Bacteriocins – novel ways for antimicrobial treatments

While rates of hospital-acquired MRSA and C. difficile have dropped, E. coli has emerged as a new threat in the fight against antibiotic resistance – Chris Penfold believes it could be treated with bacteriocins

Since the introduction of the UK Government’s policy on MRSA screening for non-emergency hospital admissions in England in March 2009, and for all admissions by March 2011, rates of hospital-acquired MRSA infections have tumbled over the past year. Combined with the general fall in Clostridium difficile infection rates, the leading source of hospital-acquired infections in the UK at present is from Escherichia coli and other related Gram negative organisms.

E. coli is normally resident in the gastrointestinal tract as a natural inhabitant that aids digestive processes, provides essential vitamins and helps stave off potentially pathogenic bacteria by occupying the same ecological niche. However, there are some strains of E.coli that are pathogenic and present added problems because they have evolved highly virulent toxins; have a wealth of antibiotic resistance genes due to the transfer of genes from other bacteria and the strong selection pressures applied in hospitals through the overuse of antibiotics; and have the capabilities of colonising and infecting other areas of the body. These bacteria represent a new challenge in the fight against antibiotic resistant microorganisms because of their increased resistance to many commonly used classes of antibiotics – such as penicillins and cephalosporins –through the generation of extended spectrum beta-lactamase enzymes (ESBLs), enzymes that are able to breakdown and inactivate all classes of beta lactam antibiotic.

There are now examples of E.coli strains such as the New Delhi metallo-beta-lactamase 1 (NDM-1) imported into the UK via medical tourism – as UK nationals visit Indian hospitals for cheaper surgery – that are resistant to carbapenem class of antibiotic (the last known effective group of drugs) and are proving virtually untreatable due to the lack of treatment options currently available. It is clear that novel strategies for the treatment of these superbugs are necessary to prevent these bugs from causing increased morbidity and mortality in a wide variety of healthcare settings.

One option is the development of bacteriocins as potential antimicrobial treatments. Bacteriocins are protein antibiotics produced by one bacterium that kill closely related bacteria to provide the surviving cells with a competitive advantage during times of environmental stresses such as nutrient depletion. They are exquisitely tuned to target only a small group of related bacteria, due to their narrow killing spectrum, and would thus not compromise the healthy microbiome in patients. Bacteriocins are produced by several species of bacteria and come in a variety of sizes and structural shapes.

Due to their microbial cytotoxicity, bacteriocins are currently receiving a lot of interest for their potential therapeutic use as antimicrobial and probiotic agents and alternative cancer therapy, as well as being of great value to the food industry where they are being exploited in order to increase food security. Probiotics are of special prophylactic value since the engineering of beneficial strains expressing bacteriocins would enable more rapid and stable establishment in the gut, and would potentially prevent the establishment of pathogenic strains taking hold. As current antibiotic treatments to fight superbugs are increasingly proving ineffective, it could be that harnessing the toxic power of bugs to fight rival bugs is going to be the way forward.

Bacteriocins are protein antibiotics produced by one bacterium that kill closely related bacteria as a method of ‘ethnic cleansing’ to provide the surviving cells with a competitive advantage during times of environmental stresses such as nutrient depletion

Bacteriocins produced by E. coli are called colicins. They are large, multi-domain proteins that bind to a specific receptor on the bacterial cell surface and then hijack bacterial transport systems to move from the outside to the inside of the bacteria, such as the cytoplasmic membrane or the cell cytoplasm where they exert killing activity by degrading DNA or RNA, or punching holes in membranes leading to leakage of cellular contents. Although colicins have excellent antimicrobial activity, it is difficult to envisage using them as potential new antibiotics because they are large protein molecules that would activate the body’s immune response to provide protection on the next occasion the colicin is used for treatment.

Nevertheless, for its unimpeded entry into the sensitive bacteria, the colicin is able to form a physical interaction with a number of proteins that are present naturally within the cell where they perform an essential function such as providing the cell with energy, supplying a structural scaffold for transport processes or having a role in the metabolism of the cell. These proteins have been called ‘Tol’ proteins because their inactivation through introducing mutations in their structure (sequence) results in tolerance of the cells to the action of the invading colicin. However, the resulting mutant bacteria are sick, unable to grow efficiently and are susceptible to many agents that otherwise would be innocuous to a healthy cell.

This offers the exciting opportunity of harnessing information gained from an understanding of how colicins or colicin domains penetrate cells and their ensuing interactions with Tol proteins for the development of novel synthetic molecules with antimicrobial activity. These small molecules with the ability to irreversibly bind to Tol proteins would inactivate the natural function of the Tol system in the sensitive cell and thus induce a bactericidal effect on the bug.

In a paper recently published in the Journal of Biological Chemistry, Chris Penfold and colleagues determined the X-ray crystal structure of the interaction of the Tol protein, TolA with the translocation domain of the pore-forming colicin, colicin A. TolA is a major protein in the E. coli cell that interacts with several different endogenous proteins to provide a structural scaffold for the cell, permit energy transfer from one cell compartment to another and contribute to cellular homeostatis (well-being). It also interacts with several different exogenous proteins including colicins and viruses through multiple interaction sites and has been classified as a promiscuous hub protein.

Hub proteins participate in a number of protein interactions that control the formation of large protein complexes and biochemical pathways that define all cellular processes and are vital to the viability of all cells. Preventing hub proteins such as TolA from initiating the formation of protein complexes through the creation of a physical barrier by the binding of colicin or alternative ligands could prevent the cells from growing normally and establishing a hardy infection, and ultimately prove beneficial to the development of new therapeutic treatments.

The work shows that colicin A binds to TolA across a relatively small surface area whose interface region consists of five interacting amino acid residues that stabilise the interacting complex. This offers the possibility of developing novel synthetic substrates with similar structural properties that would bind TolA and disrupt its normal functions within the cell. Furthermore, it was also shown that the interaction could only be prevented by mutating all five amino acids simultaneously suggesting that any resistance mechanisms developed by the bacteria in response to the inactivating substrate could prove challenging and less likely. It has also been shown that viruses that infect E. coli – bacteriophage – also enter the sensitive bacterial cell through an interaction with TolA. Comparison of the binding regions of colicin A and bacteriophage with TolA showed that colicin A binds on the opposing side of TolA to that of bacteriophage indicating potential for the development of a wide range of substrates with capabilities of disrupting TolA at a variety of locations either independently or in combination.

We have known of the immunological limitations of using colicins for the treatment of E. coli infections for a long time, but through an understanding of how these molecules are able to enter sensitive E. coli cells alternative strategies for killing of bacteria, such as the interference of cell processes, might be feasible in the future.

The Author:

Chris Penfold, Course Director MSc in Molecular Medical Microbiology, School of Molecular Medical Sciences, University Park, Nottingham,