CBD.

Overall, the RMSD value of zero as seen in all the first binding (pose 1) of all the OMPs considered in the study reveals a strong stability between CBD and the OMPs of Salmonella Typhimurium LT2.

The poses illustrated include both the 3D and the 2D poses, showing the various important binding residues and dynamics of the interactions as shown in Fig. 3. The 2D pose showed that CBD interacts with various key residues in OmpA. Essential and notable associations include conventional hydrogen bonds with ARG120, a Pi-Sigma interaction with ARG116, and lastly, a Pi-Alkyl interaction with PHE115. We have also observed an additional hydrophobic interaction of OmpA with LEU123. Furthermore, the 3D pose illustrates the spatial arrangement of CBD within the binding pockets of OmpA. The presence and positions of key residues such as ARG120, ARG116, and PHE115 are to stabilize the CBD molecules, as supported by the RMSD value of zero shown in Fig. 9. The interpolated charges visualized in the 3D pose showed positive areas (blue) and negative areas (red) potential of the interaction. The 3D pose of CBD with the receptor showed the interactions of CBD with the entire OmpA protein, and suggested that the binding site is located within a well-defined pocket, where CBD fits snugly. We showed the protein backbone in a cartoon representation, highlighting an overview of the binding environment.

The 2D pose of CBD with OmpC reveals essential interactive residues such as Tyr114, Ser127, and Asn120, whiles the 3D illustration shows that CBD engages with OmpA within the binding pocket using hydrogen bonds and hydrophobic interactions. Additionally, the 3D pose also shows an electrostatic potential of the binding location, emphasizing on the charge distribution that facilitates the binding. The CBD molecule is stably positioned in the OmpC binding site, showing a strong interaction, and the ability of the CBD to affect the proteins function in Salmonella Physiology. Generally, the results showed the potential of CBD as a potent antibacterial activity targeting these essential outer membrane proteins, and its ability to disrupt the proteins functions.

Here in Fig. 10, we demonstrated the various interactive poses for the best binding affinity of CBD with OmpD and OmpF of Salmonella Typhimurium LT2. The poses illustrated include both the 3D and the 2D poses, showing the various crucial binding residues and dynamics of the interactions as shown in Fig. 9. The 2D pose showed that CBD interacts with various key residues in OmpD and OmpF. Essential and notable interactions of CBD with OmpD include Pi-stacked interaction with PHE105, Pi-alkyl interactions with TYR108 and PHE111, and an alkyl interaction with LEU103. While the notable interactions between CBD and OmpF include Pi-Pi T-shaped interaction with TYR265 and an alkyl interaction with LEU313. Furthermore, the 3D pose analysis of both OmpD and OmpF showed the spatial distribution/arrangement of CBD within the binding pockets. For OmpD, the essential residues such as PHE105, TYR108, PHE111, and LEU103 are located to stabilize the CBD molecule. The interpolated charges are highlighted with positives (blue) and negatives (red) potential, whiles the key residues contributing to CBD interaction with OmpF includes TYR265 and LEU313. The 3D pose of CBD to OmpF’s receptor displays the binding pockets that accommodate CBD, with significant contacts from the residues, and also shows the binding sites location relative to the rest of the protein.

Similar to Figs. 9 and 10 above, we have also showed interactions of CBD with the new outer membrane C (NompC) and OmpX of Salmonella Typhimurium LT2 in both their 2D and 3D poses, as well as their 3D poses in the receptors. The 2D pose of CBD with NompC shows several key residues interacting with CBD, with the notable ones been a conventional hydrogen bond with LYS37, a Pi-cation interaction with ARG58, and Pi-alkyl interactions with TYR35, ALA16, and VAL18, while the key residues with notable interactions between CBD and OmpX includes a conventional hydrogen bond with ILE158 and Pi-alkyl interactions with VAL43, VAL161, VAL163, and MET47. Furthermore, the 3D poses reveal the spatial arrangements of CBD within the binding pockets of both NompC and OmpX. For NompC, the key residues within the binding pockets include LYS37, ARG58, ALA16, and VAL18 positioned to stabilize the CBD molecule, whiles the key residues involved in the binding of CBD to OmpX are ILE158, VAL43, VAL161, VAL163, and MET47. The charge distributions around the binding site is visualized for both NompC and OmpX with highlighted areas as positives (blue) and negatives (red) potential. Their receptor configurations show the association of CBD with the OmpX and NompC receptor. The illustrations showed a snugly fitting of CBD within the well-defined pockets in the binding sites as shown Fig. 10.

After a thorough assessment of the 2D and 3D poses as shown in the above Figures (9, 10, and 11), we extended our assessment to include the binding interactions characterized by distance, interaction category, and interaction types. Table 1 shows a detailed overview of the assessment based on the mentioned parameters. The assessment reveals that the main residues employed in the binding of CBD to the various outer membrane proteins include hydrogen bonds, hydrophobic interactions, and electrostatic interactions. Furthermore, we showed that the distances of the interactions generally ranged from 2 to 5 Å, suggesting a close and possibly strong interactions. We showed that hydrophobic interactions were predominant, especially involving alkyl and Pi-alkyl interactions. These suggest that CBD may exert its effects by binding to locations on the proteins, potentially mutilating their function and contributing to its general antibacterial influence.

