Isolation of Microbes possessing magnetosomes and their potential role in Drug delivery
Bhavana Bhat1, Sahana Pai2, Manisha Panda3, Aadrika3, Kumari Anjali3, Aswatha Ram H N4 Aravinda Pai5, Venkatesh Kamath B.6*
1Department of Pharmacy Management, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.
2PG student, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.
3UG students, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.
4Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.
5Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.
6Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.
*Corresponding Author E-mail: vk82biochem@rediffmail.com
ABSTRACT:
Magnetotactic bacteria (MTB) are also known as magnetic bacteria. They were discovered by the scientist Salvatore Bellini in 1965. These bacteria can produce nano sized bacterial magnetic particles under normal temperature and pressure. Bacterial magnetic particles are ultrafine magnetic particles having the diameter of 50-100 nm. They are produced by a genus of bacteria called Magnetospirillum magneticum. These magnetic structures act as compass needle permitting the bacteria to migrate through redox potential with Earth’s magnetic field. Therefore, they are called polyphyletic group of bacteria. Although this unique group of micro-organisms were discovered several decades ago, the mechanism and medical application of the magnetic particles have not been clarified yet. Currently, it has been claimed that these bacteria have been widely used in biochemistry, microbiology, mineralogy, limnology, biophysics and geology. But, there is a need to unravel the world of these magnificent organisms to learn about the mechanism of magnetism therapy in medical field. The present review article gives an insight on the application of magnetosomes in modern biological and medical sciences.
KEYWORDS: Microbiology, magnetic properties, broth medium, movement.
Salvatore Bellini in 1962 first discovered and described these exceptional organisms when he observed a group of bacteria swam towards North Pole and thus named them “magneto sensitive bacteria” and named the behaviour “magnetosensitivity”1. Blakemore, in 1975, renamed them as “magnetotactic bacteria” and the phenomenon as “magnetotaxis”. Identification of MTB paved way towards some serious researchin the field of microbiology, geology, mineralogy, crystallography, physics, oceanography and even astrobiology2.
2. Morphology and Properties:
Magnetotactic bacteria, a diverse group of prokaryotes are morphologically, metabolically and phylogenetically different3.
Falling under Gram negative bacteria, these organisms move along earth’s magnetic field because of intracellularly synthesized magnetic particles called magnetosomes. Magnetosomes are specialized organelles for magnetic navigation that consists membrane-enveloped, nano-sized crystals of a magnetic iron mineral; they are formed by a diverse group of magnetotactic bacteria4.
They are aquatic and move by propulsion because of presence of helical flagella. Even though flagella have less protein, they move faster than Escherichia coli, possibly due to morphological properties and directions because of magnetosome’s order.
MTB produce two minerals, iron oxide and iron sulphide. Generally, fresh water MTB’s produce magnetite and lake and saltwater MTB’s produce magnetite/gregite. Electron microscopy studies show that MTB can produce inorganic fine crystals both intracellulary and extracellularly. They also produce calcium carbonate, calcium phosphate, selenium, magnetite by biological process. They are formed with definite morphology at particular locations in highly precise manner. The origin of magnetotaxis seems to be monophyletic, that is through same ancestor to all MTB, however, horizontal gene transfer of magnetosome genes may also seem to play vital part in their distribution.
3. Collection, Isolation and Testing:
The most suitable site for collection of MTB is pond or in slow running stream having soft muddy sediment layer.
Step 1 – The suitable site is place where the depth of water is between 10- 100cms. Using clear, screw top containers, the upper most layer of sediment is collected. The sediment is scooped along with water till one-third volume is filled. It is submerged until filled with water following which the container is capped tightly. The MTB is made viable for weeks to months in cool and shaded place and then taken to lab.
Step 2 – In the lab, the cap is loosened and left by covering the container so as to minimize evaporation. Here, it is placed in dark area and covered with aluminium foil. The sediment as well as fine particles, if present, are allowed to entirely settle down the container by leaving the sample intact for few days. MTB prefer an undisturbed environment.
Step 3 – For isolating the bacteria, the equipment’s used are sterilized by rubbing them with alcohol in tissue chamber. The wire loop is sterilized following which marine mud water samples are poured into the iron marine agar medium. The medium is then kept in refrigerator for growth.
Step 4 - After sufficient growth of bacteria, they are transferred to marine broth medium for isolation. Small samples from previous medium is added to new media. The outside of tube is sterilized to prevent further contamination of bacteria other than MTB. Growth is usually observed in marine broth by a week.
Testing for magnetic property:
Marine broth medium is prepared and poured on to petri dish. A line is marked at the bottom of each petri dish and isolates are streaked in the marked area, allowing movement of bacteria to be observed. To check for magnetic properties, copper wire wrapped nails are created and its end is attached to 1.5volts battery. The magnetic strength of wire varies along with the polarity. Magnets are also designated with positive and negative charge to understand how polarity plays a role in movement of bacteria. The magnets are attached to the cover of petridish and the one showing best result is selected to test its conducting ability. Certain petri dishes are not attached to any magnets that act as control.
Isolating magnetotactic bacteria:
In about 48 hrs, bacteria with attached magnet starts to cross the streaked lines. Using a loop, the moving bacteria is transferred to petri- dish having marine medium. The petri dish is then used to detect bacteria’s electric potential.
