Isolation and Analysis of Extracellular Vesicles from Lactic Acid Bacteria

Isabel Myers, Coastal Carolina University

Presentation Abstract

Lactic acid bacteria have probiotic properties and are found within the microbiome of the human gut having a significant influence on human health. Extracellular vesicles (EVs) are produced by probiotic bacteria of the gut and are thought to be involved in intercellular communication with other bacteria and with the host. EVs are produced by prokaryotic bacteria and derived from the outer membrane in Gram-negative bacteria. The thick layer of peptidoglycan present in Gram-positive bacteria was thought to prohibit release of EVs. Recent studies have suggested that autolysin may allow EVs to be released from Gram-positive bacteria. The goal of the study was to develop an isolation procedure and observe the contents of the isolated EVs from lactic acid bacteria. It was hypothesized that the protein and RNA content of the EVs could provide greater insight into the mechanisms of intercellular communication. Streptococcus thermophilus was grown in culture. The growth cultures were centrifuged to pellet the cells. The supernatant was filtered to remove the remaining bacterial cells. The filtrate was concentrated by ultrafiltration. The retentate was presumed to contain EVs, and gel electrophoresis was run to determine protein content. Scanning electron microscopy was used to observe the membrane surface of cells isolated from growth cultures and to look for budding EVs. The protein content observed during the process of isolation is indicative of the presence of EVs. Future studies include identification of the protein and RNA content of the EVs as well as further development of methods to improve isolation techniques.

 
Apr 12th, 4:00 PM Apr 12th, 6:00 PM

Isolation and Analysis of Extracellular Vesicles from Lactic Acid Bacteria

Lactic acid bacteria have probiotic properties and are found within the microbiome of the human gut having a significant influence on human health. Extracellular vesicles (EVs) are produced by probiotic bacteria of the gut and are thought to be involved in intercellular communication with other bacteria and with the host. EVs are produced by prokaryotic bacteria and derived from the outer membrane in Gram-negative bacteria. The thick layer of peptidoglycan present in Gram-positive bacteria was thought to prohibit release of EVs. Recent studies have suggested that autolysin may allow EVs to be released from Gram-positive bacteria. The goal of the study was to develop an isolation procedure and observe the contents of the isolated EVs from lactic acid bacteria. It was hypothesized that the protein and RNA content of the EVs could provide greater insight into the mechanisms of intercellular communication. Streptococcus thermophilus was grown in culture. The growth cultures were centrifuged to pellet the cells. The supernatant was filtered to remove the remaining bacterial cells. The filtrate was concentrated by ultrafiltration. The retentate was presumed to contain EVs, and gel electrophoresis was run to determine protein content. Scanning electron microscopy was used to observe the membrane surface of cells isolated from growth cultures and to look for budding EVs. The protein content observed during the process of isolation is indicative of the presence of EVs. Future studies include identification of the protein and RNA content of the EVs as well as further development of methods to improve isolation techniques.