Investigation of novel genetic mutations for antibiotic resistance, intracellular upregulated Salmonella genes within host cells, and the mechanisms of yqiC-induced host innate immunity for combating Salmonella infection (2/3)

Salmonella is one of the most important enteropathogens for pediatric diarrhea. However, increasing antibiotic resistance in Salmonella has become a global problem. Hence, we conduct this study to investigate novel genetic mutations for antibiotic resistance, intracellularly upregulated Salmonella genes within host cells, and the mechanisms of yqiC-induced host innate immunity for combating Salmonella infection. First, our pilot study using wide genome sequencing discovered a novel point mutation in parC that leads to g.1307delA in the clinical ciprofloxacin resistant Salmonella strain. We found 3 reported point mutations (gyrA g.248C.T, parC g.170C>G, and parE g.1031G>T) related to ciprofloxacin resistance in the ciprofloxacin resistant strain, but parC g.170C>G was also present in the ciprofloxacin sensitive strain. Another reported point mutation gyrB g.2044del was found in the negative control. Therefore, we plan to generate the recombinant mutants of these point mutations for validating their phenotypes. In addition, in vitro tests for developing antimicrobial resistance to recombinant human β-defensins and commonly used antibiotics will be performed. Second, our preliminary RNA microarray study showed that certain Salmonella genes were significantly upregulated after Salmonella had invaded human intestinal Caco-2 cells for 18 hours. The top ten genes are ybfA (encoding hypothetical protein), SL1344_0345 (encoding putative outer membrane efflux lipoprotein), bioC, nrdI, bioA, bioB, fepA, SL1344_0575 (encoding hypothetical protein), bioF, and entD. These Salmonella genes are responsible for bacterial virulence against host defense. Therefore, we plan to generate their recombinant gene-depleted and complemented strains for realizing the roles of these genes in vacuolization and its fusion with lysosome within host cells. Host cytosolic sensors (e.g. Nod-like receptors) binding with their encoded proteins will be identified. Third, bacterial virulence genes affect host immunity. For the first time, we found that the yqiC mutant of Salmonella Typhimurium was significantly attenuated in bacterial colonization and invasion in four human cells, and yqiC is required for postinfection production of interleukin-8 and human β-defensin-3 (hBD3) in human colonic epithelial cells. In addition, we demonstrated that yqiC negatively regulates type-1 fimbriae genes to manipulate its downstream virulence factors including flagella, Salmonella pathogenicity island (SPI)-1 and SPI-2. Furthermore, chromatography revealed that yqiC is indispensible for biosynthesis of menaquinone, a mediator in the bacterial electron transport chain. Thus, we plan to further confirm its phenotype in human intestinal tissue using human intestinal polarized in vitro organ culture, and to examine whether yqiC influences the expression of the other antimicrobial peptides (e.g. cathelicidin, hBD1, hBD2, and hBD4). The underlying mechanisms related to cellular signaling pathways, involvement of Toll-like receptors, and respiratory electron transfer chain will be thoroughly investigated. Taken together, discovery of novel mutations in Salmonella antibiotic resistance genes facilitates detection of more antibiotic resistant strains in human and animals. Meanwhile, recombinant human β-defensins are promising alternatives to currently used antibiotics for combating the problem in Salmonella antibiotic resistance. Better understanding of the mechanisms regarding the functions of the intracellularly upregulated Salmonella genes within host cells facilitates development of anti-Salmonella strategies. Any transcriptional and translational inhibitors to yqiC expression can be developed into novel antimicrobials of Salmonella.