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Profiling lethal factor interacting proteins from human stomach using T7 phage display screening
Albin Cardona-Correa1, Carlos Rios-Velazquez1
1Department of Biology, College of Arts and Sciences, University of Puerto Rico‑Mayagüez, Mayagüez 00681‑9000, PR, USA.
Abstract:
The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents.
Insights
Researchers identified new protein interactions involved in gastrointestinal anthrax pathogenesis. This study used T7 phage display to find potential therapeutic targets for Bacillus anthracis infection.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bacillus anthracis lethal factor (LF) is a metalloproteinase causing cell apoptosis.
- Gastrointestinal (GI) anthrax mechanisms and molecular pathways of LF-induced tissue damage remain unclear.
- Previous studies noted gastric ulceration and bacterial growth in Peyer's patches.
Purpose of the Study:
- To identify proteins involved in GI anthrax pathogenesis.
- To investigate protein-protein interactions with Bacillus anthracis lethal factor (LF).
- To elucidate molecular pathways for potential therapeutic development.
Main Methods:
- Utilized human stomach T7 phage display (T7PD) cDNA libraries to identify protein ligands.
- Employed wild type and mutant LF (E687A) to differentiate interaction sites.
- Performed in silico analysis of 124 identified clones from 194 interacting phages.
Main Results:
- Identified 10 LF-interacting peptides, including pepsin A3 pre-protein (PAP).
- PAP demonstrated affinity to both wild type and mutant LF at concentrations as low as 1 µg/ml.
- Selected candidates included proteins from lipase, peptidase-A1, and cation transport families.
Conclusions:
- Findings contribute to understanding GI anthrax molecular pathogenesis.
- Identified PAP as a potential interacting partner of LF.
- This research aids in developing novel therapeutic strategies against anthrax.
