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The Bacterial Surface Expression of SARS Viral Epitope using Salmonella typhi Cytolysin A
Piao, Hong-Hua,Seong, Ji-Hyoun,Song, Man-Ki,Kim, Youn-Uck,Shin, Dong-Jun,Choy, Hyon-E,Hong, Yeong-Jin The Korean Society for Microbiology 2009 Journal of Bacteriology and Virology Vol.39 No.2
The cytolysin A (ClyA) is a 34 kDa pore-forming cytotoxic protein and expressed by some enteric bacteria including Salmonella typhi. This toxin is transported on the bacterial surface and secreted without posttranslational modification. Using the surface display of ClyA, the expression vectors for 193-aa immunogenic antigen of spike protein (termed S1E) from severe acute respiratory syndrome coronavirus (SARS-CoV) were constructed. The vectors carried a gene encoding S. typhi ClyA conjugated to S1E at the C terminus (termed ClyA-S1E) and asd gene in pGEM-T and pBR322, named pGApLCS1E and pBApLCS1E, respectively. An asd-mutated E. coli transformed with these vectors could grow without diaminopimelic acid (DAP), indicating that they were stably maintained in such mutants. ClyA-S1E recombinant proteins from these vectors were expressed on the surface of the attenuated S. typhimurium deficient of global virulence gene regulator, ppGpp. However, they did not show the hemolytic activity on the blood agar plate and cytotoxicity against HeLa cells. To examine whether bacteria expressing ClyA-S1E induced the immune response against S1E, S. typhimurium deficient of ppGpp and Asd was transformed with these vectors and orally immunized in mice. In the western blotting against GST-conjugated S1E using the immunized mouse sera, it was shown that the significant band was detected in the mouse serum by the bacteria transformed with pGApLCS1E but not with pBApLCS1E. It indicates that the immune response producing antibody was dependent on the expression level of ClyA-S1E. Therefore, ClyA delivery system can be used for SARS vaccine development.
Su-Mi Choi,Jae-Ho Jeong,Hyon E. Choy,Minsang Shin 한국미생물학회 2016 The journal of microbiology Vol.54 No.8
Enteropathogenic E. coli causes attaching and effacing (A/E) intestinal lesions. The genes involved in the formation of A/E lesions are encoded within a chromosomal island comprising of five major operons, LEE1-5. The global regulator H-NS represses the expression of these operons. Ler, a H-NS homologue, counteracts the H-NS–mediated repression. Using a novel genetic approach, we identified the amino acid residues in Ler that are involved in the interaction with H-NS: I20 and L23 in the C-terminal portion of α-helix 3, and I42 in the following unstructured linker region.
Initial Characterization of yliH in Salmonella typhimurium
Park, Kyung-Hwa,Song, Mi-Ryung,Choy, Hyon-E. The Microbiological Society of Korea 2007 The journal of microbiology Vol.45 No.6
Using microarray analysis, we determined those Salmonella genes induced at the entry of stationary phase, and subsequently discovered that uncharacterized yliH was induced most dramatically. We set out to establish the molecular mechanism underlying the stationary phase induction of yliH under the standard culture condition, LB with vigorous aeration, by analyzing its promoter activity in various mutant backgrounds, lacking stationary phase ${\sigma}$, $RpoS^-$, or stringent signal molecules ppGpp, ${\Delta}relA$ ${\Delta}spoT$. It was found that the stationary phase induction of yliHp was partially dependent on rpoS but entirely dependent on ppGpp. DNA sequence analysis revealed that the Salmonella yliH gene is composed of 381 base-pair nucleotides, with overall amino acid sequence revealing 76.38% amino acid identity and 88.98% similarity with Escherichia coli yliH, although no motif from data base was noted for its possible role. Recently however, it has been reported that yliH in E. coli was implicated in biofilm formation and motility by repressing these activities (Domka et al., 2006). We have constructed a mutant Salmonella deleting yliH gene by allele replacement and examined its phenotype, and found that the yliH in Salmonella more or less affects motility and adherence by enhancing these activities. The effect on biofilm formation in Salmonella was uncertain. Moreover, addition of cloned yliH of E. coli into Salmonella did not reduce motility or adherence. Taken together, it appears that the pathways implicating yliH for biofilm formation and motility in E. coli and in Salmonella are somewhat different.
Caveolae-mediated entry of <i>Salmonella typhimurium</i> into senescent nonphagocytotic host cells
Lim, Jae Sung,Choy, Hyon E,Park, Sang Chul,Han, Jung Min,Jang, Ik-Soon,Cho, Kyung A Blackwell Publishing Ltd 2010 AGING CELL Vol.9 No.2
<P>Elderly individuals have an increased susceptibility to microbial infections because of age-related anatomical, physiological, and environmental factors. However, the mechanism of aging-dependent susceptibility to infection is not fully understood. Here, we found that caveolae-dependent endocytosis is elevated in senescent cells. Thus, we focused on the implications of caveolae-dependent endocytosis using <I>Salmonella typhimurium</I>, which causes a variety of diseases in humans and animals by invading the eukaryotic host cell. <I>Salmonella</I> invasion increased in nonphagocytotic senescent host cells in which caveolin-1 was also increased. When caveolae structures were disrupted by methyl-β-cyclodextrin or siRNA of caveolin-1 in the senescent cells, <I>Salmonellae</I> invasion was reduced markedly compared to that in nonsenescent cells. In contrast, the over-expression of caveolin-1 led to increased <I>Salmonellae</I> invasion in nonsenescent cells. Moreover, in aged mice, caveolin-1 was found to be highly expressed in Peyer’s patch and spleen, which are targets for infection by <I>Salmonella</I>e. These results suggest that high levels of caveolae and caveolin-1 in senescent host cells might be related to the increased susceptibility of elderly individuals to microbial infections.</P>
Generation of Minicells from an Endotoxin-Free Gram-Positive Strain Corynebacterium glutamicum
( Jin Young Lee ),( Hyon E Choy ),( Jin Ho Lee ),( Geun Joong Kim ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.4
Drug delivery systems (DDSs) incorporating bacterial minicells have been evaluated as a very powerful tool in view of biocompatibility. However, limited studies have been carried out on these systems, mainly using minicells from Salmonella sp. and Escherichia coli. Thus, we generated a new minicell-producing strain from an endotoxin-free Corynebacterium glutamicum by the inactivation of genes related to cell division. The two knockout strains, ΔparA and Δncgl1366, showed distinct abilities to produce minicells. The resulting minicells were purified via sequential antibiotic treatments and centrifugations, which resulted in reproducible yields.
Shin Dong-Jun,Choy Hyon E.,Hong Yeongjin The Microbiological Society of Korea 2005 The journal of microbiology Vol.43 No.3
In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.