<em>In-vitro</em> Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Sourav Ghosh; Udit Kumar Das; Sumit Rakshit; Anirban Banerjee

doi:10.3791/69454

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

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07:58 min

January 2, 2026

DOI: 10.3791/69454-v

Sourav Ghosh¹, Udit Kumar Das¹, Sumit Rakshit¹, Anirban Banerjee¹

¹Department of Biosciences and Bioengineering,Indian Institute of Technology Bombay

Overview

This protocol describes a method to ubiquitinate Streptococcus pneumoniae using mammalian cell lysate or pure ubiquitin enzyme complex. The study assesses the bacteriolytic activity of the purified VCP/p97-UFD1-NPLOC4 protein complex, highlighting its role as a cytosolic defender against pathogens.

Key Study Components

Area of Science

Neuroscience
Microbiology
Immunology

Background

VCP/p97 is known to play a role in cellular defense mechanisms.
Understanding its interaction with pathogens can inform therapeutic strategies.
The study utilizes advanced imaging techniques to analyze these interactions.
Ubiquitination is a key process in immune response regulation.

Purpose of Study

To investigate the role of VCP/p97 in the immune response against Streptococcus pneumoniae.
To explore the therapeutic potential of VCP/p97 in pathogen defense.
To develop methods for studying ubiquitin-dependent immune effectors.

Methods Used

Ubiquitination of Streptococcus pneumoniae using mammalian cell lysate.
Application of purified VCP/p97-UFD1-NPLOC4 protein complex.
Super resolution microscopy for detailed imaging.
Cryo-electron microscopy and cryo-electron tomography for structural analysis.

Main Results

Demonstrated the bacteriolytic activity of the VCP/p97 complex.
Revealed insights into the organization of host effectors on pathogens.
Provided a foundation for future therapeutic applications.
Highlighted the importance of ubiquitination in immune responses.

Conclusions

VCP/p97 serves as a critical component in the immune defense against pathogens.
The study opens avenues for bio-engineering strategies targeting VCP/p97.
Further research is needed to fully understand its mechanisms and applications.

Frequently Asked Questions

What is the role of VCP/p97 in the immune response?

VCP/p97 acts as a cytosolic defender against various pathogens, playing a crucial role in immune defense mechanisms.

How does ubiquitination affect Streptococcus pneumoniae?

Ubiquitination modifies the bacteria, influencing their recognition and destruction by immune effectors.

What techniques are used in this study?

The study employs super resolution microscopy, cryo-electron microscopy, and cryo-electron tomography.

What are the potential therapeutic applications of this research?

The findings may lead to new strategies for bio-engineering therapies targeting VCP/p97 in pathogen defense.

Why is understanding VCP/p97 important?

Understanding VCP/p97 can provide insights into immune responses and potential treatments for infections.

What is the significance of the bacteriolytic activity observed?

The bacteriolytic activity indicates the effectiveness of VCP/p97 in combating bacterial infections.

This protocol describes a method to ubiquitinate Streptococcus pneumoniae using mammalian cell lysate or pure ubiquitin enzyme complex, followed by treatment with purified VCP/p97-UFD1-NPLOC4 protein complex to assess its bacteriolytic activity, enabling the study of ubiquitin-dependent immune effectors.

Our work unveils how VCP or p97 acts as a cytosolic defender against multiple different pathogens. This unlocks the potential of VCP or p97 for therapeutic applications and bio-engineering strategies. In addition to routine biochemistry, super resolution microscopy, cryo EM, cryo ET, are utilized to figure out how these host effectors organize on pathogen to destroy them.

To begin, grow the streptococcus pneumonia are six strain serotype two overnight at 37 degrees Celsius with 5%carbon dioxide in Todd Hewitt yeast medium. Inoculate one part of the overnight culture into 10 parts of fresh Todd Hewitt yeast medium, and incubate at 37 degrees Celsius with 5%carbon dioxide until the optical density at 600 nanometers reaches 0.4. Then mix 900 microliters of the streptococcus pneumonia culture with 600 microliters of 50%glycerol and store the glycerol stock at minus 80 degrees Celsius.

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