Jong Earn Yu

Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis.

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD(50) value of E. tarda CK164 in fish (2.0 × 10(8) colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10(7) CFU/fish). The LD(50) of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry.

Role of sigma factor E in regulation of Salmonella Agf expression

Expression of thin aggregative fimbriae (Agf) in Salmonella, which is responsible for bacterial cell adhesion to surfaces, aggregation, and formation of biofilms, is regulated by a complex mechanism. In order to identify gene(s) involved in the expression of Agf, the TnphoA transposon was introduced into Salmonella typhimurium χ8505 for random mutagenesis. Colonies showing a change from wrinkly-rough morphology to the smooth form were screened for candidates. Through multiple selection processes, a mutant, named S. typhimurium CK167 was selected as the final candidate. Analyses of the nucleotide sequences of TnphoA insertion site identified the insertion in rpoE gene. S. typhimurium CK178, a defined rpoE deletion mutant on χ8505, exhibited the same colony morphology as seen in CK167. The S. typhimurium CK178 strain expressed significantly reduced amounts of AgfD and showed modulated biofilm formation, demonstrating the role of RpoE in AgfD expression. To the best of our knowledge, this is the first report demonstrating that RpoE acts as a regulator in the expression of Agf in Salmonella.

Edwardsiella piscicida lacking the cyclic AMP receptor protein (Crp) is avirulent and immunogenic in fish

Abstract Edwardsiella piscicida is a Gram‐negative pathogen that generally causes lethal septicemia in marine and freshwater fish. We generated a E. piscicida CK216 &Dgr;crp mutant to investigate various biological roles related to this organism, including pathogenesis. Lack of Crp in CK216 was demonstrated by immunoblotting using a Crp‐specific antibody. Compared to the parental strain, the mutant exhibited changes in three biochemical phenotypes, including ornithine decarboxylation, citrate utilization, and H2S production. Complementation of crp deletion in trans rescued the phenotype of the parental strain. This study proved that hemolytic activity in E. piscicida is controlled by Crp. In addition, significantly reduced motility of E. piscicida CK216 was observed, which resulted from a lack of flagella synthesis. To examine the virulence in fish, E. piscicida cells were injected into the goldfish (Carassius auratus) via intraperitoneal route. The LD50 of CK216 was 9.25 × 108 CFU, while that of the CK108 parental strain was 9.24 × 105 CFU, attenuated 1000 fold in goldfish. Fish immunized with CK216 elicited IgM responses. Moreover, 80% of goldfish immunized with 1 × 106 CFU survived after administration of a lethal dose (1 × 107 CFU) of virulent E. piscicida CK41, suggesting the potential for E. piscicida CK216 to serve as a live attenuated vaccine in aquaculture. HighlightsThe Crp regulates multiple genes including virulence factors in E. piscicida.The E. piscicida &Dgr;crp exhibited significant attenuation in fish.Administration of E. piscicida &Dgr;crp induced E. piscicida specific immune response.Protection against virulent E. piscicida challenge was demonstrated.

Characterization and Identification of an Agar-Degrading Motile Bacteria Strain

환경에서 분리된 CK214 균주는 1.5% (w/v) agar가 포함되어 있는 LB 평판배지에서 빠르게 이동하는 특징을 가지며, agar 고체평판배지 위의 CK214 균주의 집락 주위로 움푹한 투명환이 관찰되었다. 이 균주는 단일 탄소원으로 agar만이 첨가된 최소 배지에서 잘 자랐으며, DNS 법을 이용하여 CK214 균주의 외부추출성분이 agar 분해활성을 가진다는 것을 확인하였다. CK214 균주는 다양한 농도의 agar (0.5, 1.0, 1.5 2.0% w/v)가 포함된 고체평판 배지에서 swarming 운동을 하였다. CK214 균주를 동정하기 위해 그람염색과 현미경 관찰, 생화학적 분석(API), 16S rRNA 염기서열분석에 기초한 계통발생학적 분석을 수행하였다. 이를 통해 CK214 균주는 그람 양성의 간균으로, Paenibacillus 속에 포함되었으며 Paenibacillus lactis MB 2035와 가장 가까운 연관성을 보이는 것을 확인할 수 있었다. 또한 CK214 균주는 agar 고체표면에서 주모성의 편모를 형성하는 것을 투과 전자 현미경(TEM)을 통해 관찰하였다. CK214 균주의 agarase 활성과 운동성의 연관성에 관한 앞으로의 연구를 위해 transposon random mutagenesis에 의한 agar 분해활성 결손 돌연변이주를 구축하였다. 【A bacterial strain, CK214, exhibiting high motility on an LB agar (1.5%, w/v) surface was isolated from the environment. The formation of unusual agar shrinking around colonies on agar plates was observed. The strain grew on minimal media containing pure agar as a sole carbon source. The cell-free culture supernatant of CK214 generated a reduced form of sugar in the in vitro reaction with the use of pure agar as a substrate, suggesting the secretion of an agar-degrading enzyme. The CK214 strain showed swarming motility on the solid media containing a wide range of concentrations of agar (0.5, 1.0, 1.5, 2.0% w/v). Various tests, including Gram staining, API analysis, and phylogenetic analysis based on 16S rDNA sequences identified that the CK214 strain was a G(+) rod-shaped bacterium grouped in genus Paenibacillus. Electron microscopic analysis demonstrated that the P. CK214 strain is peritrichously flagellated. Through transposon random mutagenesis, several agar-degrading activity defective mutants (ADMs) were generated. These mutants will be used in the future experimentation for the study of the correlation between agar-degrading activity and motility.】