Table 1

CBD Binding Interactions for *Salmonella* Typhimurium LT2 outer membrane proteins
	OmpA				OmpC
Name	Distance	Category	Type	Name	Distance	Category	Type
A:ARG120:N - :UNK0:O	3.15	Hydrogen Bond	Conventional Hydrogen Bond	:UNK0:H - A:ALA129:O	2.12	Hydrogen Bond	Conventional Hydrogen Bond
A:ARG116:CG - :UNK0	3.35	Hydrophobic	Pi-Sigma	A:PHE88 - :UNK0	4.40	Hydrophobic	Pi-Pi Stacked
A:ARG68 - :UNK0	4.99	Hydrophobic	Alkyl	A:ALA129 - :UNK0	4.86	Hydrophobic	Alkyl
A:ARG120 - :UNK0	4.93	Hydrophobic	Alkyl	A:ALA129 - :UNK0:C	4.41	Hydrophobic	Alkyl
:UNK0 - A:LEU69	4.65	Hydrophobic	Alkyl	A:PHE88 - :UNK0	4.22	Hydrophobic	Pi-Alkyl
A:PHE115 - :UNK0	5.00	Hydrophobic	Pi-Alkyl	A:TYR131 - :UNK0:C	5.31	Hydrophobic	Pi-Alkyl
				A:TYR149 - :UNK0	4.69	Hydrophobic	Pi-Alkyl
				A:TYR149 - :UNK0:C	4.01	Hydrophobic	Pi-Alkyl
	OmpD				OmpF
Name	Distance	Category	Type	Name	Distance	Category	Type
A:PHE105 - :UNK0	3.72	Hydrophobic	Pi-Pi Stacked	:UNK0:C - A:TYR265	3.71	Hydrophobic	Pi-Sigma
:UNK0:C - A:LEU103	5.30	Hydrophobic	Alkyl	A:TYR265 - :UNK0	5.18	Hydrophobic	Pi-Pi T-shaped
A:PHE105 - :UNK0	5.20	Hydrophobic	Pi-Alkyl	A:LEU313 - :UNK0	5.51	Hydrophobic	Alkyl
A:TYR108 - :UNK0	4.91	Hydrophobic	Pi-Alkyl
A:PHE111 - :UNK0	3.96	Hydrophobic	Pi-Alkyl
A:PHE111 - :UNK0:C	4.63	Hydrophobic	Pi-Alkyl
	OmpX				NompC
Name	Distance	Category	Type	Name	Distance	Category	Type
:UNK0:C - A:PHE116	3.54	Hydrophobic	Pi-Sigma	A:LYS37:NZ - :UNK0:O	3.22	Hydrogen Bond	Conventional Hydrogen Bond
A:ALA40 - :UNK0	4.79	Hydrophobic	Alkyl	:UNK0:H - A:ALA16:O	2.22	Hydrogen Bond	Conventional Hydrogen Bond
A:VAL163 - :UNK0	5.12	Hydrophobic	Alkyl	A:ARG58:NH1 - :UNK0	3.51	Electrostatic	Pi-Cation
:UNK0:C - A:ARG159	4.00	Hydrophobic	Alkyl	A:LYS37 - :UNK0	4.33	Hydrophobic	Alkyl
:UNK0:C - A:VAL161	5.43	Hydrophobic	Alkyl	A:ARG58 - :UNK0	4.41	Hydrophobic	Alkyl
:UNK0 - A:MET47	5.15	Hydrophobic	Alkyl	:UNK0:C - A:VAL18	4.30	Hydrophobic	Alkyl
:UNK0 - A:ILE158	4.77	Hydrophobic	Pi-Alkyl	A:TYR35 - :UNK0	5.21	Hydrophobic	Pi-Alkyl
:UNK0 - A:VAL161	4.50	Hydrophobic	Pi-Alkyl	A:TYR35 - :UNK0:C	5.32	Hydrophobic	Pi-Alkyl

Outer Membrane Proteins of Different Strains of Salmonella enterica at 90% Sequence Similarity

In this study, we have also carried out a comparative study of the outer membrane proteins (OMPs) extracted from 50 strains of Salmonella enterica with significant public health interest as displayed in Fig. 12. We achieved this by aligning the sequences of the strains and identifying their similarity based on a 90% similarity threshold. We have highlighted the conserved regions across the different strains depicting the significant conservation in the OMP sequences is illustrated in Fig. 12. The blue highlighted regions are the similarity threshold and the conserved areas. These conserved regions might play an essential role maintaining the structural integrity and function of the outer membranes, which is crucial for the bacteria’s association with the host environment, and its capacity to causes infections.

Additionally, we have employed phylogenetic analysis to illustrate the evolutionary relationships among the OmpA proteins across the varied strains of Salmonella enterica based on a 90% sequence similarity as shown in Fig. 13. The phylogenetic tree gives details into the genetic diversity and likely evolutionary trajectory of the OmpA protein within the 50 Salmonella strains. The tree illustrates distinct clusters, showing the presence of closely associated OmpA sequences among the Salmonella strains. We have observed major clusters at different points along the tree, with varying branch lengths representing the genetic distances between the sequences. Predominantly, several clades are composed of specific strains, indicating a high degree of similarity in the OmpA sequences within those strains. For instance, the “SALET” clusters appear several times throughout the tree, showing a common evolutionary origin (genetic conservation among these strains). The length of the branches is indicative of the degree of genetic divergence among the OmpA sequences. Longer branches represent significant evolutionary changes while shorter distances represent minimal/lesser genetic variations or evolutionary changes. Some of the sequences such as "PPQ29386.1OMPA_SALTY" and "Q8CZT5.1OMPA_SALTI," are considered as outliers, suggesting their unique evolutionary paths, thus are those with significant genetic divergence from other sequences in the tree. The outliers may also suggest strains with unique adaptations or evolutionary histories. Furthermore, the topology of the tree indicates a possible evolutionary relationships and common ancestors among the OmpA sequences among the Salmonella strains. The clustering pattern also suggests a pattern of horizontal gene transfer events, selective pressure or genetic recombination that have shaped the evolution of OmpA in these Salmonella strains.

