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Bacterial toxins manipulate proteins that serve as signalling switches in the host cell

Are bacteria intelligent? Of course not. However, some bacteria have amazing survival strategies that appear almost brilliant. One such example is bacteria that live in symbiosis with predatory nematodes that infest insect larvae and feed on their victims. The bacteria wait in the worm’s gut until their symbiotic partner has crept into the larvae, then they are released and kill the insect with an ingenious toxin cocktail. The table is therefore set for both worm and bacterium. Two enterobacterial representatives, Photorhabdus asymbiotica and Photorhabdus luminescens, use the mechanism in order to propagate. Prof. Dr. Dr. Klaus Aktories, director of the Institute of Experimental and Clinical Pharmacology and Toxicology at the University of Freiburg is studying the bacteria closely. Photorhabdus asymbiotica is a human pathogen that leads to skin infections. Researchers in the team led by Professor Aktories and Dr. Thomas Jank recently discovered a mechanism of one of the protein toxins that Photorhabdus asymbiotica bacteria use to exploit the metabolic pathways of insects and human host cells for their own purposes.

Prof. Dr. Dr. Klaus Aktories and Dr. Thomas Jank are focused on bacterial toxins. © private

A few years ago, a Photorhabdus species was discovered in skin ulcers of gardeners in the USA and Australia. The human pathogen was isolated and named Photorhabdus asymbiotica in the assumption that it occurred in this case without living in symbiosis with threadworms. Although this was quickly revealed to be an error, the name stuck. Photorhabdus asymbiotica is another insecticide bacterium that lives in symbiosis with the threadworm Heterorhabditis. “The worms creep into insect larvae where they disgorge the bacteria,” says Prof. Dr. Klaus Aktories from the Institute of Experimental and Clinical Pharmacology and Toxicology at the University of Freiburg. “The bacteria produce toxins and kill the larvae, which then serve as a source of food for both worms and bacteria.” Aktories’ research group is specifically interested in these bacterial toxins. The researchers want to find out how the toxins enter the cells and which structures they target. They also want to understand the exact mechanism of action that the pathogen uses to kill its host.

Attacking a regulatory switch

PaTox exerts its toxic effect on the cytoskeleton (red) of Galleria mellonella moth larvae by attaching a sugar molecule (N-acetylglucosamine) to Rho. © Prof. Dr. Dr. Klaus Aktories, University of Freiburg

As soon as Photorhabdus asymbiotica has managed to enter the insect larvae thanks to its symbiotic worm partner, the bacterial toxin PaTox (Photorhabdus asymbiotica toxin) is released into the target cells, which are thought to be the haemocytes in the blood-like larval fluid. The haemocytes are part of the victim’s immune system. PaTox binds to a receptor and is endocytosed by the haemocytes. The acidic environment of the endosomes leads to a conformational change in PaTox. Part of the toxin enters the membrane and the other part, the toxic enzyme component, is transported into the cytosol where PaTox modifies the so-called Rho proteins that are involved in the regulation of the cytoskeleton, which in turn controls phagocytosis and cellular migration. Obstruction of cellular processes that regulate the cytoskeleton leads to the death of both cell and insect. 

Rho proteins are small GTPases and important regulators of cellular signal transduction. GTPases can change from their active GTP-bound into their inactive GDP-bound forms by hydrolysis of GTP. GTP-bound Rho can activate further effectors, e.g. the Rho kinase enzyme that regulates the cells’ actin cytoskeleton. The PaTox toxin interferes with the hydrolysis of Rho-GTP into inactive Rho-GDP. PaTox does this by attaching a sugar residue (N-acetylglucosamine) to Rho, thereby setting the entire switch to “off”. The protein can no longer activate effectors and cannot be activated itself either. “Nothing works any more. Although larvae have quite a good defence system, they are unable to evade these bacterial mechanisms,” said Aktories. “The modification compromises the larvae’s immune cells, which can no longer phagocytose and destroy the bacteria.”

Aktories, a medical doctor and pharmacologist, and his colleague Dr. Thomas Jank were able to show that phagocytosis is impeded by the toxin once it is inside the cell. However, an unexpected finding for the researchers was that PaTox can only transfer a sugar residue to the Rho protein when Rho is present in its active form. Using receptor-coupled larval G-proteins and a sophisticated indirect mechanism, the researchers were able to show that the bacterial toxin first turns Rho into an active state before switching it off completely. This process enables the worm and the pathogen to propagate and infest new larvae before the switch is eventually set to off.

Bacteria with a kind of hypodermic needle

Similarities in the sequence or three-dimensional structure of certain proteins often lead to researchers finding out that the activity of proteins is due to a similar mechanism. In cooperation with Prof. Dr. Carola Hunte from the Centre for Biological Signalling Studies (BIOSS) at the University of Freiburg, Aktories and his team were able to crystallize the PaTox glycosylation domain that is essential for transferring sugar residues to Rho.

Another common feature of many toxins is their target: they go to the site in the organism where a molecule is the pivotal point of important cell functions. “Many toxins exert their effect by targeting Rho as it is easier to switch something on and off in a cell by directly targeting the switch itself,” explains Aktories.

The most recent example of this is the newly identified Photorhabdus luminescens Tc toxin mechanism. P. luminescens is closely related to P. asymbiotica. The toxin of the bacteria also targets Rho and specifically modifies and activates it. However, in this case, Rho is switched on, and therefore the switch off mechanism does not function.

But what is really spectacular is the way in which the toxin is transferred into the cell. In collaboration with Dr. Stefan Raunser from the Max Planck Institute for Molecular Physiology in Dortmund, Aktories elucidated this particular mechanism. The toxin machinery is relatively big, consisting of three subunits, TcA, TcB and TcC. The TcA unit binds to the insect cell, forming a channel that injects the toxic component inside the channel into the host cell just like a hypodermic needle. A protein chain consisting of 48 amino acids is stretched like a spring. When it retracts, energy is released that catapults the channel through the membrane and releases the toxin inside the cell. This mechanism seems to be a unique pore-forming toxin complex.

Learning from evolution

Since receptor binding domains can be easily manipulated or exchanged with others, they are popular targets for medical applications. Likewise, the switch functions of Rho proteins, which are also used by many other toxins, are useful for potential therapies. Aktories envisages the possibility of designing chimeric toxins by combining the components of different toxins in such a way that the bacterial toxins are directed to and destroy specific cancer cells. “Rho proteins play a key role in carcinogenesis and tumour metastasis,” says Aktories. “And the toxin we’ve discovered is one that is able to inactivate Rho proteins.” The huge advantage of Aktories’ assumption is that only one molecule – the enzyme with the toxic component - is needed to reliably do what it is supposed to do.

Further information:

Prof. Dr. Dr. Klaus Aktories
Institute of Experimental and Clinical Pharmaceology and Toxicology
University of Freiburg
Albertstr. 25
79104 Freiburg
Tel.: +49 (0)761 / 203 - 5301
Fax.: +49 (0)761 / 203 - 5311
E-mail: klaus.aktories(at)pharmakol.uni-freiburg.de
Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/bacterial-toxins-manipulate-proteins-that-serve-as-signalling-switches-in-the-host-cell