viralovector

[viralovector]

Shine bright like a phage

Recently, diamonds have been the center of attention, not because wedding season is upon us but rather because a group of scientists have developed fluorescent nano-diamond (FND)-bacteriophage conjugate as a tool in identifying specific strains of bacteria. This turns out to be of upmost importance in clinical diagnostics and in testing the potency of food.

Fluorescent nano-diamonds as its name suggests, are nano-sized diamonds that have fluorescent properties due to atomic defects or impurities. These nanoparticles are increasingly being used as a diagnostic and imaging tool in fuelling new discoveries in interdisciplinary fields spanning biology, chemistry, physics and material sciences. But why? FNDs have excellent long-term imaging properties, are non-toxic and most importantly are able to accept surface modifications to be further functionalized. This last property has been vastly exploited to conjugate the FND with biological entities such as bacteriophages.

Bacteriophages are far from being promiscuous – most of them are picky when it comes to bacteria. However, the specificity of bacteriophages is a tunable property with the adequate genetic tools. Bacteriophages are equipped with “keys” to gain access to specific bacterial cells. These “keys” are found on their outer membrane called the capsid. A relevant example is the bacteriophage lambda, which can infect Escherichia coli bacterial strain only if the latter has the specific complementary “lock”, in this case, lamB, on its outer membrane. These “keys” are therefore a major target to genetic modifications to manipulate the bacteriophages. 

For instance, to conjugate the FND with the bacteriophage, Trinh and his colleagues have modified both the nanoparticle and the virus. They have functionalized the FND with streptavidin, a protein known to have a very strong affinity for biotin (also known as vitamin B7). In an aim to exploit this strong attraction, they have genetically modified the bacteriophages so that once the latter take over the cellular machinery of the host, they produce viral progenies with biotinylated “keys” on their capsids. However, this genetic modification is reliant on a protein catalyst in the host. In other words, the efficiency of the formation of the conjugate system is heavily dependent on the activity of the protein catalyst. These modifications have been summarized and simplified in Figure 1. 

Figure 1. Schematic diagram representing the conjugation of the bacteriophage to the FND via a biotin-streptavidin bond.

First, to verify that the conjugate system was working, the scientists used genetically modified fluorescent bacteriophages. They could therefore visualize both the phages and the FNDs at the same time using different light filters. Firstly, they mixed the fluorescently-labelled bacteriophages with streptavidin-bound FNDs in a ratio of 1:50 to promote the production of phages conjugated to single FNDs. By overlapping the two images from the two light sources, they could determine whether the spots resulting from the fluorescent phages were localized at the same positions as the spots resulting from the FNDs (Figure 2). The co-localization of the two fluorescent spots from the phage and the FND, indicates that they were successful at generating the conjugate system.

Figure 2. Co-localization of phage and FND. A genetically modified fluorescent bacteriophage (cyan) attached to biotin and the fluorescent nano-diamond (FND) (red) attached to streptavidin co-localize to the same spot (circled), when conjugated together. The fluorescent phage pointed by the arrow represents an unbound phage.

Trinh and his colleagues also wanted to make sure that the specificity of the bacteriophages towards lamB coated E.coli was not compromised by the conjugated system. They tested the FND-bacteriophage complex on a mixture of E.coli expressing lamB on their outer membranes and E.coli with no lamB (lamB-). To differentiate between the two, the lamB- was genetically modified to express a fluorescent yellow protein. The results show that the presence of the FND on the lambda bacteriophage, does not seem to affect the specificity of the bacteriophage towards E.coli. A crucial conclusion from this test is that the FND-bacteriophage conjugate complex is able to target a specific cell in a population. This selection could be extended to more complex mixtures of bacteria, from the environment or from a clinical isolate.

Figure 3. Specificity of the FND-bacteriophage conjugate for E.coli expressing lamB. The FND-bacteriophage conjugate (cyan) is adsorbed on the E.coli expressing lamB (black) and not adsorbed on the E.coli with no lamB (green yellow). The red spots represent unbound FNDs.

