DR PETR LEIMAN Macromolecular machinery

Dr Petr Leiman discusses his latest research into the of a special in bacterial that is surprisingly analogous to structures used by to attack enemy cells

systems of some bacteria; what are these carrying a T6SS organelle can fire it repeatedly and what is their function? by reassembling the organelle again and again, thus sending numerous toxins out and The type VI secretion system (T6SS), along into other cells. On the other hand, R-type with other systems related to phage tails, pyocins, which are similar to bacteriophages, breach the membrane of the target with have to break the original cell to come out the help of a special spike-shaped . and become active. A cell essentially has to Some of these spikes carry a sharp tip protein. sacrifice itself in order to release about 200 Most of the tip proteins contain the PAAR pyocin particles, which will then diffuse out amino acid sequence motif and form a smooth and kill other cells not related to the original extension of the spike. In T6SS, the PAAR mother cell. Each pyocin particle is then able repeat proteins often contain additional toxic to kill its target cell in a single shot-type of domains, whereas in phages they make the tip action; unlike T6SS, there is no recycling. sharper and equip it with a metal close to Interestingly, the targets of R-type pyocin- the membrane-attacking apex. T6SS is known like particles are not limited to prokaryotes. to deliver toxic proteins into neighbouring R-type pyocin-like particles are used by certain competitor cells, and the association of these species of Serratia and Photorhabdus bacteria domains with a PAAR repeat domain that to kill insect larvae. forms the tip of the membrane-piercing spike, What is the current focus of your research? is a brilliant way in which these machines have What are your hopes for the next stage of evolved to be efficient. this research? Bacterial viruses, or bacteriophages, have a special organelle called a tail with which This investigation is focused on both At present, our understanding of how phage they attach to a host cell and deliver T6SS and R-type pyocins. How do these tail-like work is very fragmented. their genetic material into its cytoplasm. assemblies work together to aid infection of Some of our ideas are borrowed from The tail is a complex multicomponent host cells? macroscopic analogies, such as those of the ‘nanomachine’ created by a precise extended spring and membrane piercing. arrangement of dozens of different protein The target of T6SS and R-type We do not know whether these are really molecules. Bacteria employ similar pyocins are different. T6SS is primarily an inter- applicable. Neither do we know how much organelles to attack and kill competitors or species weapon (eg. Pseudomonas aeruginosa energy is stored in a tail-like structure or how hostile neighbours, such as other bacteria against Escherichia coli) whereas R-type pyocins much of that energy can be used for doing or cells of the human immune system. One are used in intra-species competition (eg. by actual work – such as translocating proteins of the most fascinating things about these different strains of P. aeruginosa). In principle, across a membrane – or even how much nanomachines is how much they resemble P. aeruginosa can attack its neighbours with its energy is lost in vibrations and heating of machines from our macroscopic world. A T6SS and pyocins at the same time. the surrounding solvent. The answers to phage tail-like structure can be compared to these questions, which lie at the interface of an extended spring, which contracts at the You have drawn comparisons between these physics and chemistry, are to be found in the right moment to pierce the membrane of systems and bacteriophage tail structures; structure of the phage tail-like organelles that the target cell. what are the similarities and differences we hope to obtain. More importantly, with between these structures? the atomic resolution description of a phage Your work explores proline-alanine- tail-like nanomachine, there will be a wealth of alanine-arginine (PAAR) repeat proteins, T6SS is a tubular organelle located inside biological questions and medical applications which are found in the type VI secretion the cytoplasm of the predator cell. A cell to be discovered.

WWW.RESEARCHMEDIA.EU 51 DR PETR LEIMAN An offensive organelle

A team of researchers based in the Laboratory of Structural and at the École Polytechnique Fédérale de Lausanne, Switzerland, is conducting cutting-edge research into the structure and function of macromolecular machines involved in the infection of bacterial and eukaryotic cells

Bacteriophage phi92 with its membrane- attacking spike protein highlighted in red. This 3D structure was obtained by aligning thousands of 2D images of individual particles that were preserved in their native state by cryo-fixation and imaged with an electron microscope.