Goldfish, Carassius auratus, as an infection model for studying the pathogenesis of Edwardsiella piscicida

This study demonstrates the feasibility of using goldfish as an infection model to investigate the pathogenesis of Edwardsiella piscicida. Goldfish were found to be susceptible to acute E. piscicida-induced disease and died in a dose-dependent manner. E. piscicida was further shown to replicate rapidly in the head kidneys and livers of infected goldfish from 1 d post-injection, and bacteria numbers were significantly decreased 5 d post-injection. Immune responses were successfully induced in goldfish injected with E. piscicida strains and 60% of goldfish inoculated with an attenuated E. piscicida strain were found to survive subsequent injection with a pathogenic strain. The results of differential leukocyte count experiments suggested that leukocytes were immediately recruited as an innate immune response against the infection. Thus, this well-characterized goldfish species is a suitable infection model for studying E. piscicida pathogenesis, and might be applicable to research on other fish diseases.

Effects of Salt Concentration on Motility and Expression of Flagellin Genes in the Fish Pathogen Edwardsiella tarda

E. tarda, a fish pathogen, can survive in seawater under relatively high salt conditions as well as in fish under physiological salt conditions. Bacterial growth under different salt concentrations may influence the expression of genes involved in bacterial structure and physiology. The growth rate of E. tarda culture in high salt (3.5% NaCl) was similar to that in low salt (1.0% NaCl, physiological salt concentration). Interestingly, the strain moved much faster in low salt conditions than in high salt conditions. Electron microscopic observation demonstrated that the bacterial cells grown in high salt had less or no flagellation. Obvious flagellation was observed in the parental strain E. tarda CK41 grown in low-salt condition. Two putative genes coding flagellin were identified in the E. tarda genome sequences. The amino acid sequence comparison of each gene revealed 93% identities. A flagellin gene was PCR amplified and cloned into a cloning vector. Using an E. coli protein expression system, a part of flagellin protein was overexpressed. Using the purified protein, an anti-flagellin antibody was raised in the rabbit. Immunoblot analyses with flagellin specific antibody demonstrated that E. tarda CK41 expressed falgellin in low salt conditions, which is consistent with the results seen in motility assay and microscopic observation. This is the first report of salt regulated flagella expression in E. tarda.

Roles of Glyceraldehyde-3-Phosphate Dehydrogenase in Edwardsiella tarda Pathogenesis

A research group demonstrated that the 37 kDA protein of Edwardsiella tarda, a causing causative agent of edwardsiellosis in fish, exhibited high antigenicity in Japanese flounder. The research group also showed that the N-terminus amino acid sequences of the 37 kDa protein were mapped to the N-terminus of GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Using degenerated primer sets based on the known N-terminus sequence, the corresponding E. tarda DNA was amplified and cloned. The nucleotide sequences of the cloned gene revealed high homology with a bacterial gene for GAPDH, as we was expected. The amino acid sequence of E. tarda GAPDH (etGAPDH) revealed a <70% similarity with GAPDH proteins in other Enterobacteriaceae. With the application of artificial protein overexpression system in Escherichia coli, the recombinant etGAPDH (rGAPDH) was produced and purified. In this study, Using the purified rGAPDH, the etGAPDH specific polyclonal antibody has been was generated using the purified rGAPDHin this study. The immunoblotting analyses demonstrated that the location of the GAPDH protein is located with the association of is associated with the envelops of E. tarda. The rGAPDH was administrated into Japanese flounder via IP route for evaluation of the protective ability. Although the specific antibody titer against etGAPDH was increased about 3-fold after 4 weeks post-vaccination, the survival rates of vaccinated Japanese flounder and the control group with wild type E. tarda was were 12.5% and 0%, respectively. Our results indicated that rGAPDH is immunoreactive antigen but that it will not generate protective immunity in Japanese flounder.