Again, we have employed phylogenetic analysis to illustrate the evolutionary relationships among the OmpC proteins across the varied strains of Salmonella enterica based on a 90% sequence similarity as shown in Fig. 14. The phylogenetic tree gives details into the genetic diversity and likely evolutionary trajectory of the OmpC protein within the 50 Salmonella strains. The tree illustrates distinct clusters, showing the presence of closely associated OmpC sequences among the Salmonella strains. We have observed major clusters at different points along the tree, with varying branch lengths representing the genetic distances between the sequences. Predominantly, several clades are composed of specific strains, indicating a high degree of similarity in the OmpC sequences within those strains. For instance, the “SALET” clusters appear several times throughout the tree, showing a common evolutionary origin (genetic conservation among these strains). Some of the sequences such as "A0A6G6BLP9" and "A0A6G8XCJ0," are considered as outliers, suggesting their unique evolutionary paths, or are those with significant genetic divergence from other sequences in the tree. The outliers may also suggest strains with unique adaptations or evolutionary histories. Furthermore, the topology of the tree indicates a possible evolutionary relationships and common ancestors among the OmpC sequences among the varied Salmonella strains. The clustering pattern also suggests a pattern of horizontal gene transfer events, selective pressure or genetic recombination that have shaped the evolution of OmpC in these Salmonella strains.

Similarly, we have employed phylogenetic analysis to illustrate the evolutionary relationships among the OmpD proteins across the varied strains of Salmonella enterica based on a 90% sequence similarity as shown in Fig. 15. The phylogenetic tree gives details into the genetic diversity and likely evolutionary trajectory of the OmpD protein within the 50 Salmonella strains. The tree illustrates distinct clusters, showing the presence of closely associated OmpD sequences among the varied Salmonella strains. Predominantly, several clades are composed of specific strains, indicating a high degree of similarity in the OmpD sequences within those strains. For instance, the “SALET” clusters appear several times throughout the tree, showing a common evolutionary origin (genetic conservation among these strains). Some of the sequences such as "A0A5GNBJ0" and "A0A5GN86L1," are considered as outliers, suggesting their unique evolutionary paths, or are those with significant genetic divergence from other sequences in the tree. Those outliers may also suggest strains with unique adaptations or evolutionary histories. Furthermore, the topology of the tree indicates a possible evolutionary relationships and common ancestors among the OmpD sequences among the varied Salmonella strains. The clustering pattern also suggests a pattern of horizontal gene transfer events, selective pressure or genetic recombination that have shaped the evolution of OmpD in these Salmonella strains.

Similarly, we have employed phylogenetic analysis to illustrate the evolutionary relationships among the OmpF proteins across the varied strains of Salmonella enterica based on a 90% sequence similarity as shown in Fig. 16. The phylogenetic tree gives details into the genetic diversity and likely evolutionary trajectory of the OmpF protein within the 50 Salmonella strains. The tree illustrates distinct clusters, showing the presence of closely associated OmpF sequences among the varied Salmonella strains. We have observed major clusters at different points along the tree, with varying branch lengths representing the genetic distances between the sequences. For instance, the “SALET” clusters appear several times throughout the tree, showing a common evolutionary origin (genetic conservation among these strains). Some of the sequences such as "A0A4747NEL1" and "A0A378DWC8," are considered as outliers, suggesting their unique evolutionary paths, or are those with significant genetic divergence from other sequences in the tree. The outliers may also suggest strains with unique adaptations or evolutionary histories. Furthermore, the topology of the tree indicates possible evolutionary relationships and common ancestors among the OmpF sequences among the varied Salmonella strains. The clustering pattern also suggests a pattern of horizontal gene transfer events, selective pressure or genetic recombination that have shaped the evolution of OmpF in these Salmonella strains.

Finally, we have employed phylogenetic analysis to illustrate the evolutionary relationships among the OmpX proteins across the varied strains of Salmonella enterica based on a 90% sequence similarity as shown in Fig. 17. The phylogenetic tree gives details into the genetic diversity and likely evolutionary trajectory of the OmpX protein within the 50 Salmonella strains. The tree illustrates distinct clusters, showing the presence of closely associated OmpX sequences among the varied Salmonella strains. The “SALET” clusters appear several times throughout the tree, showing a common evolutionary origin (genetic conservation among these strains). "A0A7T8FEP1" and "A0A3NIL4," are outliers, indicating their unique evolutionary paths, or are those with significant genetic divergence from other sequences in the tree. This may also suggest strains with unique adaptations or evolutionary histories. The clustering pattern also suggests a pattern of horizontal gene transfer events, selective pressure or genetic recombination that have shaped the evolution of OmpX in these Salmonella strains.

Using OmpD as a query protein, we provided a comparative analysis of the different OMPs of Salmonella Typhimurium LT2 with a summary of the findings shown in Table 2, and Fig. 18. The summary of results in Table 2 shows various proteins along with various matrics. There was no data available on RMSD, TM-score and aligned residues for OmpD. However, OmpD has sequence length of 362 and 346 modeled residues. The RMSD values for OmpF were 3.72, with a TM-score of 0.11, 2% identity, and 31 aligned residues. The sequence length of OmpF is 136, and all residues were modeled. OmpX has an RMSD value of 5.22, a TM-score of 0.24, 9% identity, and has 74 aligned residues. OmpX also has a sequence length of 338, with modelled residues been 178. The OmpC has an RMSD value of 0.72, a TM-score of 0.51, 72% identity. OmpC has 177 aligned residues, 338 sequence length and 178 modelled residues.

