INTRODUCTION:

The plant derived hydro-distillated essential oil¹are well acknowledged for their antibacterial activity², which are of course the secondary metabolites, and are included under the category of herbal drugs³and are part of pharmaceuticals⁴also. The phytochemistry⁵of essential oils have inferred about a compendium comprising enormous number of constituents, with bactericidal activity. The in vivo studies also infers about its pharmacological⁶performances. Whereas, target oriented, mechanism of action of the said antibacterial agents is of prime importance in essential oil Biology. For this purpose, authors have carried out pertinent research ^7-15and had inferred about the primary and associated target molecules, being interacted with the neat plant essential oil or its active constituents, leading the disruption of bacterial cell¹⁶.

The essential oils in toto or its active metabolites have enough molecular efficiency to denature the bacterial cell appendages like flagella, cell wall synthesizing/ catalyzing enzymes, Inter space enzymes, Intracellular enzymes, DNA associated enzymes and more so unbalancing the chemi- osmotic channel, inducing stress, by interacting with stress response factors and so on. Here is the description of some prime targets which are liable to essential oils or its components, which are reported in brief.

1. The Plant secondary metabolite bacterial targets:

1.1 Flagella as the target:

Flagella is the locomotory appendage of motile bacteria like Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Helicobacter pylori, Vibrio cholerae, Campylobacter jejunii, Listeria monocytogenes, Proteus mirabilis, Bacillus spp. etc. The flagella (Figure 1) is a polymer of protein subunits namely flagellin, which is inserted into the bacterial cell wall, having mechanical potency, derived from proteasome motors, which are fitted into the bacterial cell wall. Using the mechanistic principle and energized by ATP, can give substantial force to bacterial cell for swimming in water based environment. Bacteria may able to resist the impact of antibacterial agents and survives. Flagellum-mediated motility plays diverse roles in the pathogenesis and progression of EHEC O157:H7 infection¹⁷.

Figure 1: A typical Gram negative bacteria with a tuft of unipolar flagella

There was a report¹⁸ on repurposing some natural components, deposited in respective libraries, and was succeeded with recognition of antimotilins. Researchers ¹⁹were scientifically optimistic about the functionality of Ginkgetin, a natural biometabolite to regulate the flagella-formation genes (flhC, flhD, fliC, fliM), as a result of which the said compound could inhibit the motility of bacteria, Escherichia coli, of course without interfering its metabolism. Further, a contribution²⁰had acclaimed about the natural Tanshinones, which could be the inhibitors of the Biogenesis of the Type 3 Secretion System Needle of Pseudomonas aeruginosa for Antibiotic Therapy.

1.2 Cell Wall synthesizing proteins/ enzymes as targets:

Pathways (cell wall and protein synthesis) are considered as common targets for antibacterial agents. Considering specific views²¹, Cell wall hydrolase (CWH), which is involved in Peptidoglycan layer synthesis may be included in the target panel. In addition to this, Uridinediphosphate N-acetyl muramic acid (UDP NAM) is a critical intermediate in bacterial (PG) biosynthesis. As the primary source of muramic acid that shapes the PG backbone, modifications installed at the UDP NAM intermediate can be used to selectively tag and manipulate this polymer via metabolic incorporation²². A, flavonoid “Rutin”, was been screened for it’s interaction with peptidoglycan synthesizing enzymes, Mur ligases, MraY, FemX, FemA, FemB, and PBP2, using bioinformatics tools²³.

1.3 DNA associated enzymes as targets:

DNA associated enzymes are one of the promising enzymes, being enlisted as bacterial targets. Fluoroquinolones are the first line of antibiotics²⁴which interact with the DBA, Gyrases, the DNA topology assembler (Figure 2). Isoflavonoid and Furanochromone, the natural compounds were proved to be potential agents to inhibit the Gyrases²⁵. It was²⁶predicted that about the interacting poses between the StreptomeDB, with the Gyr A subunit of DNA gyrase.

Figure 2: The sketch of Gyrase enzyme, a composited form of Gyrase A and Gyrase B, for introduction of negative supercoiling to the replicating DNA

1.4 Ribosome assembly as antibacterial targets:

The ribosome assembly events and its associated biomolecules had attracted the researchers, since a long time. Erythromycin, the macrolide class of antibiotic, can have interaction with the assembling subunit, leading stalling of RNA precursor units and dismantling the protein synthesis machinery ²⁷. Most of the natural antibiotics inhibit the peptidyltransferase center (PTC) on the 50S subunit ²⁸.

1.5 Bacterial cell division associated Proteins as target:

Bacterial cell division^29-31is the vital event in the life of a bacteria. The cell division is laid by a partition wall which is regulated by a cluster of proteins which are actively involved in partitioning process. Researchers have taken enough interest to study the biology as well as molecular events positioned at the side of cell division. FtsZ^32-36is the leader protein which bounds to the cell partition site and undergoes a cell polymerization, accelerated by GTPase which is thought to be FtsZ itself which binds with GTP initiates hydrolysis. FtsZ is a key component being associated from the initiation and ingrowing cell wall until the septal closure. Researchers had reviewed that FsZ could undergo self-assembling and forming a ring of bacteria cell division (Figure 3).

Figure 3: The formation of ring at the midcell position of a dividing Rod shaped bacteria. a: The MreB proteins forming protofillaments in prohalves of bacterial cell; b: The protofillament of FtsA and c: The Protofilament of FtsZ. Arrows indicating the position of constriction.

