INTRO To bacteriophage
In part 1, I touched on:
- What a bacteriophage is.
- The abundance of phage in the environment.
- The evolution of phage.
- The significance of phage in scientific advancements and medicine.
- The scientific history of phage and their discovery.
Part 2: Phage Biology
In part 2 I aim to cover:
- Phage morphology.
- Their replication cycle and how our diet can influence this.
- The phageome in disease states.
- Phage genetic composition.
Cup of tea at the ready cause this is going be a long one!
Phage are most characteristically famous for their strange icosahedral (20 faced) head or capsid which contains their genetic information (single or double-stranded DNA or RNA). Their capsid is connected to a long tail or body with spidery leg-like appendages.
Structurally they are diverse, but the most studied and the most abundant population is the tailed dsDNA phage belonging to the order Caudovirales.
In the lumen of the gut there is 1 VLP (virus-like particle for every bacterial cell and most of these VLPs are dsDNA.
Tailed phage have three major components:
- The capsid (head) where the genome is packed
- The tail (body) which serves as a pipe through which their genetic material is ejected and travels through from the head into host
- The adsorption apparatus or adhesive system which recognizes and binds to a host cell and is equipped to penetrate its host cell wall.
A remarkable thing about phage is their symmetrical geometry. Their highly organized structure boasts the wonders of the configurations inherent in nature. An artwork of protein origami, their molecules fold into brilliantly balanced but functional patterns.
X-ray crystallography resolved the first structure of the phage capsid at 3.6 Å (angstrom) resolution. 1 angstrom is one ten-billionth of a meter or 0.1 nanometers
Its structure, which is so efficient and evolved for its task, resembles a lunar lander designed for the first Apollo missions.
Their remarkable capsid symmetry is broken at the junction that connects the head to the tail – the head-to-tail interface (HTI). The HTI is composed of a dodacameric (12) portal protein (PP) at the base of the capsid and serves as a valve for the release of their genome.
The portal protein and the tail-like body forms a pipe through which the virus injects its genetic material into the host.
I find the techniques involved in uncovering phage and virus morphology so interesting. Joining a recent online virus webinar it was epic to see scientists talking about their work on phage morphology and capsid structure.
One particular researcher who caught my attention was Kristin Parent whose talk was about the recently discovered genus of “giant viruses”. I practically screenshot her whole presentation.
Although not a phage, her work on the samba virus – a giant virus isolated from the Brazilian Amazon – is an excellent case study on the work that is being done on virus morphology and how brilliantly conserved and stable viral architecture is.
Giant viruses have a conserved “stargate” or star-shaped opening at the bottom of their capsid through which their dsDNA is released. The stargate is highly stable except for in high temperatures and low pH.
In a bid to prise open the stable stargate, they boiled the nucleocapsid in acid! She described the open stargate as having an uncanny resemblance to that hatching pod monster in the Alien movie.
I don’t think regular phage would be this stable. I hear their structure is destroyed at around 58°C, the same as a regular bacterial cell.
Another scientist, Pascale Boulanger, discussed the structure of tailed bacteriophage capsids, the process of assembly of the nucleocapisd of the T5 phage and how they have potential to be engineered for nanotechnology purposes.
For information on the next virus webinar visit here.
Phage Life and replication cycle
Another fascinating aspect of phage physiology is their ability to switch states or phases of their life cycle.
Phage live a double life.
Existing as obligate parasites dependent on the presence of a bacterial host, some phage have adapted their strategy to be able to remain dormant inside the host cell while taking stock of other factors in their environment.
In other words, some phage can toggle between states – as a bacterial sniper, ruthless killer and agent of replication or as a bacterial espionage, keeping it on the down-low, piggybacking off bacterial resources, and surveying their environment until an opportunity or crisis strikes.
Traditionally, phage are thought to have 2 states:
Lytic/virulent state – most famously associated with viruses. Where a phage infects its host and immediately begins driving replication and assembly of an army of phage particles, lysing or rupturing the host cell and continuing to invade nearby bacterial hosts.
Lysogenic/temperate state – here their genetic material gets recombined with the host’s DNA forming a prophage. The incorporated phage DNA can remain dormant until it deems conditions are approprate for it to switch into a lytic state.
In the dormant lysogenic state, the phage’s DNA gets replicated in concert with the bacterium’s natural reproductive cycle. Upon division, the bacterium splits in two with a copy of the bacterial DNA as well as the phage DNA being inherited. Under certain circumstances such as environmental stress, the phage can switch to the lytic cycle, begin replicating and burst from the host cell in search of another host.
Some phage have the ability to choose between states but others have been identified to be strictly virulent, following a lytic cycle. The strictly lytic phage are of interest as antimicrobial agents (AMRs).
Although most often characterized as either lytic or lysogenic, phage have been found in the human gut to exist in other states such as pseudolysogeny, a carrier state and a state of chronic infection. We don’t know that much about the logic driving phage states in the human gut or in what ratios of states a phage population exists in, but a study which I will refer to later found that in a healthy microbiome, phage occupied a viruent, lytic state.
