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An international research team led by Professor Shannon Au Wing-ngor from the School of Life Sciences at The Chinese University of Hong Kong (CUHK) has unravelled the step-by-step assembly mechanism of a “molecular syringe” used by Helicobacter pylori (H. pylori) to induce gastric cancer. The findings could help scientists explore ways to interfere with critical assembly steps, potentially limiting the bacterium’s ability to deliver a cancer-associated protein into stomach cells. Published in the prestigious journal Nature Communications, the breakthrough provides an important foundation for developing new non-antibiotic anti-virulence strategies that disarm pathogens without driving antimicrobial resistance.

Chronic H. pylori infection is a primary global cause of gastric cancer, one of the most common and deadly cancers worldwide. To infect host cells, the bacterium relies on a complex nanoscale molecular machine called the Cag type IV secretion system (CagT4SS), which acts like a needle to inject the oncoprotein CagA into host gastric epithelial cells. This disrupts normal cellular functions and increases the risk of cancerous changes.

Reconstructing the early assembly of the core structure in vitro for the first time

A core structure of the CagT4SS, known as the central cylinder, is composed of multiple CagM, CagX and CagY proteins subunits. These protein subunits can be understood as the “parts” used to build the molecular machine. However, scientists have been unable to isolate assembly intermediates while they are forming inside living bacterial cells. As a result, exactly how these protein components organise in a specific sequence to form a complex, functional structure has remained unclear.

To address this challenge, the research team placed purified Cag proteins in a controlled laboratory environment and reconstructed the early assembly of the CagT4SS core step by step in vitro. The approach was similar to rebuilding a complex machine from its individual parts so that each stage of the assembly process could be observed. The team used three complementary techniques to examine the assembly process from different perspectives:

• Single-particle cryo-electron microscopy (cryo-EM): to capture detailed three-dimensional structures of the protein complexes;
• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): to analyse potential protein-interaction regions and conformational changes upon binding;
• High-speed atomic force microscopy (HS-AFM): to observe in real time how individual molecules connect and assemble.

Professor Au said, “By combining these advanced structural biology techniques, we were able to visualise the assembly process of this molecular machine. We also found that, during formation of the CagT4SS core module, the protein components do not work independently. Instead, they interact cooperatively to build the core structure step by step.”

Nurturing global research talent at CUHK via international collaboration

The study was conducted by Dr Chu Hoi-yee and Dr Mok Chin-yu, Ph.D. graduates in Professor Au’s laboratory and co-first authors of the paper. Their contributions, together with collaborations involving Professor Zhang Huawei of the Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences, and Professor Takayuki Uchihashi of Nagoya University, Japan, demonstrate the value of international, cross-institutional partnerships in advancing scientific discovery and CUHK’s commitment to nurturing the next generation of globally engaged scientific talent.  

In collaboration with Professor Zhang on cryo-EM studies, the team determined the structures of two key assembly intermediates: the CagM-CagX double-ring structure and the CagX-CagY periplasmic ring complex (PRC). In their dimensions and molecular organisation, these in vitro-reconstituted intermediates reproduced key structural features of the native CagT4SS. The research team then used HDX-MS to analyse protein-interaction regions and the associated conformational changes that occurs during complex formation. The results support the conclusion that these in vitro-reconstituted intermediates resemble the features found in native virulence apparatus and provide additional insights to CagM oligomerization. Complementary HS-AFM experiments conducted by Professor Uchihashi’s group at Nagoya University, captured the assembly events in real time at the single-molecule level, revealing when and where each step of the assembly process occured.

Together, the findings define the early assembly sequence of the CagT4SS core:

1. CagX proteins first self-oligomerise into a ring;
2. CagY proteins subsequently associate with CagX to form a stable periplasmic ring complex;
3. CagM then associates with CagX through the multimerization of dimers;
4. The pre-coupling of CagY enhances the stability and cooperativity of CagM association, promoting formation of the complete central cylinder.

The study showed that binding between CagM and CagX is highly cooperative. In simple terms, once one protein component attaches, neighbouring sites become more favourable for additional components to bind. CagY further enhances this cooperative effect and increases the stability of the complex.

A breakthrough paradigm: disarming rather than destroying pathogens

Conventional treatment for H. pylori infection generally involves a combination of antibiotics and acid-suppressing medication. However, increasing antimicrobial resistance may reduce the effectiveness of existing treatments. The findings provide a direction for future investigation. If critical protein interactions can be disrupted before the bacterial “molecular syringe” is fully assembled, it may be possible to limit the bacterium’s ability to deliver disease-causing proteins into stomach cells.

Professor Au added: “Previous drug development efforts have largely focused on molecular machines that are already fully formed. Our findings suggest that directly targeting the assembly process itself could offer a new strategy for weakening the disease-causing ability of H. pylori. If future research can identify ways to interfere with critical assembly steps before the apparatus is completed, it may be possible to disarm the bacterium and provide a new direction for reducing reliance on conventional broad-spectrum antibiotics.”

This anti-virulence strategy is intended not to kill the bacterium directly, but to weaken its ability to cause disease. The findings provide an important mechanistic foundation for future efforts to explore and identify compounds with the potential to interfere with CagT4SS assembly.