What are Inclusion Bodies?

Inclusion bodies are thick in size, spherical and aggregated proteins typically found inside the cells of protokaryotes thanks to the overexpression of heterologous proteins.

• Inclusion bodies comprise small particles that are freely suspended and floating in the cell wall. This is why they are also known as inclusions of the cytoplasm.
• Cell inclusions develop by a decrease in pH and an accumulation of the solubilized proteins fusion within the cell.
• They are the basic body parts that are formed during the course of infections or in cells that are infected with viruses, like rabies, herpes measles, rabies, etc.
• Inclusion bodies are abnormally formed bodies that are distinct in dimensions and shapes, and are typically seen in epithelial, nerve or endothelial cells.
• They exhibit a distinct staining ability and are generally made up of proteins.
• Inclusion bodies are chemical compounds that are not living as well as by-products of cell metabolism.
• They can be found both in prokaryotes and in eukaryotes.
• There is a variety of inclusion bodies found in various kinds of cells.
• In prokaryotic cells, they’re mostly created to store reserve material.
• In animal cells they store fats and sugars which are ready for cellular respiration. In plants they store granules of substances like glycogen, starch, and so on.
• Gas vacuoles and cyanophycean granules glycogen granules, are just a handful of examples for inclusion particles.

Features of Inclusion Bodies

• They are typically acidophilic.
• Perhaps crystallized particles of the virions.
• They represent degenerative changes caused by viral infections.
• Are composed of virus antigens found at the site of synthesizing.
• They appear as pink structures when stained the blue dye methylene blue or gypsum.

Classification of Inclusion Bodies

Inclusion bodies are divided into two kinds that are organic inclusion bodies and inorganic inclusion body.

A. The Organic Inclusion Bodies

Organic inclusion bodies usually contain either (1) glycogen granules or (2) poly-β-hydroxybutyrate (PHB)

Organic inclusion bodies usually contain either (1) glycogen granules or (2) poly-β-hydroxybutyrate (PHB)

1. Glycogen granules

Storage reservoirs for carbon like glycogen or PHB inclusion bodies are a source of materials for biosynthesis and energy. Glycogen is a molecule composed of long chains joined via glycosidic linkages and branching chains joined to glycosidic bonds. Infusing cells using an iodine solution makes the cells reddish-brown when they have large amounts of glycogen.

2. poly-β-hydroxybutyrate (PHB)

Poly-β-hydroxybutyrate (PHB) contains hydroxybutyrate molecules joined by ester bonds between adjacent molecules’ carboxyl and hydroxyl groups. Beta hydroxy is the term for the part that is part of the OH group that is in contrast to the Oxygen double-bonded group. Because the group repeats multiple times throughout the structure, it is also known as Poly-β-hydroxybutyrate

Cyanobacteria is essentially a class of bacteria which can photosynthesise , and they possess two distinct organic inclusion bodies

1. Large polypeptides containing nearly identical amounts of two kinds of amino acids, arginine and aspartic acid. The first is a basic amino acid and the other is one that is acidic that makes up the cyanophycin Granules.
2. Carboxysomes can be found in a variety of cyanobacteria as well as other CO2-fixing bacteria and are able to store nitrogen for the organism.

The gas vacuole can be described as an incredible organic inclusion body found in prokaryotic organisms. Certain marine prokaryotes rely upon it to buoy. Prokaryotes with gas vacuoles within them can regulate their buoyancy so that they remain at the appropriate depth to maintain the proper oxygen concentration, light intensity and the levels of nutrients. They can float up after the formation of new vesicles and then fall down when vesicles collapse.

B. Inorganic Inclusion Bodies

In prokaryotes there are two kinds of inclusion bodies made of inorganic substances:

1. Granules of Polyphosphate

A lot of bacteria keep phosphate, in the form of polyphosphate-based granules or the volutin crystals. It is an ester-linked, linear molecule of orthophosphates. Therefore, the volutin granules function as storage reservoirs for phosphates, which are a vital element in cell components such as nucleic acids.

They function as a source of energy in certain cells, and polyphosphate is an energy source during processes. These granules are also referred to by the name of metachromatic granules. They display a metachromatic look when stained with blue pigments like methylene blue and toluidine blue. This makes them appear reddish or in different shades of blue.

2. Sulfur Granules

Prokaryotes can use sulfur granules as storage for sulfur for short periods of time. Photosynthetic bacteria, like are able to use hydrogen sulfide to act as an electron donor. It stores sulfur that is produced in the periplasmic or in cytoplasmic globules that are specific to.

• Metachromatic Inclusions –  Some of the larger cell inclusions that may stain red with blue dyes, such as Methylene blue are referred to as metachromatic inclusions. These are granules that are composed of glycogen and starch. When iodine is present glycogen granules turn to be reddish brown and the starch granules are blue.
• Lipid Inclusions – Lipid Inclusions in diverse kinds that belong to Bacillus, Mycobacterium, Azotobacter and many other genera. They serve as storage materials for lipids. They can be seen when cells are colored with fat-soluble dyes, such as Sudan dyes.
• Sulfur Granules – They get energy by oxidizing sulfur as well as sulfur-containing compounds. They could create sulfur granules inside cells that act as a reserve of energy.
• Carboxysomes – These include the protein ribulose1. diphosphate carboxylase. Bacteria utilize these inclusions as carbon sources to fix carbon dioxide in photosynthesis.
• Magnetosomes – Some bacteria position themselves in an electromagnetic field due to magnetic particles. magnetosomes can be described as intracellular inclusion bodies, or tiny particles of iron oxide mineral known as magnetite (Fe3O4). Magnetosomes are enclosed by a thin membrane comprised of phospholipid, protein and glycoprotein. It acts as the magnetic dipole of cells that allow them to show magnetotaxis, which is the process of moving along the magnetic field of Earth. It can be found in a wide variety of aquatic species.