Testing conducting potential:
Bacteria conducting ability is tested using 1.5V battery with light emitting diode for creating closed bio circuit when two copper metals are placed, the bulb lights up. A Pasco light probe helps in analyzing the intensity of light bulb as well as the light intensity produced in marine agar in presence of MTB.
Determining polarities:
For determination of polarity, the nails are positioned at a distance from metallic surfaces. The tip of cover wire is attached to negative end of 1.5V battery following which compass is placed in front of the nail. The compass lining up negative end indicates, southern pole and if compass is lining the positive end, it indicates North Pole.
4. Correlation of MTB and earth’s magnetic field:
Cells magnetic movement is taken as the basis for interaction between MTB and the electromagnetic field. The major factor which contributes in orientation of bacterium towards the direction of magnetic field is interaction energy among bacterium magnetic movement as well as outer magnetic field. The organism swims randomly due to its thermal energy that is directly proportional to the temperature of surrounding environment.
· For the orientation of MTB towards the magnetic field, interaction energy as well as outer magnetic field should be higher than thermal energy and these energies could be compared by Langevin function, which is a mathematical equation for electromagnetism.
5. Steps involved in the formation of magnetosome:
Three steps are involved in the formation of magnetosomes:
1. Invagination of magnetosome membrane.
2. Entry of magnetite precursors into the newly formed vesicles.
3. Nucleation and growth of magnetic crystals.
Step 1- Formation of invagination in the cytoplasmic membranes is triggered by GTPase which leads to the formation of magnetosome pockets.
Step 2- This step involves entry of Fe+3 into the newly formed vesicles from external environment. In Fe+3 deficient medium, these bacteria succeed in managing the high intracellular concentration of Fe+3 ions by secreting siderophore. Siderophore, secreted by bacteria and fungi are low molecular weight high affinity iron- chelating compounds that help in transportation of iron across the cell membrane5. So, siderophore-Fe+3 complex are transported into the cell membrane and once reached the cytoplasm they are cleaved and Fe+3 ions are reduced to Fe+2 to accumulate within the bacteria. Magnetic particles and transportation of Fe+2 to bacterial magnetic particles (BMP) occurs through transmembrane transporter which has a sequence homology with a Na+/H+ antiporter. However, the complex is Fe+2/H+ antiporter helps in transportation of ions via proton gradient. These transmembrane transporter are present in cytoplasm as well as on magnetotactic membrane but in inward orientation. This arrangement of transmembrane transporter allows them to generate the efflux of Fe+2 (ferrous) ions at cell membrane and influx of the same ion at magnetotactic membrane and this step is under the control of cytochrome dependent redox system and it species specific.
Step 3- the venture of transmembrane protein in acidic and basic domains leads to the nucleation of magnetite crystals. One of the protein is Mms6 and its presence leads to the production of homogeneous crystals. There are other proteins which help in maintaining the reduced condition, oxidation of iron, partial reduction etc.
6. APPLICATIONS:
Bacterial magnetic particles (BMP’s) help in chemical coupling the antibodies on BMP’s surface which leads to the development of highly sensitive chemiliceminescence enzyme immunoassays. Combination of liposome with magnetic nanoparticle help in development of multifunctional vesicles for medical application which is used as drug delivery system and diagnostic imaging enhancers.
MTB can separate heavy metals and radionuclides during magnetic separation because of its unique magnetic, physical and optical characteristics. MTB can also be used in nanorobotics. In determination of nucleic acid, magnetite particles of MTB are made use of as carrier genes. Magnetosomes are useful in analyzing biomolecular interaction in medicine and diagnosis.
Recently, it was found that magnetosome can be used as potential drug carrier for tumor treatment. Tumors are extremely difficult to treat because the cells on the inside need to be treated by different mechanism. The area in the middle of tumor is has low oxygen concentration, so the metabolism is different. Around 55% of MTB have the capability to swim towards the low oxygen concentration and penetrate the low oxygen area of tumors when small computer controlled magnetic field is applied. MTB can swim by following magnetic field direction and their efficient activity helps in delivering drugs to tumor cells. There are various factors which affects the drug delivery to the tumor cells. These include development of deformed capillaries, heterogeneous flow of blood, high pressure between the cells and other micro environment of tumors. But the use of these microorganisms as a carrier helps in even distribution of drugs to the tumor site.
In lung cancer, MTB can be used in magnetic hyperthermia which process in which magnetic nanoparticles are directed towards tumors by alternate magnetic field. The heat produced by nanoparticles have anti-tumor potential.
7. REFERENCES:
1. Richard B. F. The discovery of magnetotactic/magnetosensitive bacteria. Chinese Journal of Oceanology and Limnology. 2009; 27(1): 1-2.
3. Suleyman Dasdagand Hava Bektas. Magnetotactic Bacteria and their Application in Medicine, Journal of Physical Chemistry and J Biophysics.2014; 4(2): 1-5.
5. Syed Sajeed Ali, N.N. Vidhale. Bacterial Siderophore and their Application: A review. Int.J.Curr.Microbiol.App.Sci.2013; 2(12): 303-312.
Received on 29.10.2019 Modified on 21.12.2019
Accepted on 12.02.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(10):5042-5044.
DOI: 10.5958/0974-360X.2020.00883.5