탄저균 내 다면발현성 조절인자 CodY의 특성

Abstract

The pleiotropic regulator CodY is a transcriptional regulator commonly found in Gram-positive bacteria, and plays a significant role in metabolic regulation and growth phase transition by repressing early stationary genes and activating carbon overflow pathway. In addition, CodY participates in bacterial virulence in numerous pathogenic bacteria including Bacillus anthracis, the etiological agent of anthrax. As for B. anthracis, CodY positively regulates anthrax toxin expression by enhancing the post-translational accumulation toxin activator AtxA via unknown mechanism. In an endeavor to elucidate the underlying mechanism, this study focuses on the transcriptional profiling of the CodY regulon in B. anthracis during virulence exertion, and on the characterization of a CodY-mediated gene.

Firstly, transcriptional profiles of codY-mutated B. anthracis during late exponential phase in which the virulence expression is maximal were observed. Both overexpression and deletion affected bacterial growth and gene expression: overexpression of CodY significantly inhibited growth and repressed overall gene expression in B. anthracis, whereas deletion mutation delayed the growth rate and repressed gene expression associated with general metabolic regulation and precursor biosynthesis. This provided transcriptional profiles of CodY regulon during growth phase transition and potential positive regulation by CodY in B. anthracis.

Next, transcriptional profiles of codY-deleted B. anthracis of the individual element required for virulence were observed under starvation. Upon iron starvation, codY-deleted B. anthracis overexpressed iron acquisition genes and underexpressed central metabolism genes, whereas the parental strain generally underexpressed carbon overflow pathway-associated genes. Upon glucose starvation, codY-deleted B. anthracis overexpressed nitrogen metabolism genes and underexpressed oxidation-reduction genes, whereas the parental strain showed underexpression of central metabolism genes. Upon carbon dioxide starvation, codY-deleted B. anthracis overexpressed ABC transporter genes and underexpressed virulence related genes, whereas the parental strain showed no significant biological processes changed. As for the integrated gene expression analysis in response to environmental deprivation, genes from the CodY regulon showed complex expression patterns in response to nutrient starvation, reflecting the hierarchical relationship between starvation response and CodY regulation. Transcriptional profiling of codY-deleted B. anthracis also displayed supportive role of CodY in gene regulation. Taken together, this transcriptional profiling demonstrated complex relationship underlying the starvation response and CodY regulation, and suggests that CodY act as a DNA binding pioneer factor to recruit various inhibitors and/or activators.

Lastly, functional characteristics of a putative NlpC/P60 endopeptidase BAS1812 identified from aforementioned transcriptional profiling studies were assessed. Deletion mutant of BAS1812 B. anthracis showed less survivability in late stationary phase under minimal nutrient condition. In addition, BAS1812-deleted B. anthracis showed defective germination and sporulation, and was more susceptible to cell wall destabilizing agent. Loss of BAS1812 also diminished the ability of B. anthracis to degrade peptidoglycan, suggesting that BAS1812 gene product is a peptidoglycan-degrading enzyme that enhances bacterial long-term survival and cell wall maintenance. As previous literatures mentioned its immunogenicity in the infected host serum, it is feasible that BAS1812 in serum represents bacteremia in the host and that BAS1812 serve as a potential biomarker for anthrax treatment and diagnosis.

Collectively, transcriptional profiles associated with B. anthracis CodY in this dissertation suggests that CodY may induce both positive and negative regulation in B. anthracis gene expression, and it acts as a pioneer factor and modulate its mode of action in response to environmental changes. Being a pleiotropic regulator, CodY regulates various genes with potential therapeutic values for anthrax diagnosis and treatment. These findings from this work provide useful information for studying molecular functions of CodY. |다면발현성 조절인자인 CodY는 그람양성 미생물에서 흔히 발견되는 전사억제인자로, 초기 정체기 유전자 발현 억제 및 carbon overflow 경로 활성화를 통해 미생물 내 대사 조절 및 성장기 전환에 중요한 역할을 수행한다. 또한 CodY는 다양한 병원균의 병원성 발현에 기여한다. 탄저균의 경우, CodY는 탄저균 내 독성활성인자인 AtxA의 번역 후 축척을 향상시켜 탄저 독소 발현을 조절하는 것으로 알려져 있으나, 정확한 기작은 밝혀진 바가 없다. 본 연구는 위의 기작을 규명하기위해, 병원성 발현 과정 동안의 탄저균 내 CodY 레귤론을 전사체분석을 통해 분석하였으며, 이 중 발견된 특이 유전자의 기능 분석을 진행하였다.