FtsZ protein is inactivated/ deformed due to interaction with antibacterial agents, there is a phenomenal chance that, the rod form of bacteria may eluded as very long rods which looks like filaments (Figure 4). This is relevant to mention that, filaments may be generated when the bacteria survives in stress condition imposed by antibacterial agents or alternation in level of nutrients, pH and also temperature. Authors^37-40have indicated that generation of filamentation in a SOS or last resort survival technique conferred by the bacterial candidates. Further, to mentioned that the bacterial in the stress condition or in the event of degradation of FtsZ protein, the filamentous form is retained but there is lacking of cell partition.

Figure 4: Thefillamentose phenotype of bacteria, generated due to cell division but without cell partitioning, because of absence of FtsZ and other cell division proteins

Quinolone groups have ability to interact with topoisomerase II, Gyrase,⁴¹inhibiting the incorporation of negative super coiling which is required to release the tensile strength of super coiled DNA. Due to this effect, the bacteria may opt for filamentous form as SOS effect. Generation of filamentous morphotype⁴²is considered as a biomarker event. Therefore, various instrumental based analysis is required to probe the modality of antibacterial agents.

1.6 Scanning Electron Microscopy for evidence based topology study:

Scanning Electron Microscopy is opted for the topological study, which clarifies the differential morphology opted of bacterial cells in presence of antibacterial agents, say the plant essential oils. In SEM, the electron wave is used as a unique medium for imaging system. The SEM⁴³uses an electron beam which is focused into a small probe and the zigzag scanned pattern, named as Rastering⁴⁴or scan over the surface of the specimen. Hence, there is display of shape or topology or morphology of cell of interest. Previously authors have contributed their findings considering plant essential oils as antibacterial agents, evidenced by scanning electron microscopic observations^45-47.

1.7 Fluorescent Microscopy for cellular demarcation and intracellular organelle study:

Fluorescent Microscopy⁴⁸is an excellent image generating microscope which uses the mechanism of the fluorescence to create the image. Typically a Fluorescent dye has the ability to re-emit light upon light excitation as it typically contains several combined aromatic groups or plain or cyclic molecules with several pi-bonds. Hence, the cellular entities⁴⁹, may be visualised using the fluorescent microscopy.

1.7.1 Fluorophores for the Cell Membrane and chromosome Staining:

Experiments dealt with membrane staining techniques, specific membrane stain fluorophores are used. Different facile methods⁵⁰ and also strategies of detecting bacteria using fluorophores were well observed^[48]. Among the cell membrane, dyes FM-464 dye (N-3-triethyl ammonium propyl) – 4-(6-) 4-(dimethyl amino) Phenyl) hexatrienyl (pgri-diniumdibromide), this sterile dye has been reported to stain bacterial cell membrane with red fluorescence (excitation emission maxima 515/640nm). The hyper structures stained with this lipophilic dyes are of immense importance for analysis, to paving a way for depth study⁵¹. Chromosome visualization being an essential, analytical step in genetic diagnostic of bacterial cell. DAPI (4’, 6-diamidino-2-phenyl indole) is a blue fluorescent DNA staining agent that can discharge approximately 20 times fold enhancement of fluorescence, upon binding to AT (Adenine-Thymine) regions of double stranded DNA.

1.8 Fluorescence Associated Cell Sorting (FACS)for enumeration of bacterial cells (both living and dead):

Fluorescent activated/ associated cell sorting is a part of flow cytometry protocol, used in robust for enumeration of bacteria of interest⁵². In addition to its ability to examine and determine the amount and expression of intracellular molecules, it can define and also characterize distinct cell types, volumes, size and also enumerating the sub-population of cell clusters. The density (of population or subpopulation) plot shows expression level and also relative number of events (density) in a given region⁵³. Figure 5 is demonstrating basic instrumentation of FACS, generating the density plots.

Figure 5: The instrumentation of FACS (Fluoroscent Associated Cell Sorting). a, the source of Laser beam; b, the fluid dynamic chamber for flow of single bacterial cell (both dead and alive); c, Forward scattering; d, Fluorescent channels: Blue, Green, Yellow, Red; e, The detector; f, monitor showing the cell population in the form of histogram

Efforts were made in past to capture the targets of bacteria under study with an objective to differentiate between viable and nonviable cells using the DNA binding fluorophorethiazole orange,⁵⁴ assessment using FACS to discriminate three different subpopulations (a) viable, (b) dead and (c) injured cells,⁵³quantitative analysis of bacterial cell populations using Bayesian mixture model, to enunciate the degree of gene expression of bacteria⁵⁵. While, a combinatorial flowcytometry along with imaging system had enabled a research group⁵⁵. From a review report, it was inferred that the lineage specific cell sorting of Salmonella could be achieved by FACS⁵⁶. It was documented that the FACS along with SEM and more over TEM could recognise the cellular disruption, membrane damage under the influence of Limonene⁵⁶.

CONCLUSION:

From this review report it may be concluded that the plant derived antibacterial agents, interacting specific targets and the instrumental used for the tracking the possible mode of action of antibacterial agents, say the herbal constituents. Hence, it is agreed that, attempts are to be made in deciphering the conceivable modality of the plant metabolites interacting with target molecules or enzymes to restrict bacterial growth using pertinent instrumentation.

ACKNOWLEDGEMENT:

This communication is part of a thesis submitted under Sambalpur University.

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Received on 08.01.2024 Revised on 24.04.2024

Accepted on 29.07.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):388-392.

DOI: 10.52711/0974-360X.2025.00060

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