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Updated: Feb 23, 2026
Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species
Published on: October 15, 2013
Investigation of host-pathogen interaction between Burkholderia pseudomallei and autophagy-related protein LC3 using
Pattamaporn Joompa1, Saranyoo Ponnikorn2, Sittiruk Roytrakul3
1Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Background:
Burkholderia pseudomallei is an intracellular bacteria causing Melioidosis, the disease widely disseminates in Southeast Asia and Northern Australia. B. pseudomallei has ability to invade various types of host cell and to interfere with host defense mechanisms, such as nitric oxide (NO). Due to the cross-talk among alternative killing mechanisms in host immune response against invading microbes, autophagy is the molecular mechanism belonging to intracellular elimination of eukaryotic cells that has been widely discussed. However, bacterial evasion strategy of B. pseudomallei and host-bacterial protein-protein interaction within autophagic machinery remain unknown.
Methods:
Here, we demonstrated the protein-protein interaction study between different strains of B. pseudomallei, including wild type PP844 and rpoS mutant, with autophagy-related protein LC3 that has been constructed, using the modified immunoaffinity hydrophobic chromatography based-technique. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was utilized for identifying the eluted proteins obtained from the established column. In addition, the expression level of gene encoding candidate protein was predicted prior to verification using real-time quantitative reverse transcription PCR assay (RT-qPCR).
Results:
LC3 recombinant proteins could be entrapped inside the column before encountering their bacterial interacting partners. Based on affinity interaction, the binding capacity of LC3 with antibody displayed over 50% readily for hydrophobically binding with bacterial proteins. Following protein identification, bacterial ATP-binding cassette (ABC) transporter periplasmic substrate-binding protein (BPSL2203) was identified as a candidate LC3-interacting protein, which was found only in B. pseudomallei wild type. Gene expression analysis and bioinformatics of BPSL2203 were validated the proteomic result which are suggesting the role of RpoS-dependent gene regulation.
Conclusions:
Remarkably, utilization of the modified immunoaffinity hydrophobic chromatography with LC-MS/MS is a convenient and reliable approach to a study in B. pseudomallei-LC3 protein-protein interaction.
Insights
This study identifies a bacterial protein, BPSL2203, that interacts with autophagy protein LC3 in Burkholderia pseudomallei. This finding advances understanding of how this pathogen evades host defenses during Melioidosis.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Burkholderia pseudomallei causes Melioidosis, a significant disease in Southeast Asia and Northern Australia.
- B. pseudomallei invades host cells and interferes with immune responses like nitric oxide (NO) production.
- Autophagy is a key cellular mechanism for eliminating intracellular pathogens, but B. pseudomallei evasion strategies and interactions with autophagy remain unclear.
Purpose of the Study:
- To investigate protein-protein interactions between B. pseudomallei and the autophagy-related protein LC3.
- To identify specific bacterial proteins involved in evading the host's autophagic defense mechanisms.
Main Methods:
- Utilized modified immunoaffinity hydrophobic chromatography to study interactions between B. pseudomallei strains and LC3.
- Employed liquid chromatography tandem-mass spectrometry (LC-MS/MS) for protein identification.
- Verified candidate protein gene expression using real-time quantitative reverse transcription PCR (RT-qPCR).
Main Results:
- Identified bacterial ATP-binding cassette (ABC) transporter periplasmic substrate-binding protein (BPSL2203) as an LC3-interacting protein in wild-type B. pseudomallei.
- Demonstrated that BPSL2203 interaction with LC3 is dependent on the RpoS regulatory system.
- LC3 recombinant proteins effectively bound bacterial proteins with over 50% binding capacity.
Conclusions:
- The modified immunoaffinity hydrophobic chromatography coupled with LC-MS/MS is an effective method for studying B. pseudomallei-LC3 interactions.
- BPSL2203 is a potential key player in B. pseudomallei's strategy to evade autophagy.
- Findings suggest RpoS-dependent regulation of bacterial evasion mechanisms against host autophagy.