There are lots of improvements to be done in this system. For example, the FND can be easily modified and therefore improved by adding a chemical modification that will react with more promiscuity. This would increase the likeliness that the bacteriophage is bound to the FND. The detection spectrum of the FND-phages could also be increased. Some bacteriophages can adsorb a broader range of bacteria. This can be exploited in distinguishing between pathogenic and non-pathogenic bacteria, which very often are similar. On the other side, the bacteriophages required genetic manipulations which may not always be a possibility with some phages. To bypass these genetic modifications, the FND can be modified with a molecule specific for the bacteriophage’s “keys” found on their capsid.

With bacterial infections on the rise and the emergence of antibiotic resistant bugs, there is a pressing issue for finding new tools aimed at detecting specific strains of bacteria. While there are very efficient tools on the market such as ELISA, it appears that this new FND-bacteriophage conjugate technique could defeat most available methods as the latter are usually associated with high costs and limited shelf-life. This paper however did not mention the cost associated with this technique. So, this peaked my interest and I found out that 5mg of FND costs about $450 which is about the same price as a ready-to-use ELISA kit. On top of that there are additional expenses associated with modifying both the bacteriophages and the FNDs. No PRESSURE but do you think it’s worth it?

- Dji

[viralovector]

Your new drug supplier

Scientists are loading filamentous bacteriophages with therapeutic drugs or genes and delivering them to the lesion location; This is what the future of drug delivery looks like. Phage nano-vehicles delivering drugs right at the doorstep.

Filamentous phages have the best qualifications for this job. They have good biocompatibility, low toxicity, high targeting specificity and most importantly they are easy to engineer. While there are other delivery systems on the market such as liposomes (lipid-bound vesicles) and antibodies, all of them have some inherent shortcomings. Liposomes, for example, which have been approved by the FDA, are easily degraded in vivo and have hindered diffusion and penetration abilities because of their large sizes. Antibodies on the other side, are rapidly cleared from the body and therefore are not the best choice in targeted drug delivery. Because of the pressing issue for new targeted drug delivery systems, scientists have paid more and more attention to bacteriophages.

The scientists behind this idea, are foreseeing two main delivery methods: One using the whole phage and one using only phage proteins. So how does it work? For the whole phage method, the filamentous phages are selected based on their ability to bind the selected target through a process called Biopanning. The chosen phage is then subjected to chemical modifications, attached to nanoparticle drugs or genetically engineering with the desired foreign gene(s). This method is known to have a far more superior “durability” when compared to spherical nanoparticles. For the second method, phage proteins are inserted into polymer nanoparticles to form phage-mimetic complexes or self-assembling nano-phages to deliver the drug or the gene. These two methods have proved to be the foundation of this delivery system.

Figure 1. The filamentous phage loaded with the desired nanoparticle drugs or the desired gene (Method 1).

This cutting-edge technology is still being developed and tested and there are only few research that have been done in this area. Ju and Sun have therefore reviewed the few recent applications of filamentous bacteriophages for targeting drug delivery in vitro and in vivo, one of the in vivo examples being about Alzheimer’s. If you think about it, this concept could also be applied to life threatening diseases such as cancer, where targeted therapies are required. While this project needs to be rapidly pushed forward, it will definitely take a while to concretize our hopes, in view of the fact that the FDA has rigorous rules pertaining to bacteriophages.

Until next time,

- Dji

[viralovector]

At the end of the eclipse

Figure 1. A representation of an infective centre or an infected gram-positive bacterium, right before cell lysis.

https://www.tinkercad.com/things/8MMQZjeWNet-terrific-vihelmo After adsorbing on the bacterium and inserting its genomic material (DNA in this case), phage proteins are produced using the host’s metabolic machinery directed by the phage’s DNA. The newly synthesized phage parts assemble into new viral progenies, which then produce lysins or lysozymes. With the damaged cell wall, osmosis causes the cell to swell and eventually burst. Figure 1 represents a snapshot right before the actual lysis. I wanted to do a 3D model for my creative post as it really depicts the process of the phage infection. You can click on the link and interact with the 3D model. The program is pretty user-friendly. However, the transparency of the bacterium is not visible in the interactive mode, not sure why :(. 

