8 Jul 2013

The strange virus from the sewage

Viruses can infect all types of organisms. Unable to multiply on their own, viruses parasitise animals, plants, bacteria and even other viruses, in order to propagate. Bacteria-killing viruses, called bacteriophages or simply phages, are the most abundant and diverse organisms on the planet. It is estimated that there are over 100 million different phages, but only about 0.0002% of phage genomes have been sequenced. 

Sewage-polluted waters, like some lakes and ponds, are a sample haven for virologists- they are filled with organic material on which bacteria thrive; and where there are bacteria, there are bacteriophages. It was in one of these bacteria broths, about 200km northwest of Vilnius, in Lithuania, that a research team led by Rolandas Meskys and Laura Kaliniene found Rak2, a phage unlike any other.
Water contaminated with sewage is a sample-haven for virologists. 
(Credit: Flickr/eutrophication&hypoxia)

Merciless lifestyle
Bacteriophages latch on to bacteria and then transfer their genetic material into them. In a matter of minutes, the bacterial cellular machines replicate and translate the phage genes into viral proteins, which assemble into hundreds of new viral particles. Merciless to their host, the new phages burst the bacterium to free themselves and move on to infect other victims.

Despite the phages' tiny size - about 100 times smaller than bacteria - with the help of high-power electron microscopes (EM), scientists can see them in quite some detail. The most abundant types of phage are by far the Caudovirales, or tailed phages, which have a maraca shape, with a head (containing the genetic information) and, as the name suggests, a tail. The phage tail is a versatile lethal weapon. First, it recognises the right bacteria host by protein matching, like a barcode reading machine. Second, it works as an anchor, firmly attaching the phage to the bacterial surface. And finally, the phage tail acts as a syringe, by piercing the bacterial cell wall and pushing viral DNA through it.

It was the shape of the Rak2 phage that first intrigued Meskys and his colleagues at the University of Vilnius. "The morphology of this phage is amazing," he says. Detailed EM images revealed that Rak2 is a tailed virus from the Myoviridae family, which typically have a contractile tail with six fibres at the end. But Rak2’s tail is very special. “The EM shows that the tail fibres contain spikes, this is only known in a few phages,” explains Meskys. Rak2’s tail structure, with its spiky fibres, resembles the tails of some myoviruses, but other features, like the absence of prongs and the intricate pattern of the spikes, set Rak2 aside from any other known phages.
Typical myovirus bacteriophage (Credit: wikipedia)

A giant phage
The other unusual thing about Rak2 is its genome- it’s huge. With about 534 predicted genes, Rak2 is the fourth largest myovirus sequenced to date, and the largest phage known to infect Klebsiella sp. bacteria, Rak2’s only host. But size isn’t everything; Rak2’s genome is truly unique. About half of its genes don’t have any similarity to other viral genes, and a significant proportion of its predicted proteins have an unknown function. The 117 genes that do encode for well-described proteins, such as tail or DNA repair proteins, show similarities to genes of different phage families, but also to some bacterial genes. Meskys says “Philogenetic analysis shows that this phage is quite mosaic, some parts [of the genome] are more similar to the Myoviridae group and other parts to the Podoviridae group, maybe there was some horizontal transfer of genes.”

Horizontal gene transfer occurs when genes ‘jump’ from one species to another. For instance, different bacteria strains can exchange antibiotic-resistance genes between them in a process called conjugation- the closest thing bacteria have to sex. Viruses can exchange genes between them and also with their host. Instead of killing their host cell, some viruses, including phages, insert their DNA into the host’s genome so it replicates with its DNA. When the viral DNA leaves the host’s genome, it can carry along some chunks of it, or, more often, it can leave some of its own DNA behind. Because several viruses can invade the same host, genes from one virus might end up in another virus’ genome. It is estimated that a whopping 8% of the human genome is made of viral DNA, and it seems that most species, from bacteria, to mammals and plants, carry viral genes in their genome. Bacteriophages are experts in this kind of inter-species gene mixing, and Rak2 appears to have an extreme mishmash genome.

