Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Studies on a Toxin/Antitoxin System in Streptococcus iniae

어류병원균 Streptococcus iniae의 toxin/antitoxin system에 대한 연구

  • Yoon, Seongyong (Department of Integrated Biological Science, Pusan National University) ;
  • Kim, Yeon Ha (Department of Integrated Biological Science, Pusan National University) ;
  • Jeun, Moonjung (Department of Integrated Biological Science, Pusan National University) ;
  • Seong, Minji (Department of Integrated Biological Science, Pusan National University) ;
  • Yoo, Ah Young (Department of Microbiology, Pusan National University) ;
  • Lee, Donghee (Department of Integrated Biological Science, Pusan National University) ;
  • Moon, Ki Hwan (Division of Marine Bioscience, Korea Maritime and Ocean University) ;
  • Kang, Ho Young (Department of Integrated Biological Science, Pusan National University)
  • 윤성용 (부산대학교 대학원 생명시스템학과) ;
  • 김연하 (부산대학교 대학원 생명시스템학과) ;
  • 전문정 (부산대학교 대학원 생명시스템학과) ;
  • 성민지 (부산대학교 대학원 생명시스템학과) ;
  • 유아영 (부산대학교 미생물학과) ;
  • 이동희 (부산대학교 대학원 생명시스템학과) ;
  • 문기환 (한국해양대학교 해양생명과학부) ;
  • 강호영 (부산대학교 대학원 생명시스템학과)
  • Received : 2019.01.09
  • Accepted : 2019.01.25
  • Published : 2019.01.30
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Abstract

Streptococcus iniae is a typical fish pathogen causing streptococcosis and it can also cause zoonotic infectious diseases. We studied S. iniae FP5228 isolated from infected olive flounder in Wando, Korea. In a study to find virulence factors in FP5228, we found that the number of live bacteria decreased dramatically in culture medium containing S. iniae FP5228 for more than 24 hr. This phenomenon was hypothesized to be related to Toxin ${\zeta}$ and Antitoxin ${\varepsilon}$ genes, components of the Toxin/ Antitoxin (TA) system on the 14 kb plasmid of FP5228. We used a protein overexpression system to identify it. The pBP1140 vector system was constructed to regulate the expression of Toxin ${\zeta}$ and Antitoxin ${\varepsilon}$ by IPTG and Arabinose. E. coli/pBP1140 strain grew slowly in early growth under toxin expression condition, and it was confirmed by microscopic observation that the strain became longer. S. iniae CK287, lacking a 14 kb plasmid of S. iniae FP5228 strain, was constructed. CK287 bacterial cells did not show rapid killing during culture, and the ability to produce biofilm was also decreased, and toxicity was weakened in cytotoxicity test and fish test. These results suggest that the TA system is involved in physiological regulation and expression of virulence factors in S. iniae FP5228.

Streptococcus iniae는 대표적인 어류병원균으로 인수공통의 질병을 일으킨다. S. iniae FP5228에 존재하는 병원성 인자를 찾고자 하는 연구과정에서 S. iniae를 24시간 이상 배양한 배양액에는 살아있는 균의 수가 급격하게 감소하는 현상을 발견하였다. 이 현상은 FP5228 균이 가지고 있는 14 kb plasmid 상에 있는 toxin/antitoxin (TA) system의 구성요소인 toxin ${\zeta}$와 antitoxin ${\varepsilon}$ 유전자가 관련이 있을 것이란 가설을 설정하였다. IPTG와 arabinose에 의해 toxin ${\zeta}$와 antitoxin ${\varepsilon}$의 발현이 조절되는 pBP1140 vector system을 구축하였다. E. coli/pBP1140 균주는 toxin이 발현되는 조건에서 초기 생육이 느려졌고, 현미경의 관찰에서 균체가 길어짐을 확인하였다. FP5228 균주가 가진 14 kb plasmid를 없앤 S. iniae CK287을 제조하였다. CK287 균은 배양 중 급격하게 사멸되는 현상을 보이지 않았고, biofilm 생성능력도 감소하였고 세포독성 시험과 물고기 시험에서 독성이 약화 된 것이 확인되었다. 이들 결과 들은 TA system이 생리적 조절 및 병원성 인자의 발현에 관련이 있음을 추정할 수 있다.

Keywords

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Fig. 1. Growth analysis of S. iniae, E. coli, B. subtilis, E. piscicida. The absorbance and viable count were measured every 12 hours. The absorbance was measured at OD600, and the number of viable cells was determined by counting the colonies formed in the BHI agar plate after serial dilution with 0.85% NaCl solution.(A)S. iniae FP5228, (B) E. coli DH5a, (C) B. subtilis subsp. subtilis, (D) E. piscicida CK41; dark gray line : OD600, gray line : viable count (CFU/ml).

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Fig. 2. Construction of overexpression vector of toxin and antitoxin in S. iniae FP5228. (A) pBP1140 vector construction process. (B) Confirmation of pBP1140 vector using PCR. M : marker, lane 1 ; antitoxin ε : 287 bp, lane 2; pBAD :: toxin ζ : 2643bp, lane 3 ; toxin ζ : 864 bp.

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Fig. 3. Growth analysis of toxin antitoxin overexpressed E. coli. The growth rate of the arabinose overexpressing strain at the early stage of growth was slower than the other conditions. (A) Growth curves measured using absorbance (OD 600nm). (B) Growth curves measured by viable count. (log CFU/ml/1,000,000), ◆ : control, ■ : IPTG induction, ▲ : Arabinose induction, × : IPTG & arabinose induction.

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Fig. 4. Morphological changes of E. coli BL21 overexpressing Toxin. Overexpression of toxin in E. coli BL21 confirmed that the cell length was prolonged. (Gram staining, ×400) ; (A) control (not induction), (B) IPTG induction, (C) Arabionse induction, (D) IPTG & arabinose induction.

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Fig. 5. Characteristic difference between wild type S. iniae (FP 5228) and plasmid-cured S. iniae (CK287). (A) Growth curves and viable counts of S. iniae FP5228 and CK287. It was confirmed that the decrease in viable cells was slightly improved in CK287.; ◆ OD600 of FP5228, ▲: OD600 of CK287, ■: CFU/ml of FP5228, ×: CFU/ml of CK287. (B) Plasmid preparation of S. iniae FP5228 and CK287.; lane 1 : FP5228, lane 2 : CK287. Plasmid size: 13,781 bp.

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Fig. 6. Comparison of biofilm formation between S. iniae FP5228 and CK287.

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Fig. 7. MTT assay between S. iniae FP5228 and CK287. In all cases, wild type S. iniae showed lower value than CK287. (n=3) (*p< 0.1).

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Fig. 8. Virulence of S. iniae strains in zebrafish. To confirm the virulence, different concentrations of bacteria were inoculated. Survival rate was measured for 12 days. ◆, FP5228 108 CFU/fish; ■, FP5228 107 CFU/fish; ▲, FP5228 106 CFU/fish; Χ, CK287 108 CFU/fish; Ж, CK287 107 CFU/fish; ●, CK287 106 CFU/fish.

Table 1. PCR primers used in this study

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