MANY OF THE world’s deadliest diseases up the Laboratory of and are caused by bacterial infections. Although Biophysics at EPFL and has an excellent immune system cells attack potentially track record of publications in high-profile threatening microorganisms, and several scientific journals. At present, he is working T6SS resembles a microscopic decades of research have led to enormous on an exciting project with the aim of advances in antibiotics, bacteria keep fighting gaining detailed information about the spear with a metal-hardened and back. New strains are evolving and developing function of two biological assemblies: the resistance to antibiotics that were previously type VI secretion system (T6SS) and R-type poisoned tip, which is surrounded effective. With bacterial drug resistance on the pyocins. As a cytoplasmic organelle – that is, by a sheath and assembled like a rise, there is a constant and urgent need to a macromolecular assembly situated in the develop new antibiotics. cytoplasm of a cell with a specific function stretched-out spring – T6SS is a complex that One promising potential weapon in the injects toxic effector proteins into target fight against infectious strains of bacteria is cells and resembles a bacteriophage tail-like bacteriophage therapy: making use of viruses cell-puncturing device. R-type pyocins, which While T6SS appears to be the most versatile that infect and replicate within bacteria. are also related to bacteriophage tails, are of all known secretion systems, it is only Bacteriophages in the Caudovirales group employed by certain bacteria to kill competing recently that researchers at the EPFL have have a tail that functions as a nanomachine; strains of the same species. uncovered key information about the way attaching the virus particle to the host cell it selects effector proteins for secretion surface, to create an opening in the membrane and subsequently delivers them into the UNDERSTANDING SECRETION SYSTEMS and deliver DNA into the host cytoplasm. This target cell. Crucially, Leiman and his team starts the virus replication cycle that can kill the Secretion systems are used by bacteria to gain – working in collaboration with Dr John host bacteria. However, before this mechanism advantage in a given environment by secreting Mekalanos from Harvard Medical School, can be safely and successfully implemented as toxic effector proteins that can inhibit or USA – have demonstrated that the structure a therapy, there is an urgent need for a better kill neighbouring cells, including cells of the of T6SS is inextricably linked to its function. understanding of how the process of virus human immune system. T6SS and R-type By utilising a range of techniques, including attachment to the host cell is regulated and pyocins consist of an internal tube and an X-ray crystallography, and whether it can be manipulated. external sheath, and function in a similar way. in vivo experiments on pathogenic bacteria, When targeting susceptible cells, the sheath the researchers have discovered a new class Dr Petr Leiman, a prominent researcher at the contracts and the internal tube pierces the of proteins: proline-alanine-alanine-arginine École Polytechnique Fédérale de Lausanne target . The similar biological (PAAR) repeat domains. These create a (EPFL), Switzerland, has made important organisation and functional mechanisms conical extension on the tip of the T6SS advances in understanding the function, of tailed bacteriophages, T6SS and R-type spike, which tapers to a one-atom diameter structure and assembly of bacteriophages pyocins, strongly imply that they share a apex. Amazingly, many such tip proteins and bacterial secretion systems. He heads common ancestor. contain a centrally positioned zinc or iron