Table 2

Structural alignments of the various Outer membrane proteins of *Salmonella* Typhimurium LT2
Entry	Chain	RMSD	TM-score	Identity	Aligned Residue	Sequence Length	Modeled Residue
OmpD.pdb	A	-	-	-	-	362	346
OmpF.pdb	A	3.72	0.11	4%	41	65	65
OmpA.pdb	A	5.12	0.11	2%	31	136	136
OmpX.pdb	A	5.55	0.24	9%	74	171	171
OmpC.pdb	A	0.72	0.51	72%	177	338	178

Figure 18 below represents the sequence alignment in showing the conserved and differing regions across the OMPs, with OmpD used as the reference sequence. OmpF reveals some conserved regions in comparison to OmpD, OmpX reveals moderate alignment with little conserved regions. OmpC and OmpD showed notable similarity in their alignment with a 72% identity as shown in Table 2 above.

Figure 19 below illustrates the KEGG pathways for both OmpC and OmpF. It represents a comprehensive network of the two-component systems that responds to various environmental stimuli and modulate various cellular processes. OmpC and OmpF are both outer membrane porins that function as osmoregulators and allow the transport of small molecules into and out of the cytoplasm through the plasmalemma (Chamachi 2021). The trajectory involves several key interconnected systems such as magnesium transport, phosphate modulation, osmoregulation, and antimicrobial peptide resistance. These interwoven pathways all play a significant role in Salmonella Typhimurium LT2 adaptive responses.

The beta-lactam resistance of Salmonella Typhimurium LT2 was explored as shown in Fig. 19 below. The figure shows a dynamic interaction of genetic, enzymatic and structural factors all contributing to resistance development. Major resistance mechanisms involve the loss or downregulation of some porins such as OmpC, OmpD, OmpF, and PhoE leading to the impairment of drug uptake. A second mechanism involves the RND family efflux pumps such as AcrAB-TolC that removes beta-lactams, minimizing intracellular concentrations. The expression of beta-lactam enzymes such as Class A BlaA, Class C AmpC is another mechanism that involves hydrolysis of the β-lactams, the expression of these enzymes are modulated by the AmpR gene. Another critical mechanism employed is through the modification of penicillin-binding proteins such as PBP1 through PBP6 as shown in the figure. This leads to the reduction in efficacy of beta-lactams. Salmonella may also change enzymes that regulate peptidoglycan biosynthesis such as MurA-MurF to aid in resistance development. The figure also revealed that there is a plasmid-mediated and genetic regulation involved in the resistance process. This also plays a significant role in disseminating the resistance traits through horizontal gene transfer.

We have used ADMETLab version 3.0 to analyze the efficacy of CBDs potency as a good drug candidate as demonstrated in Table 3. The results showed that CBD possess acceptable physicochemical characteristics and also follows the Lipinski’s rule of five, suggesting that it has good drug-likeness. Nevertheless, CBD also shows to have poor inytestinal and cell permeability indexes but interacts strongly with key transporters such as Pgp and BSEP inhibitors. The data also shows that CBD inhibits CYP2C9 enzyme. CBD also has a short half-life of 1.043 and moderate plasma clearance. Notably, CBD is non-biodegradable, which may impact environmental safety concerns.

Table 3

ADMET Results for CBD
Parameter	Score	Parameter	Score
Molecular Weight (MW)	314.22	CYP2C19 inhibitor	-
Volume	359.057	CYP2C19 substrate	+++
Density	0.875	CYP2C9 inhibitor	+++
Flexibility	0.462	CLplasma	3.51
Stereo Centers	2	T1/2	1.043
PAINS	0	Acute Aquatic Toxicity Rule	0
Lipinski Rule	Accepted	Genotoxic Carcinogenicity Mutagenicity Rule	0
QED	0.511	Non-Genotoxic Carcinogenicity Rule	0
SAscore	Easy	SureChEMBL Rule	0
Promiscuous compounds	0.114	FAF-Drugs4 Rule	1
Caco-2 Permeability	-4.746	Carcinogenicity	0.017
MDCK Permeability	-4.582	Eye Corrosion	0.004
PAMPA	---	Drug-induced Neurotoxicity	0.246
Pgp inhibitor	+++	hERG Blockers	0.539
Pgp substrate	--	hERG Blockers (10um)	0.882
OATP1B1 inhibitor	++	NR-AhR	-
OATP1B3 inhibitor	-	NR-AR	---
BCRP inhibitor	+	NR-AR-LBD	---
MRP1 inhibitor	++	NR-Aromatase	++
BSEP inhibitor	+++	NR-ER	-
CYP1A2 inhibitor	---	Hematotoxicity	0.448
CYP1A2 substrate	---	NonBiodegradable	1
BBB	+++	AMES Toxicity	0.386

For the classification endpoints, the prediction probability values are transformed into six symbols: 0-0.1 (---), 0.1–0.3 (--), 0.3–0.5 (-), 0.5–0.7 (+), 0.7–0.9 (++), and 0.9-1.0 (+++).

