<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

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

시험관 내 박테리아 유비퀴틴화 재구성 및 VCP/p97 매개 제거

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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

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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.

이 프로토콜은 포유류 세포 용해액 또는 순수 유비퀴틴 효소 복합체를 이용해 폐렴 구균을 유비퀴틴화하는 방법을 설명하며, 이후 정제된 VCP/p97-UFD1-NPLOC4 단백질 복합체로 처리하여 박테리오리네이션 활성을 평가하여 유비퀴틴 의존성 면역 효과 연구를 가능하게 합니다.

저희 연구는 VCP 또는 p97이 여러 병원체에 대해 세포질 방어자로서 어떻게 작용하는지 밝혀냅니다. 이로 인해 VCP 또는 p97의 치료 응용 및 생명공학 전략 잠재력이 열립니다. 일상적인 생화학 외에도, 초해상도 현미경, 크라이오 EM, 크라이오 ET를 이용해 이 숙주 효과체가 병원체에 어떻게 조직되어 파괴하는지 규명합니다.

우선, 연쇄상구균 폐렴을 37도 섭씨에서 5% 이산화탄소를 토드 휴잇 효모 배지에서 하룻밤 동안 재배하세요. 하룻밤 배양액의 일부를 신선한 토드 휴잇 효모 배지 10부에 접종하고, 37도 섭씨에서 5% 이산화탄소와 함께 600나노미터에서 광학 밀도가 0.4에 도달할 때까지 배양합니다. 그 다음 연쇄상구균 폐렴 배양 900마이크로리터와 50% 글리세롤 600마이크로리터를 섞어 영하 80도에서 저장합니다.

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