52 INTERNATIONAL INNOVATION INTELLIGENCE STRUCTURE, FUNCTION AND ASSEMBLY OF MACROMOLECULAR MACHINES INVOLVED IN INFECTION OF BACTERIAL AND EUKARYOTIC CELLS OBJECTIVES • To describe the structure of a contractile tail-like nanomachine at the atomic level of detail and understand the trigger of the contraction event The central spike complex of a T6SS organelle. The PAAR repeat domain is in yellow; the spike is in red, • To design a contractile tail-like organelle green and blue; and the toxic domains are in orange. with a desired function that can be The structure was obtained by X-ray crystallography controlled and manipulated and molecular modelling. KEY COLLABORATORS Dr Mikhail Shneider, Shemyakin- Ovchinnikov Institute of Bioorganic atom. Furthermore, tip proteins often carry type of antimicrobial. Leiman’s laboratory Chemistry, Russia extension domains with toxic function. T6SS collaborates with AvidBiotics Corp (a small therefore resembles a microscopic spear with biotech company based in San Francisco, Professor John Mekalanos, Harvard Medical a metal-hardened and poisoned tip, which is USA), which showed that replacing one of the School, USA surrounded by a sheath and assembled like a pyocin-component proteins could cause the Professor Ian Molineux, The University of stretched-out spring. If a bacterium carrying pyocins to kill a different bacterial target cell. Texas at Austin, USA T6SS is attacked – by immune cells, for example – the spring-like sheath contracts and the toxic To explore the possibility of manipulating Dr Dean Scholl, AvidBiotics Corporation, USA spear is driven out of the bacterium, piercing organelles, the researchers are working the target cell’s membrane and poisoning it. to obtain more information about their Professor Z Hong Zhou, Electron Imaging The researchers have hypothesised that the highly complex structures. With each tail- Center for NanoMachines, USA shape and structure of the tip is related to the like organelle consisting of 1 million Professor Marek Basler, University of target that can be attacked. or more, one of the team’s objectives is to Basel, Switzerland comprehend the structure at the atomic level of detail: “We want to understand how this Professor Dominique Belin, University of INNOVATIVE METHODS huge structure behaves in solution and how Geneva, Switzerland The team is utilising a range of innovative it changes its conformation upon interaction FUNDING methodologies and equipment to achieve with the target cell surface,” explains Leiman. their research aims, as undertaking structural “This information will make it possible to Swiss National Science Foundation (NSF) studies of these macromolecular machines is control the function of T6SS. Furthermore, École Polytechnique Fédérale de a highly technical procedure that requires a we are trying to harness the efficiency of the Lausanne (EPFL) hybrid approach. Firstly, X-ray crystallography pyocin particle to retarget it from its natural is employed to determine the structure of the host, Pseudomonas aeruginosa, to other types CONTACT individual component proteins; and electron of pathogenic bacteria.” of specimens, preserved in their Dr Petr Leiman native state by cryo-fixation, is used to ascertain Assistant Professor FUTURE APPLICATIONS larger complexes or the whole assembly. Next, Laboratory of Structural Biology and the data from both of these techniques are Looking ahead, the hope is that the research Biophysics combined to give a detailed, high-resolution conducted in Leiman’s laboratory will have École Polytechnique Fédérale de Lausanne description of the nanomachine as a whole. clinical implications. Organelles such as T6SS BSP-415 Preparing the best possible sample at this and R-type pyocins could prove to be promising CH-1015 Lausanne stage is vital because it directly impacts the weapons in the fight against infectious bacteria Switzerland quality of the structural data. – and could perhaps be used to complement or even replace traditional antibiotics. If the T +41 79 538 7647 Conducting these experiments involves a high parts of the organelles most effective at killing E [email protected] level of expertise, with the successful study bacteria can be isolated, a whole new class of http://lbbs.epfl.ch of large biological complexes necessitating antibacterial agents could even be created. specialist knowledge of X-ray crystallography, PETR LEIMAN has Master’s degrees in electron microscopy, biochemistry, molecular To facilitate the transition of their research Applied Physics and Mathematics from biology and . Indeed, when it comes to into clinical practice, Leiman and his team are Moscow Institute of Physics and Technology selecting the most important proteins from a currently focusing on building a more robust and Shemyakin-Ovchinnikov Institute of large pool, the researchers often have to make understanding of T6SS. While knowledge of Bioorganic Chemistry, Moscow, and a PhD in educated guesses by drawing on their vast this system is incomplete and fragmented, it is Structural Biology and Biophysics from Purdue swathes of existing knowledge. known that T6SS can secrete a large number of University, USA. toxic effector proteins that kill many different types of cells. Drawing on their extensive EXPLOITING ORGANELLES knowledge and expertise, the researchers at In addition, Leiman and his colleagues are EPFL aim to design molecules that can regulate seeking to discover how to manipulate the activity of T6SS by turning it on or off. organelles. For example, if the properties These experiments, conducted with ingenuity of R-type pyocins can be exploited, there and enthusiasm, herald exciting opportunities is a chance they could be used as a new for treating a range of diseases.

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