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 plays 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. In addition to this, 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. 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, inflammasome activation and BMDM death was also completely abolished when flagellin or SPI1 deficient strains of Salmonella were used. These results indicate that during acute Salmonella systemic infection severe inflammation occurs mainly through NAIP/Caspase 1/GSDMD axis induced by the combination of both flagellin and T3SS. However, coagulation could also be induced also by factors other than flagellin and T3SS present in SPI1 that contributes to lethality.