Of course, bacteria have evolved sophisticated mechanisms for resisting phage advancements and fighting back! So understanding phage state is further complicated by phage-host dynamics.
Being obsessed with the notion of symbiogenesis and endosymbiosis – cells or even DNA becoming lodged or permanent residents in a foreign cell – I needless to say find prophages very intriguing.
Prophages, the modules or segments of phage DNA that get incorporated into the bacterium’s chromosome, are extremely common in bacteria. I gleamed some important bullet points from the paper bacteriophages benefit from generalized transduction regarding their behaviour and the mutualism occurring between them and bacteria:
- Almost half of all bacterial genomes carry prophages
- Pathogens are more likely to be lysogenic than non-pathogens
- The common human pathogen Staph. aureus carries between 1 and 4 prophages, while all Salmonella species have prophages.
- Prophages can negatively impact the host through
- disrupting genes during insertion,
- obviously through lysis when toggling into lytic cycle and
- by a metabolic cost.
- However, a mutualism occurs where the prophage provides benefits to the host cell:
- prophages provide immunity from subsequent attacks from related phages by expressing phage repressor protein that controls the transition between temperate and lytic replication
- expressing a variety of adaptive genes
- expressing virulence factors which aid the host, if it is pathogenic, in colonizing its target and increasing its pathogenicity
Temperate phage Dominate Crohn’s disease state
A number of studies have shone a light on the virome portion of the human microbiome and have illuminates connections between phage life cycles and disease states.
Inflammatory bowel diseases such as IBD and Crohn’s, are associated with decreased bacterial diversity and decreased numbers of certain Firmicutes and Bacteroides.
Recent studies have shown the virome to also be a marker of disease states and of microbiome stability or churn (the turnover of organisms in the biome).
A whole-virome sequencing study sequenced samples from 61 healthy controls, 27 subjects with Crohn’s disease, and 42 with Ulcerative colitis and aligned them against.(n = 7,605) known viral sequences as well as a database of unknown viral sequences or viral dark matter.
A picture that emerged was that the core virome – the clustered viral sequences of taxonomic rank shared across healthy individuals – disappeared in Crohn’s disease.
They also found that healthy individuals were dominated by virulent phage whereas disease cohorts were dominated by lysogenic phage.
One can postulate that in a healthy individual their phage population is active and engages a high rate cell turnover – killing bacteria and multiplying, whereas in an inflamed or unhealthy microbiome environment, phage toggle into a dormant or environmental espionage state.
It is thought that phage mostly attack bacteria when they are highly abundant in their environment. Perhaps inflammation causes reduced or muted population numbers which has a knock-on effect on the phage population.
Food Induces bacteriophage state
Chemicals such as mytomycin c are commonly used in the lab to induce the lytic cycle in phage. Mytomycin C is a chemotherapeutic which causes DNA damage and would be commonly used to “induce” prophage i.e. get them to jump out of the lysogenic cycle, and enter the lytic cycle.
By stressing the bacterial cell, this causes the phage to “jump out” or cause the phage DNA to be replicated by the bacterial host cell’s machinery.
It’s pretty wild that this segment of phage DNA can “jump out” or become expressed under certain conditions in the host. such as when it senses the bacterial cell is under stress, replicating at a slower rate than is optimal. or if there’s an unfavourable ratio of chemical constituents inside the cell. It makes you wonder what segments of our DNA are becoming expressed under certain conditions. This train of thought would be better explored in the field of epigenetics.
This comprehensive study looked at the effect of 117 commonly consumed foods and chemical additives on the induction of prophages in bacterial hosts
By counting the number of VLPs (virus-like particles) using flow cytometry in bacterial cultures before and after the addition of a long list of foods and chemicals, they found that some additives induced the lytic phage state due to a massive increase in the number of VLPs and others induced a prophage state from seeing less VLPs in the culture than before the addition.
They tested these ingestible compounds on:
- Bacteroidetes (Bacteroides thetaiotaomicron strain VPI-5482)
- Firmicutes (Enterococcus faecalis)
- Staphylococcus aureus
- Pseudomonas aeruginosa (Phylum Proteobacteria)
Known prophage inducers are soy sauce, nicotine, sunscreen and antibiotics such as ciprofloxacin.
The study found:
Several compounds reduced the number of VLPs relative to the control. Rhubarb (−62%), Fernet (57%), coffee Arabica (−49%), and oregano (−44%) reduced the number of VLPs in all bacterial species. Several other compounds suppressed VLPs in individual species. Pomegranate (−89%), grapefruit seed extract (−89%), toothpaste (−88%), and cinnamon (−88%) reduced VLP production in B. thetaiotaomicron. Kombucha reduced E. faecalis VLPs by 44%.
This study showed that diet influences our microbiome composition not only through feeding our bacteriome but also by shifting bacteriophage life cycle.
As mentioned in Part 1, phage vary wildly in genomic composition. No two isolated phage have been genetically similar, and the small pool of sequenced phage are highly varied.
:Lateral/horizontal gene transfer both describe the acquisition of genetic material from another organism without being its offspring. It often can include the transfer from organisms of another species.
Bacteria and viruses’ promiscuous methods of gene transfer can confer many advantages in evolution for their species through acquisition and selection of beneficial genes from the wider gene pool.