Enjoy!

[viralovector]

Forging the perfect weapon against GRAM-NEGATIVE BACTERIA

Scientists are taking advantage of a phage-encoded weapon, called lysin, as an alternative therapeutic to fighting bacterial infections. By tweaking this molecule, they have been able to design this game-changing drug, that is predicted to alleviate this antibiotic crisis. What makes it so attractive to scientists, is its high specificity and high effectiveness. While, a single bacteriophage takes more than 30 minutes to blow up its host, a lysin molecule on the other side, takes seconds to do so in vitro. Furthermore, it has been proven that contrary to antibiotics, treatment with lysins do not affect our inner bacterial ecosystem, thanks to their high specificity. This makes lysin an ideal candidate for therapeutic purposes. Now let's rewind a little bit to understand this fundamental weapon. 

Over millions of years of evolution, bacteriophages have perfected two main methods of exploiting bacterial cells: through either a lytic cycle or a lysogenic cycle. In the lysogenic cycle, the viral DNA/RNA is incorporated in the host's genome and passed on to subsequent generations. In the lytic cycle, the lysin released by the bacteriophage, eats off the most important bacterial protective layer, the peptidoglycan, and unleashes the viral progenies. This peptidoglycan is a crucial structural polymer surrounding bacterial cells; It is the door to entering the bacteria and the key is lysin. 

Lysins are part of a big family. The variation within the lysins is based on the different right and left compositions. While the left side is the active part, that snips off the peptidoglycan, the right part (CBD) has the unique cuts that allow access to different varieties of bacteria and thus providing its high specificity. In recent years, scientists have been able to exploit this CBD to orchestrate the specificity of lysins towards different bacteria. 

Bacteria are usually divided into two groups: gram-positive and gram-negative bacteria, which differ fundamentally in the number membranes surrounding them. Gram-positive bacteria have two layers of protection, a plasma membrane and the peptidoglycan membrane while gram-negative bacteria have an extra outer membrane, peppered with lipid-sugar chains, called lipopolysaccharides (LPS). 

So, all in all, lysins are able to burst open bacterial cells, via the peptidoglycan. However, things are never as simple as it appears. It turns out that a major problem arises when lysins are exposed to gram-negative bacteria, not interiorly but externally. The extra outer membrane present in gram-negative bacteria acts as a barrier to the lysin, rendering it utterly useless (Figure 1).

Figure 1. The reason why lysin cannot lyse gram negative bacteria when exposed exteriorly. Gram negative bacteria have an extra outer membrane which prevents lysin from accessing the peptidoglycan.

Wang and his collaborators might have found a solution to this problem. They have utilized the properties of the CBD to disorient lysins to targeting gram negative bacteria. By fusing the lysin from a gram-negative bacterium (E.coli) phage, to the CBD of a gram-positive bacterium phage lysin, they have engineered this hybrid named lysep3-D8 (Figure 2). So how does it work? This extra CBD has a slight positive charge, which facilitates its binding to the lipopolysaccharide, LPS, found on the outer surface membrane of gram negative bacteria. A research in 2004 suggested that this binding increases the permeability of the outer membrane, changing its integrity. This alteration presumably facilitates the access to the peptidoglycan and therefore facilitates its hydrolysis.

Figure 2. The engineered hybrid lysin, Lysep3-D8, made from lysep3 isolated from an E.coli phage and the D8 (CBD) domain from a Bacillus amyloliquefaciens phage lysin.