EM images of Rak2 phages (Credit: PLoS ONE/Rolandas Meskys)

An alternative to antibiotics?
Meskys plans to continue working on this phage. He would like to understand the function of its unique proteins predicted by sequence analysis. “We found a gene that predicts a huge protein with no functional homology in any other phage. What is this protein doing?” he asks. There are also potential applications for Rak2. Historically, phages have been used in medicine to treat bacterial infections, such as dysentery and cholera, but with the discovery of antibiotics this approach was mostly abandoned. Now, with the dangerous rise in antibiotic-resistance bacteria strains, phage therapy is coming back in vogue.

There are many advantages for using phage therapy. Unlike antibiotics, phages target specific bacteria strains, so the ‘friendly’ bacteria in our guts are left unharmed. If bacteria become resistant to a phage, it can quickly change to overcome the new resistance, while new antibiotics take over ten years to be developedBesides, new phages targeting multi-resistant bacteria can easily be identified in sewage samples. 

Another key difference between using antibiotics and phage therapy is that, in contrast to antibiotics, which normally just prevent bacteria from multiplying, phages actually destroy bacteria. And they do it with finesse- at low dosage (phage dose is increased ‘naturally’ by replication in the bacteria) and with negligible toxicity for the human patient. 

A number of pharmaceutical companies are also developing phages for other applications, such as veterinary, agriculture, food control and drug delivery, just to name a few. “If we could identify which type of tail spikes are involved in the recognition of a specific bacteria strain, […] maybe we will be able to change the spike proteins so that the phage attacks other bacteria that are more important for medicine or food,” Meskys says.

A productive department
Meskys currently runs a research department at the Institute of Biochemistry of the University of Vilnius, the country’s capital. With six research groups working on several aspects of bacteriophage diversity and biocatalysis, the department operates as a huge lab. “If we have a particular problem to solve, we can involve different members of the department to solve it.” Meskys has a strong creative input in the department’s research and plays an essential part in getting intra-departmental collaborations going. “I am involved in all research groups […] I need someone to implement my crazy ideas,” he jokes. 

A biochemistry graduate, Meskys began his research career as a PhD student in Valdas Laurinavicius’s lab at the Institute of Biochemistry, where he later established himself as an independent researcher and finally was promoted to head of department in 2002. Despite having spent his entire career in Lithuania, Meskys started multiple international collaborations and has, in several instances, been invited to teach or visit labs in other countries. There are fruitful relations established in the department with local and foreign biotech companies. “We are cooperating in the screening for new enzymes for chemical synthesis, diagnostics, food processing etc. Our expertise is in development of new screening technologies,” he says.

Laura Kaliniene, lead author of the Rak2 study, holding a phage plate.

Research in Lithuania
The main source of research funding in Lithuania is the Lithuanian Research Council (LRC). Like many research institutions in Europe, the LRC gives priority to applied research. Most research grants are allocated to projects with potential industrial applications, or to groups with high number of publications and patent submissions. “There is pressure to show that you are achieving something,” says Meskys, but there are also smaller grants for projects “where you can do what you want,” but the competition is high.

Despite Lithuania’s fast growing economy, rising unemployment and low salaries continue pushing highly skilled Lithuanians abroad. "We are losing the bright and intelligent people, emigration is a huge problem for Lithuania.” Meskys adds that there is a ‘narrow market’ in research in Lithuania, so students prefer to do their PhDs in countries like the UK, Denmark or the USA. But there is some world-leading research in Lithuania, especially in the fields of biochemistry and laser technology (a certain type of laser produced in Lithuania accounts for 80% of the world market), and the number of biotech start-ups is on the rise; for example, Fermentas, which was bought by Thermo-Fisher in 2010, was originally a Lithuanian company. “Some research fields are well established, Vilnius University is more than 400 years old, […] in some specialities there are long traditions.”

Šimoliūnas E., Kaliniene L., Truncaitė L., Zajančkauskaitė A., Staniulis J., Kaupinis A., Ger M., Valius M., Meškys R. & van Raaij M.J. & (2013). Klebsiella Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family Myoviridae, PLoS ONE, 8 (4) e60717. DOI:

This is a modified version of my article published in Lab Times on 5-07-13. You can read it here.

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