In eukaryotic cell division, the daughter cell receives an exact copy of DNA from its parent cell during mitosis, known as vertical gene transfer. The daughter cell will directly continue the exact genetic lineage inherited. The method of replication by bacteria and viruses is more akin to the shuffling of a deck where the deck is the accessible gene pool from organisms in its environment.
One could say the evolution of eukaryotes occurred through the lateral gene transfer of bacteria to primitive eukaryotic cells e..g mitochondria, plasmids.
There are a number of methods via which phage can transfer genes laterally:
- As gene transfer agents
Temperate phages are gene brokers
Phage play a massive role in bacterial genome evolution through lateral gene transfer. Temperate phages (the ones whose genetic material gets incorporated into the host cell) have been described as brokers, providing an intracellular reservoir of new genes for the host as well as viruses that may infect the same cell.
Bacteria benefit from phage transduction
Phages are not only viruses but gene-transfer particles, making them massive drivers of bacterial evolution.
Transduction is where foreign DNA is transported or introduced into a new cell by means of a virus.
Phage partake in a number of methods of transduction. Some are precise and by necessity e.g. due to flanking bacterial DNA next to the prophage insertion site (termed specialized transduction). Others occur in a more random fashion.
Generalized transduction is where phage randomly package bacterial DNA into their capsid instead of their own. By this method, any section of bacterial DNA can get shipped to a random bacterial cell of the same species.
In this way bacteria can acquire new genes that are selected for in their environment i.e. antibiotic resistance. Here the host cell acquires adaptive genes which further protects the prophage life cycle and prophage viability.
This study, titled “Bacteriophage benefit from generalized transduction” ran a number of cell culture scenarios and found that phage provide adaptive power to the hosting bacterium through facilitating transduction of their genes.
What they did was allow a number of bacteria to replicate in an environment where some bacteria possessed an antibiotic resistance gene and others did not. Then they introduced phage into the mix, along with an addition of antibiotics.
In all simulations, except for where phage were temperate and transducing, and bacteria accepted lysogens, both the populations of bacteria and phage peetered out and died.
Genomic analysis of the surviving bacteria showed they carried a prophage (integrated phage) and the antibiotic resistance gene.
This experiment showed that “bacteria and generalized transducing phages cooperate to survive adverse conditions such as the presence of antibiotics by phage integration and transduction of antibiotic resistance genes from neighboring cells.
Even in their “dormant” lysogenic state, phage are quietly pulling the strings, transducing particles of antibiotic resistance, and influencing their environment to make bacteria do their bidding.
It is not by accident that phage are posting their host’s DNA to neighbouring cells, they are purposely trying to infer fitness to the population!
Bacteriophages are said to have a mosaic genome or display mosaicism..
What this means is that their genome is comprised of segments from different origins. This occurs directly from participating in horizontal gene transfer where segments of DNA which originated from the wider phage population get incorporated into the genome in a kind of cut-and-paste insert-non-related-lego-brick fashion.
These segments or bricks of DNA that are exchanged between phage are called “modules” and are thought to be “minimal autonomously functional units,” such as groups of genes that must function together or single proteins or even protein domains that function independently.
These segments often encode the crucial functional units needed for replication, assembly and envelope formation.
The regions with different extents of similarity have been interpreted to be genome sections with different evolutionary histories that have been horizontally exchanged among phages.
Virus taxonomy is a tighlty regulated affair. There even exists an International Committee on the Taxonomy of Viruses (ICTV) who are responsible for updating their taxonomy scheme yearly to name and classify new viruses.
From checking out the ICTV 2019 species masterlist release there are 6590 unique species of virus.
Historically phage have been categorized by their morphology, the range of hosts they occupy and their nucleic acid structure e.g. dsDNA, ssRNA. A big amendment in the field of virus taxonomy came with the announcement that the ICTV would accept metagenomic data for taxonomic proposals.
With horizontal gene transfer being the dominant mode of evolution, this creates a turbulent evolutionary history.
This slow boil of cut-and-paste horizontal gene transfer over billions of years resulted in a series of highly different and incongruent phage genomes with very few genetic similarities. This makes attempts at categorizing phage taxa from their genetic sequences very difficult.
Despite this, a small number of core genes, called viral hallmark genes, responsible for important processes such as virion morphogenesis and replication can be identified within sequences which confirm its status as a virus but does little to confer context or functionality of the wider unrecognized genes (viral dark matter).
Since the phage we have in current databases are all genetically different, database-independent bioinformatic methods of classification are required for further exploration.
Phylogenetic reconstructions are not possible to map virus relativity but a gene-sharing network approach has been suggested as a better means of classifying them with existing sequencing data.
If you are weirdly captivated by virus taxonomy like me and want to learn more in detail about proposed viral megataxonomy I highly recommend this review.
As well as through making sense of sequencing data, newly isolated phage displaying an interesting phenotype need to be thoroughly examined.
I have no doubt that further exploring phage genetics and physiology will lead to exciting new discoveries which will yield solutions for overcoming antimicrobial resistance and serve impactful biotechnology functions.