Outer membrane proteins (OMPs) are integral in the pathogenicity of Salmonella Typhimurium LT2. They are also involved in nutrient transport, host immune interaction and structural integrity of Salmonella (Futoma-Kołoch et al., 2019). It is imperative to understand how CBD interacts and reacts with these essential proteins if we are to employ CBD as an antimicrobial agent against infections caused by Salmonella Typhimurium and other pathogenic organisms. This thorough understanding will equip us with valuable insights into CBD’s capacity as an antibacterial agent. We have employed in silico analysis to substantiate the intricate interactions between CBD and various OMPs of Salmonella Typhimurium LT2. This exploration showed several significant interactions between CBD and the Salmonella Typhimurium LT2 OMPs used, and these interactions are characterized by hydrogen bonds, Pi-stacking interactions and hydrophobic interactions. We retrieved the OMPs structures in their pdb formats from https://www.rcsb.org/, and where structures were not available, we retrieved the amino acid sequences from UniprotKb (https://www.uniprot.org/), and modeled the structures using Swiss-Model (https://swissmodel.expasy.org/). Figure 1b, represents the modeled structures of OmpX and OmpD shown in their 3D models and Ramachandran plots for their outer membranes. The highlighted outer membranes are a typical structural and conformational characteristics of outer membrane proteins (Goswami et al., 2018). Figure 1b A and C showed that both OmpX and OmpD have β-sheet conformations as the predominate feature, and some regions showing α-helices. The typicality of the outer membranes is confirmed by the 3D models of the modeled structures possessing transmembrane β-barrels formed by antiparallel β-strands as shown in Fig. 1b C and D, suggesting their role in maintaining structural integrity and encouraging the selective passage of favorable molecules (Hawley et al., 2021). Furthermore, the high GMQE and QMEANDisCo Global scores for OmpX (0.66, ± 0.07), and OmpD (0.75, ± 0.05) underscores their reliability and high-quality warranting further functional studies. We also retrieved the 3D CBD structure from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Figure 1b shows the 2D structure of CBD revealing its hydroxyl groups essential for its bioactivity (Formato et al., 2020). The hydroxyl group plays a crucial role in the formation of hydrogen bonds with protein residues, and the hydrocarbon chain and the aromatic rings facilitating π-π stacking, and hydrophobic interactions, stabilizing and influencing the CBD-OMPs binding complexes (binding affinity and specificity) (Raghunathan et al., 2020). The hydroxyl groups also highlight the flexibility of the compound that allows it to fit well in varied binding pockets, activating its versatility for regulating protein function (Li et al., 2021). These properties are essential for exploring the antimicrobial properties of CBD.

OmpA and CBD exhibited a relatively strong binding affinity at -5.9 kcal/mol showing a significant interaction. Key interactions involved in were hydrogen bonds at 3.2 Å, and a hydrophobic Pi-Sigma interaction at 3.4 Å. Hydrogen and Pi-Sigma bonds play an enormous role in the stability and specificity of protein-ligand complexes. OmpA plays a significant role in Salmonella pathogenicity as they are involved in maintaining stability of the outer membrane and influencing bacterial ability to adhere and invade host immunity (Chaudhuri et al., 2019). With CBD ability to bind to OmpA albeit relatively low binding affinity of -5.9 kcal/mol suggests that CBD is capable of affecting and disrupting its function, weakening the bacterial outer membrane, minimizing its ability to adhere to and invade host immune cells, promoting its susceptibility to various stress and host immune defense (Figs. 6 & 7). This could possibly lead to a stronger immune response and clearance of the infection. With OmpC, CBD demonstrated a stronger binding affinity to the protein at -6.3 kcal/mol (Figs. 6 & 7) suggesting a robust interaction with hydrogen bond at 2.1 Å and hydrophobic Pi-Pi stacked interaction at 4.4 Å as the key interactions (Table 1). While OmpA is involved in maintaining stability, OmpC primary role is nutrient transport and permeability of the bacterial outer membrane. Thus, they are typically involved in the selective permeability of substances that are deemed safe for the bacterial while restricting the entry of potentially harmful substances (Chamachi 2021). By binding to OmpC with such an affinity of -6.3 kcal/mol, CBD could disrupt and interfere with its ability to form functional pores, restricting nutrient passage and influencing the structural integrity of the outer membrane. This limits OmpCs’ ability to function as selective membrane of favorable substances, making the bacteria prone to deleterious substances such as antimicrobial agents. Furthermore, affecting the function of OmpC could expose the bacteria to immune recognition, facilitating the clearance of infections (Mancini et al., 2020). A binding affinity of -6.2 kcal/mol of CBD-OmpD interactions (Figs. 6 & 7) suggests that CBD is capable of disrupting the basic function of OmpD of the transport of small molecules and maintaining outer membrane integrity. Some notable significant interactions were identified such as a Pi-Pi stacked interactions at 3.7 Å and a hydrophobic alkyl interaction at 5.3 Å as shown in Table 1. This suggests that CBD will likely disrupt other functions of OmpD such as increasing membrane permeability, thereby making the bacteria susceptible to environmental stress including antibiotic stress, impairing the bacteria ability to evade host immune responses, leading to a quicker clearance of the infection. The varied RMSD values for the rest of the poses as shown in Fig. 8 suggest the relative strength and weaknesses of the other binding affinities which all together may lead to malfunction of the protein function. With major interactions such as Pi-Sigma interactions at 3.7 Å and a pi-pi T-shaped interactions at 5.1 (Table 1), CBD exerts a binding affinity of -5.7 kcal/mol with OmpF. OmpF known as a poring protein forms pores/channels where the transport of small molecules occurs. OmpF also plays a critical role in maintaining outer membrane integrity (Chamachi 2021). A -5.7 kcal/mol (Figs. 6 & 7) CBD-OmpF binding affinity suggests a moderately good binding and that CBD may likely interfere with OmpF structural stability and nutrient transport, enhancing membrane permeability and susceptibility to host defenses and other hostile environments including antibiotic agents. The highest binding affinity was recorded for OmpX with a CBD-OmpX binding affinity of -6.6 kcal/mol with several key interactions such as Pi-Sigma interactions at 3.5 Å and a hydrophobic alkyl interaction at 4.8 Å. With this binding affinity of -6.6 kcal/mol as shown in Figs. 6 & 7, CDB is likely to interfere with the primary role of OmpX in maintaining outer membrane integrity, immune evasion, and biofilm formation, minimizing the bacterium persistence and resistance to treatments (Tsaplina 2024). This will subsequently enhance the efficacy of antimicrobial agents and the innate immune defense. With the consistent interaction profile OmpX showed with CBD, it suggests a stable and impactful effects on its structure. For NompC, it has a binding affinity of -6.4 kcal/mol to CBD with hydrogen bonds at 3.2 Å and 2.2 Å as the key interactions. Being a mechanosensitive porin/channel protein, NompC plays a role in the sensing of mechanical stimuli and maintaining outer membrane integrity. The ability of CBD to bind to NompC with such affinity suggests that the primary function of the protein may be disrupted and render the bacteria incapable of responding to environmental stresses, impairing the bacterium’s survival and virulence (Zhang et al., 2015). The notable flexibility (as seen in RMSD results) in CBD-NompC interactions suggests that CBD binding may lead to dynamic structural changes.

