II nd MSc. Biotechnology
A living cell dated back to the 1930s is back in fashion – “Minicells”. Its application in delivery of therapeutics, nucleic acids and other bioactive compounds to the target cell, as a damage disposal mechanism in E.Coli, makes it extremely crucial in science.
WHAT ARE MINICELLS?
Minicells are small, spherical achromosomal bacterial particles (200-400 nm). They are formed due to aberrant cell divisions at chromosome-free polar ends of rod-shaped bacteria. They were first identified in Bacilli and later reported in both Gram-negative and Gram-positive bacteria and chloroplasts. Like their parent cells, they contain membranes, peptidoglycan, ribosomes, RNA, protein and often plasmids. Since bacterial poles are chromosome free, minicells have no chromosomal DNA. As a result, minicells cannot divide or grow, but they can continue other cellular processes such as replication and transcription of plasmid DNA, translation of mRNA, ATP synthesis, etc.
Minicells are derived from mutant bacteria. The mutant phenotype is obtained by the aberrant placement of the division septum leading to formation of a division site near the pole of the cells. This results in formation of small cells which lack chromosomal DNA.
Prior to the discovery of molecular tools such as PCR and green fluorescent protein (GFP) reporters, minicells were widely used as a vehicle for studying a variety of cellular processes such as protein synthesis, viral infection and isolating high-purity plasmid DNA from bacterial cells. There is a growing interest recently in the use of minicells as a smaller medium for visualizing the role of macromolecule and as a non-proliferating vector to deliver DNA, vaccines and cancer drugs into a variety of host cells. The primary objective of the production of vaccines is to generate secure and successful delivery mechanisms capable of inducing defensive immune responses. The use of bacterial minicells offers a special, complementary solution to promoting the production of secure and efficient delivery of vaccines.
CHARACTERIZATION OF MINICELLS PRODUCING MUTANTS
Genetic and biochemical characterization of E. coli minicell-producing mutants have shown that most are defective in a multi-protein system known as the Min system, which mediates proper mid cell placement of the cell division septum. In E. coli, three Min proteins, MinC, MinD, and MinE are known to synergistically mediate the proper placement of the cell division machinery or the divisome, by inhibiting its development at sites other than midcell. FtsZ (a bacterial homolog of tubulin) is an essential component of this complex because it initiates the development of the divisome by forming a ring-like structure called the Z-ring at the potential site of division, thereby recruiting additional divisome proteins to form the complete divisome.
E. coli mutant strains deficient in the Min system cannot spatially restrict the Z-ring to midcell. Another negative spatial regulator of Z-rings is called “nucleoid occlusion,” and it inhibits Z-ring assembly in the space occupied by the chromosomes. This results in Z-rings in min mutants assemble at either midcell between separated chromosomes or at the chromosome-free cell poles. The polar division from a polar Z-ring produces a viable mother cell containing chromosomal DNA and a chromosome-less minicell. Inactivation of the Min system in some bacterial species is not sufficient for generating high minicell yields, but the overproduction of FtsZ can overwhelm the Min system and produce minicells in wild type E. coli cells.
Fig: Normal and Abnormal cell division
ISOLATION OF MINICELLS
Due to their small size (around 400 nm) minicells can be separated and purified from normal-sized cells by using two successive buoyant density sucrose gradients.
USE OF MINICELLS AS VACCINES
Minicells can be used as an excellent means to deliver vaccines. Administration of minicells is an important part of using bacterial minicells as a vaccine delivery vehicle. Minicells should be good at mucosal delivery due to the fact that bacteria have evolved to survive the hostile environment these surfaces represent. Therefore, minicells were tested for their ability to induce immune responses after mucosal vaccine delivery, and it was reported that mucosal route of administration is an efficient way of evoking immune response compared to other modes of vaccine delivery.
Using bacterial minicells to deliver vaccines have distinct advantages over other bacterial based delivery systems:
- Minicells can package a wide array of different antigenic proteins
and plasmid DNA vaccines, and the levels of both can be easily modified and manipulated.
- Minicells have a distinct safety advantage from the fact that they
are non-infectious and non-dividing. This allows minicell vaccines to be used by all patient populations such as children, the elderly and the immunocompromised.
- They contain all the adjuvant properties of their parent cells
(LPS, etc.), they do not contain a chromosome, eliminating the possibility of pathogenic reversion in this approach.
- The ability of minicells to contribute to horizontal gene transfer
to other pathogenic strains of bacteria is minimal, as it has been documented that bacterial mating into minicells is possible and not vice versa.
Vaccine delivery using minicells could potentially elicit similar responses as live attenuated pathogens, but they are much safer to use. Minicells are packaged with the component responsible for the cause of a disease (eg: proteins) or a plasmid construct encoding for that protein and delivered to the host. The limiting factors in vaccine development as a whole are in delivering antigens to the appropriate cell types and antigen processing pathways and generating T cell responses so that it will support the development of immunological memory in both B and T cells.
Although live attenuated forms potently stimulate innate immune receptors, one limitation of these bacterial vaccine platforms is their inefficient capacity to stimulate cytotoxic T-cell responses, which require the delivery of antigens to the cytosol of antigen-presenting cells. This limitation has been largely overcome by the use of type III secretion systems (T3SS) which are complex multi-protein molecular machines that deliver bacterial virulence effector proteins into the host cell cytosol.
Giacalone et al. (2007) carried out studies on delivering minicell vaccines against lymphocytic choriomeningitis virus (LCMV). LCMV nucleoprotein causes this viral infection. It was reported that minicells delivering both the heterologous protein and plasmid DNA encoding the protein induce immune responses significantly superior to those induced by minicells that carry protein or DNA alone. So they constructed a minicell vaccine to deliver both the LCMV-NP protein and a plasmid encoding LCMV-NP and inoculated in mice. It was found that the protective and immunodominant MHC class I restricted peptide epitopes identified these delivered minicell vaccines and evoked an antigen-specific CD8+ T cells response leading to production of various cytokines.
Minicells given three times via the mucosal route were able to generate cell-mediated immune responses as determined by peptide- specific target cell lysis. In short, minicells are an outstanding platform to ensure safer mode of vaccine delivery. Since minicells retain functional and often infectious properties of the parent bacteria, they promise to be useful model systems for understanding the molecular basis of bacterium-host interactions, a central process during bacterial infection.