The bacterial botulinum neurotoxins are the most toxic substances known in the world and cause the rare but serious disease botulism in humans and animals, which is accompanied by severe paralysis and can lead to death if left untreated. A total of over 40 variants (subtypes) are known, which in turn are divided into eight serotypes A to H. In addition to wound botulism and infant botulism, which are caused by bacterial colonization of wounds or the gastrointestinal tract of infants, the most common form in Germany is food-borne botulism. This is food poisoning following ingestion of botulinum neurotoxins formed in food.

In order to reach their final site of action, the motoneurons, after oral ingestion, the neurotoxins must survive the harsh conditions of gastrointestinal passage in active form. The producing bacteria provide the toxins with a series of auxiliary proteins, so-called neurotoxin-associated proteins, whose role in mediating oral toxicity researchers in the field of biological toxins have been trying to understand for decades.

It was known that the neurotoxins are encoded at the genetic level by the bacteria in one of two possible gene clusters (HA cluster or OrfX cluster). The toxin cluster of all known neurotoxins always contains a non-toxic non-haemagglutinin (NTNH), which protects the neurotoxin in a pH-sensitive complex from the harsh conditions in the gastrointestinal tract. In the same gene cluster, either the HA proteins (hemagglutinins HA70, HA17, HA33) or the OrfX/P47 proteins (OrfX1, OrfX2, OrfX3, P47) are also encoded.

The structure of the botulinum neurotoxins encoded in the HA cluster in a stable multiprotein complex of enormous size (760 kDa), which is already formed under simple cultivation conditions, has been elucidated in recent decades [1]. Furthermore, the role of HA proteins in the absorption of the toxin via the intestine after oral uptake is largely understood [2]. In contrast, the type of complex formation of the neurotoxins associated with OrfX and the role of the OrfX/P47 proteins were previously unknown. A multidisciplinary team from the Universities of California (Irvine, USA), Helsinki (Finland) and Nottingham (UK), the Hannover Medical School (Germany) and the Robert Koch Institute has now succeeded in elucidating key physiological and regulatory mechanisms of the oral toxicity of OrfX-associated botulinum neurotoxins [3]. The researchers showed how the oral toxicity of botulinum neurotoxin serotype E is mediated by interaction with the OrfX/P47 proteins at the molecular and functional level.

This study was published in Nature Structural & Molecular Biology (Gao L, Nowakowska MB, Selby K, Przykopanski A, Chen B, Krüger M, Douillard FP, Lam KH, Chen P, Huang T , Minton NP, Dorner MB, Dorner BG, Rummel A, Lindström M, Jin R. 'Botulinum neurotoxins exploit host digestive proteases to boost their oral toxicity via activating OrfXs/P47'; Nature Structural & Molecular Biology 2025; https://doi.org/10.1038/s41594-024-01479-0).

By skillfully using CRISPR/Cas9 technology to switch off the production of individual protein components of the OrfX cluster in conjunction with animal experiments, it was shown that all four proteins (OrfX1, OrfX2, OrfX3, P47) are required in addition to the neurotoxin and the protein NTNH to cause a drastic increase in neurotoxicity. The four proteins are not toxic individually or in combination with each other, but act like a “Trojan horse” to enhance the activity of the neurotoxin.

A key step in this process is the activation and proteolytic cleavage of OrfX2 by digestive enzymes that occur naturally in the host's gastrointestinal tract. After activation, up to two OrfX2 proteins form a complex with the neurotoxin-bound NTNH, which was detected using cryo-electron microscopy (cryo-EM). BoNT/E only becomes highly toxic through the formation of the high-molecular complex OrfX2-NTNH-BoNT. This molecular mechanism is in contrast to the previously known mechanism of action of HA-associated BoNT complexes, which are all naturally associated before entering the host and are highly stable against the conditions of the gastrointestinal tract.

Interestingly, the protein OrfX1, which naturally interacts with OrfX3, is also sensitive to degradation by proteases. According to the current state of knowledge in this study, the two protease-sensitive structures - the detached part of the OrfX2 protein and the OrfX1 protein - are ascribed a protective function for their respective binding partners (the protease-stable part of OrfX2 or the OrfX3 protein), which is, however, only relevant up to the time of protease degradation. However, a direct contribution of these structures to toxicity is not suspected.

This study was the first to demonstrate a structural and functional link between all four OrfX/P47 proteins and botulinum neurotoxins and their effects. Recent findings based on methodological advances in genomic analysis show that similar OrfX gene clusters are also present in many other non-BoNT-producing bacteria, especially in association with insecticidal toxins [4]. Therefore, the discovery of the role of OrfX proteins in enhancing the toxicity of botulinum neurotoxins has far-reaching implications for future insights into the modes of action of many still largely unexplored toxins.

The Department of Biological Toxins at the Robert Koch Institute (ZBS3, www.rki.de/zbs3-en) is a national reference center for high-molecular biological toxins with the following main areas of work:

• diagnostics of toxins of microbial and plant origin that can be used for bioterrorist attacks, using cell biological, genetic and serological techniques as well as chromatographic methods and mass spectroscopy,
• Consultancy laboratory for neurotoxin-producing clostridia (botulism, tetanus) and DVG consultancy laboratory for C. botulinum / botulinum neurotoxins in food,
• Creation of SOPs for diagnostics,
• provision of reference samples, reference bacterial strains and standards, provision of diagnostics,
• adapting the diagnostics to the expected test materials,
• developing strategies for detecting new and modified toxins and agents,
• research in the field of the pathogenesis of the diseases caused,
• developing strategies for prevention, decontamination and control
• interlaboratory comparisons for quality assurance in diagnostics,
• participation in the development of standard therapies.

1. Lee K, Gu S, Jin L, Le TT, Cheng LW, Strotmeier J, Kruel AM, Yao G, Perry K, Rummel A, Jin R. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity. PLoS Pathog. 2013;9(10):e1003690. doi: 10.1371/journal.ppat.1003690. Epub 2013 Oct 10. PMID: 24130488; PMCID: PMC3795040.
2. Lee K, Zhong X, Gu S, Kruel AM, Dorner MB, Perry K, Rummel A, Dong M, Jin R. Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex. Science. 2014 Jun 20;344(6190):1405-10. doi: 10.1126/science.1253823. PMID: 24948737; PMCID: PMC4164303.
3. Gao L, Nowakowska MB, Selby K, Przykopanski A, Chen B, Krüger M, Douillard FP, Lam KH, Chen P, Huang T , Minton NP, Dorner MB, Dorner BG, Rummel A, Lindström M, Jin R. Botulinum neurotoxins exploit host digestive proteases to boost their oral toxicity via activating OrfXs/P47. Nature Structural & Molecular Biology. 2025 Jan 21; https://doi.org/10.1038/s41594-024-01479-0.
4. Nowakowska MB, Douillard FP, Lindström M. Looking for the X Factor in Bacterial Pathogenesis: Association of orfX-p47 Gene Clusters with Toxin Genes in Clostridial and Non-Clostridial Bacterial Species. Toxins (Basel). 2019 Dec 31;12(1):19. doi: 10.3390/toxins12010019. PMID: 31906154; PMCID: PMC7020563.