We conducted phylogenetic analysis on fifty different Salmonella enterica strains known to be involved in several diseases to elucidate the evolutionary relationships and genetic conservation on the outer membrane proteins considered in this study. Thus, a thorough investigation of the phylogenetic relationships will unravel highly conserved regions across the fifty different Salmonella enterica strains. The conserved regions are of utmost significance because they play a critical role in the function and structure of the proteins, making them ideal targets for antimicrobial agents (Lange et al., 2007). Additionally, a deeper appreciation of the evolutionary history and divergence of OMPs among different Salmonella enterica strains demonstrates how these proteins evolve over time and adapt to varied environmental conditions and host interactions. CBD’s ability to function as a broad-spectrum antibiotic may partly be assessed through how bacteria is able to conserve target regions or proteins over time. And by targeting these conserved regions, the likelihood of resistance development can be curtailed (Hulbert et al., 2001). The study also takes into cognizance the functional significance of the conserved regions in the OMPs. Since conserved regions are less likely to undergo mutations, they are pertinent for the stability and function of the protein, making them strategic targets for disrupting bacterial processes.

The phylogenetic exploration from fifty different Salmonella enterica OMP sequences showed a high level of conservation, with a 90% sequence similarity threshold. This level of conservation hammers on the significance of these proteins as main targets for CBD activity and other antimicrobial agents, possibly enhancing CBDs effectiveness as a broad-spectrum antibiotic and minimizing the possibility of resistance development (Hulbert et al., 2001). There were distinct clusters and evolutionary relationships shown among the various OMP sequences, suggesting a common evolutionary origins and significant genetic conservation among varied strains of Salmonella enterica. Figure 13 through Fig. 17 illustrates the genetic diversity and evolutionary pathways of the OMPs. These figures reveal some distinct clusters and closely related sequences among the different strains indicating the likely common evolutionary origin and genetic conservation. This underscores their significance as targets for CBD and other antimicrobial agents.

Another dimension of this exploration was assessing the structural similarities or divergence of the outer membrane proteins to understand the conservation and diversity among these OMPs in Salmonella enterica strains. We have used OmpD as the reference structure (Hawley et al., 2021). The 3D alignment of the OMPs suggests that they have both conserved and variable regions that play paramount role in binding associations with CBD. These all points to the idea that CBD can disrupt the bacterial functions, minimizing resistance development and providing grounds for fabricating new antimicrobial therapies. Generally, our findings underscore the paramount role CBD can play against Salmonella species by targeting highly conserved regions such as the OMPs.

The modulation of phosphate uptake and metabolism is influenced by the PhBR system under low phosphate conditions. The expression of both OmpC and OmpF are modulated by the PhoBR system, revealing their intricate role in nutrient uptake (Velázquez-Sánchez et al., 2020). PhoP/PhoQ system associated with magnesium ion levels regulate the expression of various genes including those that encode outer membranes such as OmpC and OmpF. In varying magnesium concentrations, the PhoP/PhoQ system enhances the bacteria to maintain cell integrity and function. OmpC and OmpF are both part of the bacteria defense mechanisms against antimicrobial peptides (AMPs). For evasion of host immune response, the system may modulate the modification of lipopolysaccharides (LPS) and other outer membrane components (De Gaetano et al., 2023). As porins, whiles OmpF allows both small and large molecules to pass through the outer membrane, OmpC specifically allows only small molecules to pass through the outer membrane especially under high osmotic pressure. The adjustment of these porins is orchestrated by the EnvZ/OmpR system influencing their expression based on osmolality, protection, and balancing permeability as shown in Fig. 19. As shown in Fig. 12, both OmpC (Fig. 14) and OmpF (Fig. 16) are highly conserved across the fifty different Salmonella strains. This indicates that CDB could be play a crucial as a broad-spectrum antibiotic, due to its high binding affinity to these porins. Furthermore, drugs formulated to bind to highly conserved regions can potentially avoid resistance mechanisms that target more variable regions. Additionally, the higher binding affinity of CBD to these porins suggest that major regulatory systems such as PhoBR, PhoP/PhoQ, and EnvZ/OmpR might have their functions impaired leading to impairment of bacterial adaptation to stress, making them more prone to CBD and other antibiotics. CBD binding to these porins with such high affinities also places the compound in a vantage position as a potential and potent antibiotic. This is because inhibitors that prevent LPS modification or block the regulatory pathways can weaken bacterial defenses. From Fig. 19, it is clear that both OmpC and OmpF are a part and parcel of the general regulation of LPS biosynthesis which is another potent mechanism of evading the efficacy of antibiotics (Romano et al., 2023). Hence, the role of these porins as nutrient transport agents can be exploited to improve the delivery of CBD, further increasing their intracellular concentrations.