Isolation and Characterization of Plant Pathogen that Cause Soft Rot Disease in Napa Cabbage

In order to establish in vitro infection model for research of plant pathogen based on tissue softening disease in napa cabbage, eighty independent bacterial strains were isolated from the softened napa cabbage tissues. Eight bacterial isolates were primarily screened with the generation of reproducible tissue softening disease to fresh napa cabbages within 2448 hours after inoculation. Through various microbiological biochemical and morphological examinations, three Gram (-) isolates which harbor independent biological properties were finally chosen, and named as RBI, RB2 and RB6. Collective results obtained from API 20E test and analyses of VITEK 2 COMPACT and nucleotide sequences of 165 rRNA of each isolate proposed that isolates RBI and RB2 are close to the Erwinia carotovora subsp. odorifera, and RB6 is close to the Erwinia carotovora subsp. carotovora. These isolates grew optimally at with neutral pH culture condition. The isolates caused softening tissue disease with dose-dependent manner regardless of pre-surface damages of napa cabbage. Minimum dose to cause soft rot disease for RBI, RB2 or RB6 were CFU/mt CFU/ml or CFU/ml respectively. These isolates caused tissue softening disease to eggplant, paprika and napa cabbage out of 14 different tested vegetables, indicating that these isolates damages specific plant tissues. The bacterial isolates obtained in this research and in vitro plant infection model will be adapted in the understanding of the mechanism of pathogenesis by plant pathogen.In order to establish in vitro infection model for research of plant pathogen based on tissue softening disease in napa cabbage, eighty independent bacterial strains were isolated from the softened napa cabbage tissues. Eight bacterial isolates were primarily screened with the generation of reproducible tissue softening disease to fresh napa cabbages within 24～48 hours after inoculation. Through various microbiological, biochemical and morphological examinations, three Gram (-) isolates which harbor independent biological properties were finally chosen, and named as RB1, RB2 and RB6. Collective results obtained from API 20E test and analyses of VITEK 2 COMPACT and nucleotide sequences of 16S rRNA of each isolate proposed that isolates RB1 and RB2 are close to the Erwinia carotovora subsp. odorifera, and RB6 is close to the Erwinia carotovora subsp. carotovora. These isolates grew optimally at 30℃ with neutral pH culture condition. The isolates caused softening tissue disease with dose-dependent manner regardless of pre-surface damages of napa cabbage. Minimum dose to cause soft rot disease for RB1, RB2 or RB6 were 8.0×10? CFU/㎖, 10? CFU/㎖ or 4.7×10? CFU/㎖, respectively. These isolates caused tissue softening disease to eggplant, paprika and napa cabbage out of 14 different tested vegetables, indicating that these isolates damages specific plant tissues. The bacterial isolates obtained in this research and in vitro plant infection model will be adapted in the understanding of the mechanism of pathogenesis by plant pathogen.

Identification and Characterization of Genes Involved in Cysteine Auxotrophy in Salmonella typhi

In spite of long research period for Salmonella typhi, little information is known about the pathogenesis mechanism of human typhoid fever caused by S. typhi due to lack of infection model in animals. A wild-type of S. typhi Ty2 strain requires cysteine to grow on minimal media. We hypothesized that this cysteine requirement may restrict colonization of S. typhi in animals during infection process. Among the S. typhi strains carrying Salmonella typhimurium genomic library, we have isolated three S. typhi transformants growing on minimal media without cysteine. Although there were three ORFs in DNA of pBP71, the STM1490 ORF complemented cysteine auxotrophy of S. typhi. Analysis of the deduced amino acid sequence of the STM1490 homolog in S. typhi revealed that there are differences in two amino acids. Plasmids containing amino acid substitutions in STM1490 supported S. typhi growth on minimal media without cysteine, indicating irrelevance of these two amino acids to STM1490 function. These results tells us that there are other factors or systems involved in cysteine requirement of S. typhi.