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Enriching Subcellular Proteins in Leptospira Using a Triton X-114-Based Fractionation Approach
Published on: August 8, 2025
Identification of candidate host proteins that interact with LipL32, the major outer membrane protein of pathogenic
Suwittra Chaemchuen1, Suang Rungpragayphan, Yong Poovorawan
1Medical Microbiology, Interdisciplinary Program, Graduate School, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.
Abstract:
Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira spp. Rodent species are the major reservoir hosts that can excrete leptospires in their urine leading to environmental contamination. After gaining entry into the host via skin breaks and mucosa, leptospires disseminate through the bloodstream to target organs causing a wide range of disease manifestations in susceptible mammalian hosts. The crucial step of infection requires host-pathogen interactions. LipL32, the major outer membrane protein (OMP) of pathogenic Leptospira, is conserved among pathogenic leptospires, immunogenic, and expressed in target organs during acute infection in animal models. Therefore, it may play a key role in host-microbe interactions. To identify host proteins that interact with LipL32, phage display technology was employed in our study. Recombinant LipL32 was used as a target molecule for biopanning with a random heptapeptide phage library to enrich for phages expressing peptides with high affinity to LipL32. After three rounds of panning, 44 plaques of eluted phages were subjected to pyrosequencing. Six different peptide sequences were identified and used to search for matching proteins in the database. Putative proteins with potential binding to LipL32 are proteins known to be expressed on the surface of target cells of pathogenic Leptospira such as chloride channel accessory 2, glycoprotein VI, scavenger receptor expressed by endothelial cell isoform I (SREC-I), coronin 2A, laminin alpha 5, collagen XX, and prostaglandin receptor EP1. However, interactions of LipL32 with these host proteins and their role in the pathogenesis of leptospirosis requires experimental confirmation.
Insights
Researchers identified potential host proteins interacting with LipL32, a key outer membrane protein of Leptospira, using phage display. This finding advances understanding of leptospirosis pathogenesis and host-microbe interactions.
Area of Science:
- Infectious Diseases
- Microbiology
- Immunology
Background:
- Leptospirosis is a global zoonotic disease caused by Leptospira spp.
- Rodents are primary reservoirs, contaminating the environment via urine.
- Leptospires invade hosts through skin/mucosa, leading to systemic infection and diverse disease manifestations.
Purpose of the Study:
- To identify host proteins that interact with LipL32, a major outer membrane protein of pathogenic Leptospira.
- To elucidate the role of host-pathogen interactions in leptospirosis pathogenesis.
Main Methods:
- Phage display technology was employed to identify LipL32-interacting proteins.
- Recombinant LipL32 was used as bait for biopanning with a random heptapeptide phage library.
- Pyrosequencing was used to analyze eluted phages and identify binding peptide sequences.
Main Results:
- Six distinct peptide sequences with high affinity to LipL32 were identified.
- Potential host-binding partners include proteins on target cell surfaces, such as chloride channel accessory 2, glycoprotein VI, SREC-I, coronin 2A, laminin alpha 5, collagen XX, and prostaglandin receptor EP1.
Conclusions:
- Phage display successfully identified potential host proteins interacting with Leptospira's LipL32.
- Further experimental validation is required to confirm these interactions and their significance in leptospirosis pathogenesis.