Another essential pathway we considered in the KEGG analysis was the Beta-Lactam resistance. A thorough grasp of how Salmonella Typhimurium LT2 utilizes this trajectory to evade and render antibiotics less effective is imperative to designing new, novel and potent antibiotics. Beta-Lactam antibiotics including penicillins and cephalosporins are those agents designed to target the bacterial cell wall (peptidoglycan synthesis) which is crucial for bacterial survival and cell wall integrity (Vilvanathan 2021). Figure 20 demonstrates the detailed Beta-Lactam resistance pathways followed by Salmonella Typhimurium LT2. Outer membrane proteins such as OmpC, OmpF and NompC play a crucial role in the passive diffusion of minute hydrophilic molecules such as nutrients and antibiotics, into the bacterial cell. A common strategy employed by bacteria to develop resistance is effectively limiting the permeability of the outer membrane proteins to beta-lactams and other antibiotics minimizing the entry and preventing the antibiotics from reaching the target site in the cell (Ghai 2023). This significantly reduces the bactericidal effects of the said agent. Several enzymes play a crucial role in the biosynthesis of peptidoglycans. Each enzyme is involved in creating and assembling peptidoglycan precursors. For instance, during the dehydrogenation of MurB, enzymes such as UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA), UDP-N-acetylmuramate are activated/catalyzed. While the formation of UDP-MurNAc-peptide and other intermediates requires the activation/catalysis of enzymes such as MurC, MurD, MurE, MurF, and MurG. Lipid II is an important peptidoglycan precursor, and lipid II synthesis are modulated by enzymes such as Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY) and UDP-N-acetylglucosamine transferase (MurG) (Fathalla et al., 2022). In the periplasmic space (inner membrane) are where the penicillin-binding proteins (PBPs) are found. PBPs such as PBP1, PBP2, PBP3, PBP4, PBP5, PBP6, and PBP7 are involved in the final stages of peptidoglycan synthesis (Dabhi et al., 2024). Their primary role is in the catalysis of the cross-linking of glycan strands (essential for cell wall integrity). Beta-lactam antibiotics are designed to target these PBPs, curtailing their activity and disrupting the synthesis of peptidoglycans, resulting in cell lysis and death. Most bacteria have developed strategies to resist these drugs through the overproduction of PBPs, or mutating these proteins, decreasing the binding affinity of beta-lactam drugs (Jubeh et al., 2020). This strategy is commonly employed in methicillin-resistant Staphylococcus aureus (MRSA) where the bacteria employ the mecA gene to encode PBP2a (a low-affinity PBP) that enhances the bacteria to grow in the presence of methicillin and other beta-lactam drugs. To render beta-lactam drug ineffective, enzymes such as TEM, SHV, and CTX-M hydrolyses the beta-lactam ring (Neil et al., 2021). These enzymes are also involved in the spread of antibiotic resistance through horizontal gene transfer because they are often encoded on a plasmid as shown in Fig. 20. This is particularly true in extended-spectrum beta-lactamases (ESBLs) and carbapenemases (Neil et al., 2021). The use of efflux pumps such as the AcrAB-TolC (a well characterized resistance-nodulation-deviation (RND)) is another common strategy used by enterobacteriaceae to expel toxic substances including antibiotics out of the bacterial cell wall, contributing to multidrug resistance. efflux pumps actively pump out beta-lactam antibiotics, reducing the intracellular concentrations of the drugs, reducing their efficacy (Du et al., 2014). In Fig. 20, the pathway also highlights the role of regulatory proteins such as MarA, SoxS, and Rob that control the expression of resistance genes. Thus, allowing bacteria to adapt to the presence of antibiotics. Whiles AmpR modulates the inducible expression of AmpC beta-lactamase genes, Mecl represses the expression of mecA gene in the absence of beta-lactams this information is essential in developing strategies to curtail antibiotic resistance, because they can be influenced by several environmental factors (Araos et al., 2017). We have included the beta-lactam resistance pathway in this study because of the similarities that CBD shares with beta-lactam drugs. Thus, both CBD and beta-lactam drugs can disrupt the bacterial cell processes. With the high binding affinity of CBD to the outer membrane proteins may interfere with the bacterial cell envelop, increasing the permeability to other antimicrobial agents. Beta-lactam drugs employ similar working principles by targeting cell wall synthesis, binding to and inhibiting PBPs resulting in cell death. We suggest that CBD might be more potent than the beta-lactam drugs because of their CBDs additional anti-inflammatory and immunomodulatory effects. Beta-lactams have no direct influence on the hosts immune system. While resistance mechanisms to beta-lactam drugs are well-documented such as beta-lactamase production, efflux pumps, and PBP modifications, stringent resistance mechanisms of resistance development are yet poorly understood. In fact, a study published in nature by Abichabki et al suggests that the bacteria strain they tested against CBD has not failed to develop resistance to CBD (Abichabki et al., 2022). Our lab however has created a CBD-resistant Salmonella strains and further analyzed the resistance mechanisms. We found that LPS, ergosterol, myristic, palmitic acid were some structural medications that were used. In terms of genetics, we noticed that genes such fimA, fimZ, OmpC, Spy protein among several other genes were upregulated in the resistant strains (Ibrahim et al., 2023). This gives us a general idea of the possible mechanism regimens Salmonella and perhaps other pathogens may employ to resist CBD.

