The Effects of Dictyostelium discoideum Phosphatase 2A Subunit B56 and Superoxide Dismutase C on the Ingestion and the Killing of Live Bacteria Klebsiella aerogenes

Authors

Victor Osorio CastilloFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biochemistry

First Advisor's Name

Lou W. Kim

First Advisor's Committee Title

committee chair

Second Advisor's Name

Manuel A. Barbieri

Second Advisor's Committee Title

committee member

Third Advisor's Name

Xiaotang Wang

Third Advisor's Committee Title

committee member

Fourth Advisor's Name

Mauricio Rodriguez-Lanetty

Fourth Advisor's Committee Title

committee member

Keywords

biochemistry, molecular biology

Date of Defense

6-19-2023

Abstract

Multidrug-resistant bacteria (MDRB) are a critical risk to public health. The challenge of MDRB emergence and its quick spread has been worsened by the failure to develop or discover novel antibiotics and a drug approval decrement. Drugs to treat MDRB infections are limited, expensive, or non-existent. Alternative non-antibiotic therapies, such as Host-direct therapy, have been developed in response to these problems. Host-direct therapy improves the host's defense response by using drugs or proteins. An effective innate immune response for pathogen elimination is phagocytosis. Phagocytosis is the endocytosis of solid particles. Dictyostelium discoideum is an excellent model organism to study phagocytosis. During its unicellular stage, Dictyostelium utilizes a phagocytosis mechanism similar to those found in mammalian cells. In Dictyostelium, the receptor fAR1 mediates the chemotaxis, phagocytosis of bacteria, and the activation of PKB and ErkB proteins. These proteins are regulated by phosphatase 2A (PP2A) subunit B56. Our laboratory has investigated two pathways that affect PKB: SodC/Ras/PI3K/PKB and PP2A/B56/PKB. The present study aims to determine the effect of SodC/Ras/PI3K/PKB, and PP2A/B56/PKB pathways and PKB and ErkB proteins in the bacterial ingestion and inactivation of live bacteria Klebsiella aerogenes. In this study JH10 (wild-type), sodC^-, Ax3 (wild-type), and psrA^- (knockout B56) cells were used. Phagocytosis and killings were performed in the presence or absence of LY294002 using 2 x 10⁶ Dictyostelium cells and 2 x 10⁴ Bacteria. Further western blots in the presence or absence of the PI3K inhibitor LY294002 were performed for cell lines Ax3 and psrA^- using Anti-MAPK and Anti-pPKB antibodies. sodC^- and its wild-type cell (JH10) did not show a significant difference in their engulfment (P > 0.09) and killing (P > 0.08), psrA^- significantly increased its engulfment and bactericidal activity compared with WT cells (AX3) (PpsrA^- cells significantly increase their bactericidal activity (PpsrA^- after an LY294002 treatment was statistically significant (Pin psrA^- and Ax3 cells showed an insignificant increase (P>0.36 and P>0.1).

In conclusion, PP2A/B56 is essential for the proper uptake and elimination of Klebsiella aerogenes.

Identifier

FIDC011214

Recommended Citation

Osorio Castillo, Victor, "The Effects of Dictyostelium discoideum Phosphatase 2A Subunit B56 and Superoxide Dismutase C on the Ingestion and the Killing of Live Bacteria Klebsiella aerogenes" (2023). FIU Electronic Theses and Dissertations. 5352.
https://digitalcommons.fiu.edu/etd/5352