Author ORCID Identifier

https://orcid.org/0000-0002-7433-3129

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department/School/Program

Chemistry

First Advisor

Dr. Yinan Wei

Second Advisor

Dr. Zhenyu Li

Abstract

Antibiotic resistance is one of the major global issues in the field of public health and medicine. Good antibiotic candidates need to be selectively toxic, inhibit cellular target, and effectively penetrate and accumulate in bacterial cells. The last factor is a formidable barrier in the development of antimicrobials effective in Gram-negative bacteria, due to the presence of two layers of cell envelope. The first half of my thesis focuses on understanding the permeation of small molecules through this formidable cell envelope, distribution inside the cell of Gram-negative bacteria, and design of novel methods to make small molecules effectively cross the cell envelope. The second half of my thesis focuses more on the crosstalk between Gram-negative bacteria and host immune system during systemic infection and sepsis. More specifically we studied the contribution of inflammasome activation and pyroptosis during pathogenesis of Salmonella systemic infection.

In the first project, I studied the accumulation and distribution behavior of fluoroquinolone class of antibiotics inside Gram-negative bacteria using E. coli as a model. Although several studies have been focused regarding the correlation between compound’s cellular accumulation and their effectiveness against Gram-negative bacteria but no correlation between accumulation of antibiotics and their efficacy has been observed. In this study, we measured the concentration of nine fluoroquinolones accumulated in the subcellular compartments of E. coli. Good correlation between the MIC and the cytoplasmic accumulation, but not whole cell accumulation, was observed using a pair of isogenic wild type and drug-efflux deficient strains. Our results supported the explanation that the efficacy cannot be determined by the whole cell accumulation alone. Accumulation in the target region as well as the intrinsic potency determines the effectiveness of an antimicrobial compound.

In the second project, I explored whether conjugation of biotin to small molecules can increase the permeation of small molecules through the Gram-negative cell envelope. We used a florescent molecule pair, Atto565 and Atto565-biotin as model compounds and studied their permeation behavior in E. coli. Our results indicated that biotinylation helped the molecule Atto565 to cross the outer membrane of E. coli through OmpC porin.

Moreover, in the third project, I studied how the inflammasome activation and pyroptosis play a role in pathogenesis of Salmonella systemic infection. We found that Salmonella systemic infection causes severe inflammation as indicated by very high plasma concentration of pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α. Furthermore, it also caused disseminated intravascular coagulation (DIC) as indicated by increased prothrombin (PT) time and plasma thrombin-antithrombin (TAT) levels. Deficiency of caspase 1 protected the mice from Salmonella induced inflammation, coagulation and death during acute systemic infection. Similarly, deficiency of NAIP and GSDMD significantly reduced the Salmonella induced inflammation in vivo. Use of flagellin and Salmonella pathogenicity island 1 (SPI1) region knockout strains of Salmonella induced significantly less cytokine release in the plasma, however, could not protect from the coagulation and lethality.

In vitro studies showed that deficiency of Caspase 1, NAIP and GSDMD also protects the bone marrow derived macrophage’s (BMDM’s) death upon Salmonella infection during early phase of infection. In vitro, inflammasome activation and BMDM death was also completely abolished when flagellin or SPI1 deficient strains of Salmonella were used during early but not late phase of infection. During late phase of infection, caspase 11 and NLRP3 dependent pyroptosis had major contribution. These results indicate that during acute Salmonella systemic infection under flagellin and SPI1 expressing conditions, severe inflammation occurs mainly through NAIP/Caspase 1/GSDMD axis. However, coagulation could also be induced also by factors other than flagellin and SPI1 that contributes to lethality. Flagellin and SPI1 independent coagulation was protected by NLRP3 and caspase 11 deficiency.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.318

Funding Information

This study was supported by:

National Institutes of Health/ National Institutes of Allergy and Infectious Diseases Grant 1R56AI137020

National Institutes of Health/ National Institute of Allergy and Infectious Diseases Grant 1R21AI142063-01

National Institutes of Health/National Heart, Lung and Blood Institute Grant R01 HL142640

National Institutes of Health/ National Institute of General Medical Sciences Grant R01 GM132443

National Institues of Health Grant R01 HL146744

National Science Foundation Grant CHE-1709381

Available for download on Thursday, August 10, 2023

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