Presentation Title

Magnetic Isolation of Pure Phagosomes

Faculty Mentor

Dr. Cheryl Okumura

Start Date

17-11-2018 12:30 PM

End Date

17-11-2018 2:30 PM

Location

HARBESON 23

Session

POSTER 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Group A Streptococcus is a gram-positive bacterial pathogen which is known to cause severe infections such as necrotizing fasciitis, and subsequent conditions such as rheumatic heart disease. We and others have shown that although Group A Streptococcus (GAS) is phagocytosed by macrophages and that normal phagosome-lysosome fusion occurs, it can resist degradation by lysosomal enzymes and the mechanisms behind this are not known. We hypothesize that GAS alters the proper assembly of key protein complexes that are vital for degrading contents within the phagolysosome. To determine whether these complexes are assembling properly and to determine other GAS-mediated changes, we are undertaking a proteomics analysis of GAS-infected phagolysosomes. Phagolysosomes were isolated from the cell by lysis with nitrogen cavitation followed by density gradient centrifugation or magnetic isolation techniques. The phagolysosomes were fed iron oxide particles to magnetize them and allow them to be separated from the rest of the cell lysate. Overall, the magnetic techniques proved to be more successful in separating the phagolysosomes from the rest of the whole cell lysate. However, upon further analysis by Western blot, we determined the phagolysosome fraction was heavily contaminated by other organelles and nuclear material. Our current work focuses on high stringency washes to decrease the contamination. Once a more pure sample of phagolysosomes is obtained, it can be analyzed by mass spectrometry to determine the protein content of the phagolysosomes. With a better understanding of the composition of GAS-infected phagolysosomes, we may be able to decipher how GAS manipulates the phagolysosomal environment for bacterial survival. These proteins in turn can be developed as targets of effective treatments for GAS infection.

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Nov 17th, 12:30 PM Nov 17th, 2:30 PM

Magnetic Isolation of Pure Phagosomes

HARBESON 23

Group A Streptococcus is a gram-positive bacterial pathogen which is known to cause severe infections such as necrotizing fasciitis, and subsequent conditions such as rheumatic heart disease. We and others have shown that although Group A Streptococcus (GAS) is phagocytosed by macrophages and that normal phagosome-lysosome fusion occurs, it can resist degradation by lysosomal enzymes and the mechanisms behind this are not known. We hypothesize that GAS alters the proper assembly of key protein complexes that are vital for degrading contents within the phagolysosome. To determine whether these complexes are assembling properly and to determine other GAS-mediated changes, we are undertaking a proteomics analysis of GAS-infected phagolysosomes. Phagolysosomes were isolated from the cell by lysis with nitrogen cavitation followed by density gradient centrifugation or magnetic isolation techniques. The phagolysosomes were fed iron oxide particles to magnetize them and allow them to be separated from the rest of the cell lysate. Overall, the magnetic techniques proved to be more successful in separating the phagolysosomes from the rest of the whole cell lysate. However, upon further analysis by Western blot, we determined the phagolysosome fraction was heavily contaminated by other organelles and nuclear material. Our current work focuses on high stringency washes to decrease the contamination. Once a more pure sample of phagolysosomes is obtained, it can be analyzed by mass spectrometry to determine the protein content of the phagolysosomes. With a better understanding of the composition of GAS-infected phagolysosomes, we may be able to decipher how GAS manipulates the phagolysosomal environment for bacterial survival. These proteins in turn can be developed as targets of effective treatments for GAS infection.