Based on the place of origin of the inclusion body, whether in the nucleus, cytoplasm or both cell organelles, we are able to classify inclusion bodies into one of the categories below:

• The inclusions of cells in the intranuclear space.
• Infection inclusion bodies.
• Intracytoplasmic inclusion body.
• The physiological inclusion bodies.

Inclusion bodies can be present in a bacterium or eukaryotic cell in the form of cystic lesions, fungal infections, virus-infected cells, bacterial infections, autoimmune diseases, neoplasms and blood dyscrasias.

Examples of inclusion bodies

1. In red blood cells

The normal red blood cell doesn’t contain cytoplasmic inclusions. However, it can be observed in the context of certain hematologic conditions. There are three types that red blood cells are included in:

Developmental organelles

• Howell-Jolly bodies: Howell-Jolly bodies are small round fragments of the nucleus, resulting from karyorrhexis or nuclear degradation of the reticulocyte’s late phase and stain with Wright’s stain in reddish-blue.
• Basophilic stipplings: Basophilic stipplings stipplings can be coarse or fine, with a deep blue-purple staining which appears on erythrocytes dried stain of Wright’s.
• Pappenheimer bodies – are also known as siderotic granules that are small irregular, dark-staining small granules which are located close to the edges of an erythrocyte’s young cells in the form of a Wright stain.
• Polychromatophilic red cells – Polychromatophilic red cells are young red cells that do not contain nucleus, but contain RNA.
• Cabot rings – Cabot rings are a ring-like structure and are often found in erythrocytes during megaloblastic anemia as well as in cases of severe anemias, poisoning and in dyserythropoiesis. the erythrocytes are destroyed prior to being released from bone marrow.

Abnormal hemoglobin precipitation

• Heinz bodies – Heinz bodies circular bodies with refractile inclusions that are not visible on a Wright’s stained film. They can be identified using the staining of supravital with basic dyes.
• Hemoglobin H inclusions – Hemoglobin H inclusions Alpha Thalassemia, inclusion bodies that are greenish-blue occur in many erythrocytes when 4 drops of blood are placed incubated with 0.5mL of Brilliant Cresyl Blue in 20 minutes, at 37 degrees Celsius.

Protozoan inclusion

• Malaria
• Babesia

2. In bacteria

Polyhydroxyalkanoates (PHA) are produced by bacteria as inclusion bodies. Size of PHA Granules is limited to E. coli, due its size. Bacterial cell inclusion bodies are not as numerous intracellularly when compared with eukaryotic cells.

3. In viruses

Some examples of viral inclusion bodies in animals are

a. Cytoplasmic eosinophilic (acidophilic)-

• Downie bodies in cowpox
• Negri bodies in rabies
• Guarnieri bodies in vaccinia, variola (smallpox)
• Paschen bodies in variola (smallpox)

b. Nuclear eosinophilic (acidophilic)-

• Type A of cowdry bodies within Herpes simplex virus as well as Varicella Zoster virus
• Torres bodies covered in yellow fever
• Type B of cowdry body in the adenovirus and polio

c. Nuclear basophilic-

• Cowdry bodies of type B in Adenovirus
• “Owl’s eye appearance” in cytomegalovirus.

d. Both nuclear and cytoplasmic-

• Measles bodies of Warthin-Finkeldey in meas HIV/AIDS

Some examples of viral inclusion bodies found in plants include aggregations made up of virus particles (like those of Cucumber mosaic virus) and the aggregation from viral proteins (like the inclusions that are cylindrical in potyviruses). Based on the species of plant and the plant virus family, the inclusions are found in mesophyll cells, epidermal cells and stomatal cells if the tissue of plants is stained.

4. In white blood cells

Inclusions of immunoglobulin known as Russell bodies can be found in normal plasma cells. Russell bodies form clumps in large quantities, causing them to push cells’ nuclei towards the edge. the cell is then referred to as Mott cell. Mott cell.

Prevention of inclusion bodies formation

Inclusion bodies typically consist from denatured aggregates of inactive proteins. Even though, the renaturation of inclusion bodies can result in the solubilisation of and recuperation of proteins active However, the process remains extremely speculative, uncertain and low-efficiency. There have been a variety of techniques created over time to avoid from the development of inclusion body. These techniques include:

• Utilizing weaker or less powerful promoters in order to slow the rate at which proteins are expressed.
• The utilization of low copy number plasmids
• Chaperone is a chaperone-related gene that co-expresses (such as DnaK-DnaJ and GroES-GroEL)
• The use of certain E. coli strains such as (AD494 and Origami)
• The target protein is fused to the partner that is soluble
• Reduce the temperature of the expression

Diseases Involved with Inclusion Bodies

• Inclusion of body myositis that can affect muscle cells.
• Amyotrophic lateral Sclerosis, which causes motor neurons to be affected.
• Lewy inclusions in dementia that affect the brain’s neurons.

References

• Kumar, D. (2011). Comprehensive Biotechnology || Protein Refolding/Renaturation. , (), 765–784. doi:10.1016/b978-0-08-088504-9.00136-7 
• Singh, A., Upadhyay, V., Upadhyay, A.K. et al. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact 14, 41 (2015). https://doi.org/10.1186/s12934-015-0222-8
• https://www.frontiersin.org/articles/10.3389/fmicb.2014.00056/full
• https://www.slideshare.net/suramyababu1/inclusion-bodies
• https://www.vedantu.com/biology/inclusion-bodies