We employed ADMETLab to analyze various parameters of CBD to ascertain its safety as a potential drug candidate, taking into account the ADMETs (adsorption, distribution, metabolism, excretion and toxicity) as elaborated in Table 3 (Dong et al., 2018). We found that CBD possesses several properties suitable for drug development, though there are some notable limitations, and potential safety concerns. CBD has molecular weight of 314.22 and a volume of 359.057, and a density of 0.875. It is moderately flexible (0.462). CBD is seen to have a favorable Quantitative Estimation of Drug-likeness (QED) score of 5.22, suggesting its capacity to a drug candidate. Nevertheless, the ADMET data shows that CBD has poor intestinal and cell permeability with a Caco-2 permeability index of -4.746 and a MDCK permeability index of -4.582. Furthermore, CBD indicates a poor permeability in PAMP assays suggesting that it may encounter some challenges in effectively absorbing and permeating through cellular membranes, limiting its bioavailability. Our lab previously published a review paper suggesting how carbon nanodots may be employed to mitigate this shortcoming of bioavailability issues of CBD (Ibrahim et al 2023). Additionally, CBD also interacts with key transporters such as P-glycoprotein (Pgp) inhibitors, MRP1 inhibitors, and BSEP inhibitors, posing substantial impact on CBD’s transport and efflux mechanisms (Lempers et al., 2016). This will in turn influence its distribution and resistance. With CBD higher affinities for the OMPs, combined with these key transporters, CBDs retention, efficacy, and ability to overcome drug resistance may be higher. Nonetheless, the administration of CBD requires careful monitoring to manage possible side effects associated with transporter inhibition. The ADMET data also suggested that CBD can inhibit CYP2C9 enzyme. This is a strong suggestion of a possible drug-drug interaction involving major pathways. However, CBD does not inhibit CYP1A2, or other main cytochrome p450 enzymes. CBD’s short half-life of 1.043 hours and moderate plasma clearance (CLplasma) of 3.51 indicate that it can be cleared rapidly from the body. This rapid clearance can impact dosing convenience and patient compliance but also offers the advantage of quickly resolving any side effects upon discontinuation. Alternative delivery methods or extended-release formulations may be explored to maintain consistent therapeutic levels without frequent dosing (Daughton et al., 2023).

The high prevalence of antibiotic resistance is a global health canker that threatens the effectiveness of antibiotics, which have played crucial role in treating bacterial infections for centuries. Antibiotic resistance occurs when bacteria devise strategies to overcome or withstand the influence of antibiotics, reducing or curtailing the effectiveness of standard treatments. This phenomenon leads to persistent infections and increased risk of spread to other bacteria through horizontal gene transfer and other mechanisms. There have been several efforts curated to curb this incidence. Nevertheless, the development of novel, new and potent antibiotics has failed to march up with the emergence of resistance bacteria, making it a challenging and desolate endeavor for pharmaceutical companies in terms of research and development, regulations and economics. The entire search for novel antibiotic therapies is challenging, and humanity is likely going into dark days where every minute of infection looked like a death penalty. This is why we have shifted our perspectival search for potent antimicrobial agents towards natural products such as CBD and other natural compounds. A thorough understanding of the interaction between CBD and OMPs will provide scientific evidence and lay the grounds for the further exploration of CBDs’ potential in disrupting bacterial function. As OMPs are primarily involved in bacteria ability to transport nutrients, maintains its structural integrity, and enhance immune system evasion, agents that bind to and disrupt these functions are crucial to be employed in antibiotic therapy. Our exploration reveals that CBD has strong binding affinities to several or all the OMPs screened here for Salmonella Typhimurium LT2. OmpX and NompC specifically stood out as the those that have the highest binding affinities with CBD. Notable interactions were orchestrated by hydrogen bonds, Pi-stacking interactions, which both play a crucial role for the stability and specificity of protein-ligand complexes. The highly conservative regions and high similarity threshold of 90% of the fifty different Salmonella enterica strains further suggests that CBD can act as a broad-spectrum antibiotic. Thus, the phylogenetic assessment provides detail understanding into the genetic diversity and evolutionary landscape of OMPs, which inform the development of targeted antimicrobial agents in a conundrum of finding solutions to the ever persistence antibiotic resistance development.

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding:

This research was funded by United States Department of Education, Title III-HBGI-RES.

Acknowledgments:

The Authors acknowledge Alabama State University, C-STEM for supplies and Laboratory space. The authors also acknowledge receiving funding from United States Department of Education, Title III-HBGI-RES.

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The authors declare no competing interests.

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Cannabidiol Interactions with Outer Membrane Proteins in Salmonella Typhimurium LT2

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Abstract

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Introduction

Methods

Protein Selection and Preparation

Ligand Preparation

Interaction Analysis

Phylogenetic Analysis

Comparative Structural Analysis

Pathway Mapping

Data Analysis and Interpretation

Results

Discussion

Conclusion

Declarations

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References

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