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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2012)

The Disulfide Bond Formation (Dsb) System is important for maintaining the structure and function of periplasmic proteins. Although the biochemistry of various Dsb proteins has been characterized in E. coli, the potential link between this system and virulence mechanisms of pathogenic Gram-negative bacteria has not been elucidated. Deletion mutations generated in dsb genes demonstrated multiple virulence defects in Salmonella enterica serovar Enteritidis, which were in turn rescued by complementation. Analyses revealed that two members of the Dsb system, DsbA and DsbC, were required for invasion of epithelial cells, a process mediated by the Type III Secretion System (T3SS) encoded by Salmonella Pathogenicity Island 1 (SPI-1). The Dsb system was also required for colonization and virulence in vivo. Strains expressing T3SS effectors tagged with a FLAG epitope were transduced with the dsb mutations, and analyses of the supernatant fractions indicated that the Dsb system was involved in the secretion of SipA and SipC, which are critical for ruffling of the host cell membrane and subsequent internalization of the bacteria. A bacterial two-hybrid assay was performed to identify Dsb substrates among the T3SS needle complex proteins, and preliminary results indicated direct binding of the Dsb system with SpaO, a T3SS sorting platform protein. These results suggested that the formation and isomerization of disulfide bonds are critical steps in the function of the T3SS which contributes to the pathogenesis of Salmonella.

    Cover page: A Role for the Dsb System in Type III Secretion and Virulence in Salmonella
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2013)
    Abstract Background Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. Results We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. Conclusion Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology.
      Cover page: Release of extracellular ATP by bacteria during growth
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      External guide sequence (EGS) RNAs are associated with ribonuclease P (RNase P), a tRNA processing enzyme, and represent promising agents for gene-targeting applications as they can direct RNase-P-mediated cleavage of a target mRNA. Using murine cytomegalovirus (MCMV) as a model system, we examined the antiviral effects of an EGS variant, which was engineered using in vitro selection procedures. EGSs were used to target the shared mRNA region of MCMV capsid scaffolding protein (mCSP) and assemblin. In vitro, the EGS variant was 60 times more active in directing RNase P cleavage of the target mRNA than the EGS originating from a natural tRNA. In MCMV-infected cells, the variant reduced mCSP expression by 92% and inhibited viral growth by 8,000-fold. In MCMV-infected mice hydrodynamically transfected with EGS-expressing constructs, the EGS variant was more effective in reducing mCSP expression, decreasing viral production, and enhancing animal survival than the EGS originating from a natural tRNA. These results provide direct evidence that engineered EGS variants with higher targeting activity in vitro are also more effective in reducing gene expression in animals. Furthermore, our findings imply the possibility of engineering potent EGS variants for therapy of viral infections.
        Cover page: Inhibition of Murine Cytomegalovirus Infection in Animals by RNase P-Associated External Guide Sequences
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2019)
        External guide sequences (EGSs) signify the short RNAs that induce ribonuclease P (RNase P), an enzyme responsible for processing the 5' termini of tRNA, to specifically cleave a target mRNA by forming a precursor tRNA-like complex. Hence, the EGS technology may serve as a potential strategy for gene-targeting therapy. Our previous studies have revealed that engineered EGS variants induced RNase P to efficiently hydrolyze target mRNAs. In the present research, an EGS variant was designed to be complementary to the mRNA coding for human cytomegalovirus (HCMV) major capsid protein (MCP), which is vital to form the viral capsid. In vitro, the EGS variant was about 80-fold more efficient in inducing human RNase P-mediated cleavage of the target mRNA than a natural tRNA-derived EGS. Moreover, the expressed variant and natural tRNA-originated EGSs led to a decrease of MCP expression by 98% and 73%-74% and a decrease of viral growth by about 10,000- and 200-fold in cells infected with HCMV, respectively. These results reveal direct evidence that the engineered EGS variant has higher efficiency in blocking the expression of HCMV genes and viral growth than the natural tRNA-originated EGS. Therefore, our findings imply that the EGS variant can be a potent candidate agent for the treatment of infections caused by HCMV.
          Cover page: Inhibition of human cytomegalovirus major capsid protein expression and replication by ribonuclease P-associated external guide sequences
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