The researchers predicted that this new designed lysin will be primarily effective against E.coli. Additionally, since part of Lysep3-D8 is highly similar to another gram-negative bacterium (P. aeruginosa) phage lysin, Wang and associates raised the possibility that the hybrid might also be effective against P. aeruginosa. To verify these strong hypotheses, Wang and his colleagues decided to put the hybrid to the test.

The effectiveness of the lysep3-D8 hybrid was tested on one E.coli strain (CVCC1418). As indicated in Figure 3, the growth was significantly inhibited in the presence of the hybrid lysin, in the first four hours, when compared to the negative controls, for which no inhibition of growth was predicted. These results suggested that the engineered lysin is potent against CVCC1418 at low concentrations.

Figure 3. The cell density for each treatment on CVCC1418 over a period of 48 hours, with the colors representing the cell counts/ml.

However, significant regrowth was observed for the lysep3-D8 treatment after the first four hours. The researchers were able to prove that this regrowth was not due to a decrease in the activity of the lysin. Furthermore, the cell density for the hybrid treatment remained significantly lower as compared to the controls. In other words, the hybrid was indeed partially effective at lysing the E.coli. But how about other bacterial strains?

To verify the antibacterial spectrum of the hybrid lysin, Wang and associates repeated the experiment on 14 other strains and clinical isolates of E.coli, as well as other gram positive and negative bacteria, including Pseudomonas aeruginosa (gram-negative), Streptococcus (gram-positive) and Klebsiella pneumoniae (gram-negative). The effectiveness of the hybrid lysin towards the different bacterial strains and species is summarized in Figure 4. These results suggest that the hybrid is reasonably potent over a fairly broad spectrum of bacteria.

Figure 4. The bacteriostatic spectrum of Lysep-D8.

Considering these scattered results, we cannot make any generalized conclusions with respect to the effectiveness of the hybrid towards any particular bacterial species. However, for now this research is encouraging as they have been able to target some gram-negative bacteria externally using lysins! While loads of work remain to be done in improving the efficacy of this weapon, there is still some hope that engineered lysins could possibly serve as an alternative to antibiotics in the future. Something to bear in mind though is that the research in lysin works hand in hand with the discovery of bacteriophages. The more bacteriophages are discovered, the more lysins are isolated and the more potential there is for mix and match to design other tailored lysins. This exciting field is still in infancy and I am certain that in a couple of years, the number of discoveries in that sphere will boom. However, how long will it take for natural selection to take over and wipe out the sparse efficacy of these engineered weapons? Will all these research in that direction be even worth it?

This opens the door to further discussion…

- Dji 

[viralovector]

Goodbye allergies?

Peanut butter… spring… For some people, these things bring back wonderful childhood memories, for others they are the culprits behind missing out on time in the great outdoors or on enjoying a good PB&J sandwich. The figure below shows the remarkable distribution of prevalent food allergies in the world. In other words, allergies are not to be taken for granted. However, new advances in the understanding of phage therapy has led to new perspectives for their use in controlling anomalous immune responses, such as asthma, atopic dermatitis or food allergies.

http://www.cbc.ca/natureofthings/features/map-of-allergies-around-the-world

90% of food allergies in the world

Our immune system is very well orchestrated, equipped with an artillery of defence mechanisms. From B cells to antibodies to mast cells, every single component has an essential role. Some B cells are responsible for detecting allergens - an substance that causes and immune response. They consequently produce antibodies which bind mast cells triggering the release of histamines. Too complicated? Let's put it into perspective. By accident, you end up consuming a cake containing traces of nuts that triggers an allergic reaction: itching, inflammation, difficulty in breathing and increased heart-rate. These physiological symptoms are triggered by the release of histamine. This is why some people take anti-histamines or even the magic EpiPen, which are both very often associated with other side effects, in case of allergic reactions.

