Research Summary: Vibrio cholerae cytolysin (VCC), a pore-forming toxin of cholera pathogen, engages novel innate receptor assembly (TLR1/4) to trigger inflammation and death, critical for bacterial pathogenesis.
Novel TLR Signalling Orchestrates Inflammation and Cell Death
Author interview: Ms. Shraddha Gandhi is an immunologist with a background in Biotechnology. She earned her Bachelor’s degree in Biotechnology from Biyani Girls’ College, Rajasthan University, Jaipur, followed by a Master’s in Biotechnology from the Central University of Rajasthan (CURAJ). In 2017, she began her doctoral studies in the Department of Biological Sciences at the Indian Institute of Science Education and Research (IISER) Mohali, under the joint supervision of Dr. Arunika Mukhopadhaya and Dr. Kausik Chattopadhyay. Her research focuses on various aspects of host-pathogen interactions.
Lab: Dr Arunika Mukhopadhaya and Dr Kausik Chattopadhyay, Indian Institute of Science Education and Research (IISER) Mohali
What was the core problem you aimed to solve with this research?
Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin (β-PFT) secreted by the Gram-negative bacterial pathogen V. cholerae, the causal organism of the notorious human diarrheal disease cholera. Pore-formation results in the disruption of the host cell membrane permeability barrier function. Further, we and several others found that by acting as a pathogen-associated molecular pattern (PAMP), VCC engages innate pattern recognition receptors (PRRs) to induce pro-inflammatory responses in innate immune cells, such as monocytes, macrophages, and mast cells. Based on this background, in this study, we have addressed several fundamental questions related to the interaction between VCC and the host immune cells. We started with how VCC acts as a PAMP to trigger immune responses in dendritic cells (DCs), which not only contribute to the innate immunity but also shape the adaptive immunity. We observed that VCC activates pro-inflammatory signalling in DCs, and found that it engages novel TLR1/4 heterodimer as PRR, in contrary to conventional TLR heterodimers. Our findings revealed that VCC is also differentially recognized by both conventional TLR2/6 heterodimer and TLR1/4 heterodimer in the macrophages, suggesting that the same ligand/PAMP VCC can be differentially recognized by different TLR combinations in a cell-type-specific manner. Further, we observed that in both macrophages and neutrophils, TLR1/4 are the preferred heterodimer for the induction of innate immunity in the form of inflammation. Our study further demonstrated that VCC-induced TLR pathways are critical for not only inflammation but also VCC-induced cell death and mortality in mice following V. cholerae infection, and TLR1/4 heterodimer is the major player in this context. By elucidating the mechanisms of VCC recognition and signalling, our study fills major gaps in understanding how this toxin modulates immune responses and contributes to disease severity, highlighting the importance of novel TLR assemblies in host-pathogen interactions.
How would you explain your research outcomes (Key findings) to the non-scientific community?
Our body’s security system relies on vigilant guards called immune cells. These cells have special alarm sensors known as pattern recognition receptors (PRRs) that constantly scan for danger signals from harmful invaders like bacteria. Toll-like receptors, or TLRs are the important groups of such PRRs that work as homo or hetero-dimers.
Vibrio cholerae, the bacterium that causes cholera, produces a weapon called VCC, a pore-forming toxin. VCC acts like a molecular drill, punching holes in our cells that, in turn, cause cellular damage and trigger our immune system’s alarm.
Our recent work uncovered that VCC is specifically detected by a unique, not yet discovered, combination of gatekeepers: the TLR1 and TLR4 (TLR1/4) assembly, in key inflammatory cells like dendritic cells, macrophages and neutrophils. When TLR1/4 assembly detect VCC, they set off a chain reaction that leads to inflammation and cell death.
This is significant because the body uses different combinations of TLRs to recognize and respond to specific threats. By understanding how VCC activates the unusual TLR1/4 combination as the main sensor, we opened the immense possibility to explore new and targeted strategies to fight cholera and further have drawn the attention of the world towards the usage of this novel innate receptor assembly as a possible mechanism of host-system modulation and therapeutic target for other bacterial pathogens as well. Our study underlines the necessity of more studies towards understanding the role and collaboration of innate sensors on the aspect of host-pathogen interaction to understand more about the pathogen and how they manipulate our cellular responses, and how we can curate specific therapeutic strategies by limiting as many side effects as possible.
This study provides novel insights into how VCC, apart from its conventional membrane-damaging action, activates the inflammatory, death responses.
What are the potential implications of your findings for the field and society?
Our findings highlight that the immune cells actively respond to bacterial threats, using clever tactics rather than remaining passive targets. Our finding that VCC employs different TLR combinations in different cell types (TLR1/4 in DCs and both TLR1/4 and TLR2/6 in macrophages) provides insights into the complexity of immune recognition and how the immune system activates its responses based on the cell type and the specific threat. Furthermore, the discovery of a novel TLR heterodimer opens up new insights into the broader principles of innate immune sensing, with potential implications for recognizing and responding to other bacterial ligands beyond VCC. We opened up the immense possibility to explore new and targeted strategies to fight not only cholera but also brought the idea in the field towards the usage of this novel innate receptor assembly as a possible mechanism of host-system modulation and therapeutic target for other bacterial pathogens as well. Further, our findings compel the researchers to study more towards understanding the role of innate sensors in relation to weapons and arsenals in terms of pathogen-associated molecular patterns (PAMPs) in the aspect of host-pathogen interaction. This study will help to understand more about how pathogens manipulate host cellular responses, and the information will help us to generate precise therapeutic strategies.
What was the exciting moment during your research?
Every result in my research journey, whether positive or negative, became a source of excitement and learning. I learned from my supervisor, Professor Arunika Mukhopadhaya and Professor Kausik Chattopadhyay, that negative results also provide a great insight and motivation, like the positive ones. Early in our work, we were surprised to find that VCC was not recognized by the usual TLRs, which led us to the unexpected and fascinating discovery of unusual TLR heterodimer formation in response to VCC. The microscopic visualisation of immune cell death and mice-based studies to understand the necessity of TLR1/4-mediated signalling were the most interesting experiments. Our work’s acceptance for publication in PLOS Pathogens was the most exciting moment during this journey. My supervisors’ unwavering encouragement played a crucial role in my growth, and throughout this journey, I formed incredible friendships. Although research can be challenging, the knowledge gained is much more than the difficulties faced. While making discoveries is exciting, the collaborative effort, overcoming obstacles, and continuous learning made this experience deeply fulfilling.
Paper reference: Gandhi, S., Puravankara, S., Mondal, A. K., Chauhan, A., Yadav, S. P., Chattopadhyay, K., & Mukhopadhaya, A. (2025). Vibrio cholerae cytolysin induces pro-inflammatory and death signals through novel TLR assembly. PLoS pathogens, 21(4), e1013033. https://doi.org/10.1371/journal.ppat.1013033
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