This paper highlights the different effects of phage therapy on those inflammatory responses. Phage therapy has been proved to down-regulate allergen-induced inflammation both in vitro and in vivo through different biochemical pathways. Any side effects to this therapy? Well there are no definite answers. Very often though, the introduction of foreign particles or organisms in the body can cause an allergic response. However, no allergic reactions have yet been associated to phages.

That paper struck my attention as I am myself a victim to atopic dermatitis - a type of allergic hypersensitivity. Just imagine getting rid of all allergies; no more worrying about nuts, gluten, lactose, no more inhalers. Could this be the end of allergies? Well maybe...

- Dji

[viralovector]

Phages and bacteria: a complicated relationship

CRISPR is well known for being a gene editing tool. However, most of us are not aware that CRISPR is part of the prokaryotic defence mechanism against phages. The CRISPR system consists of incorporating part of the viral genome as a way of remembering each specific virus, similar to a barcode. The bacteria can then mount a strong response for subsequent invasions from that same virus. As a result, the phages have to change their game plan and thus feel pressured to evolve. What is very interesting is that the CRISPR system has limited storage, just like a hard-drive. At one point, the bacteria have to get rid of 'old' viral genomic barcodes to make space for new ones.

This study done by Deem and Han shows that the interplay between phages and bacteria can have three phases: one that drives the extinction of the phages, one where the phages win and one where both bacteria and phages coexist. The occurrence of these three phases depends entirely on how often bacteria encounter phages and the mutation rates in both genomes. Let's take a step back and understand this fundamental point with an example. If a bacterium encounters multiple phages in a certain amount of time, it has higher chances of having a copy of the viral genomic material; therefore, more likely to drive the phages to extinction. Hence, the interaction between phages and bacteria is highly unpredictable.

What is great about this paper/article is that it underlines the fact that the path to phage therapy will be laborious. The factors discussed in this paper just add up to the list of elements to consider before phage therapy can be approved globally. I do encourage you to dedicate a few minutes of your time to this article. The actual paper does have a lot of convoluted formulas but for most part it is quite neat.

Until next time,

- Dji

[viralovector]

About me

Hello everyone

My name is Djihane (also known as Dji) and I come from Mauritius, a tropical island next to Madagascar. I am in my fourth and final year of my biochemistry degree at the University of New Brunswick, Fredericton. Since I came here about 3 and a half years ago, I lost track of the amount of times people ask me: why UNB though? My answer: I don’t know. My mom wanted me to. I guess it was the furthest place I could go.

For those who do not know me, I am a quirky, introvert and awkward person. Some of my hobbies include drawing, cooking, baking and playing my guitar. I also enjoy any kind of sports but mainly tennis, badminton and soccer. As cliché as it might sound, I love travelling. So far, I have been to France, UK, Germany, UAE and Canada. I am also looking forward to attending the Game of thrones concert this spring; I am a huge fan of the soundtracks and the show. However, my favourite show will always remain The Simpsons.

A picture of one of my dog that I miss so much. I have been told she looks like Dobby from HP

Enough about me now. Let’s talk about the good stuff: MY BLOG. My blog is going to be centered around bacteriophages.  I personally think that viruses are amazing, standing at the frontier between living and non-living. Being a huge fan of drawings and symmetrical shapes, I have always been fascinated by viral morphologies; bacteriophages more specifically. In my opinion, phages convey the imagery of robotic spiders grasping their prey with hooks, needles or teeth and eventually taking their life. It is just fascinating how such infinitesimal biological entities, with barely any organelles, are able to rule over the prokaryotic world.

Throughout this semester, I will emphasize on the medicinal and biochemical aspect of bacteriophages. This includes the mechanisms that they employ to invade bacteria. Moreover, I will talk about the resurgence of phage therapy in the treatment of bacterial infections in this post antibiotic era. It is undeniable that there is a long way to go for the approval of phage therapy by the FDA. However, I think that we should remain hopeful that phages will one day be a definite alternative to antibiotic treatments.

I hope you are as excited as I am to learn more about the amazing abilities of these masterminds.

- Dji