첫째로, CodY 돌연변이주에서 병원성 발현이 최고인 성장 말기 동안의 전사체 분석을 진행하였다. 과발현변이주 및 결핍변이주 모두 세포 성장과 유전자 발현에 변화를 보였으나 다소 차이가 있었다. CodY 과발현 시, 성장이 크게 저해되었으며 탄저균 내 유전자 발현이 전반적으로 억제되었다. CodY 결핍변이의 경우, 성장 속도가 저해되었으나 성장에는 큰 장애는 없었으며, 대사 및 전구물질 생합성과 관련된 유전자의 발현이 억제되었다. 이를 통해 탄저균 내 성장기 전환 동안의 CodY 레귤론의 전사 패턴에 대해 알 수 있었으며, 추가로 CodY의 전사활성인자로서 기능할 수 있음을 확인하였다.

그 다음으로, codY 결핍변이주에서 병원성과 관련된 환경 요인 기아에 의한 전사체 변화를 관찰하였다. 철 이온 결핍 시, 탄저균 내 carbon overflow 관련 유전자 발현이 전반적으로 감소하였다. 반면에 결핍변이주에서는 철 획득 관련 유전자 발현은 증가하나, 주요 대사 경로의 유전자 발현은 감소하였다. 포도당 결핍 시, 탄저균 내 주요 대사 경로의 유전자 발현이 전반적으로 감소하였다. 반면에 결핍변이주에서는 질소 대사 관련 유전자 발현은 증가하나, 산화환원 반응 관련 유전자 발현은 감소하였다. 유산소 성장 시, 탄저균 내에서는 통계적으로 유의미한 유전자 종류를 발견할 수 없었다. 반면에 결핍변이주에서는 ABC 수송 단백질 관련 유전자 발현은 증가하며, 병원성 관련 유전자 발현은 감소하였다. 환경 요인에 의한 전체 발현 패턴 변화 분석을 통해 CodY 레귤론 내 유전자들은 최소 하나 이상의 기아에 발현이 변화되는 것을 확인하였으며, 이는 기아 반응과 CodY 조절 간의 계층 조절을 반영한다. 또한 CodY 전사체 분석에서 CodY의 유전자 발현 조절 내 보조역할을 볼 수 있었다. 종합하였을 때, 본 전자체 연구는 기아 반응과 CodY 유전자 발현 조절 사이의 긴밀한 관계를 확인할 수 있으며, CodY가 선구인자로서 활성인자 및 억제인자를 불러들이는 역할을 제시한다.

마지막으로, 전사체 연구에서 발견한 펩티드내부가수분해효소 BAS1812의 특성을 분석하였다. BAS1812의 결핍변이주는 최소 배지에서 배양할 시, 정지상 말기에서의 세포 생존률이 감소하였다. 또한 결핍변이주는 아포 형성과 아포 발아에서 장애를 보였으며, 세포벽을 불안정시키는 인자에 더 민감하게 반응하였다. 또한 BAS1812의 결핍변이는 탄저균의 펩티도글라이칸 분해능이 감소하였으며, 이는 BAS1812 유전자 산물이 탄저균의 세포 생존과 세포벽 유지보수에 관여하는 펩티도 글라이칸 분해효소이라 추측할 수 있게 한다. 이전 문헌에서 BAS1812이 감염된 숙주 내 면역원성을 가지고 있음을 미루어볼 때, 혈청 내 BAS1812의 존재는 숙주 내 균혈증이 있음을 의미하며, 이는 BAS1812의 탄저균 치료 및 진단에 활용가치가 있음을 제시한다.

본 연구에서의 탄저균 CodY 전사체 분석을 통해, CodY는 탄저균 내 유전자 발현을 억제 및 활성화할 수 있으며, 개척 인자와 유사하게 작용하여 환경 변화에 따라 발현 작용 기작을 조절하는 것을 알 수 있다. 다면발현성 조절인자로서 CodY는 의학적 활용가치가 높은 다양한 유전자들의 발현을 조절한다. 본 연구 결과는 향후 CodY의 분자적 기능 분석 및 탄저 치료 신기술 개발에 이바지할 것으로 기대된다.; The pleiotropic regulator CodY is a transcriptional regulator commonly found in Gram-positive bacteria, and plays a significant role in metabolic regulation and growth phase transition by repressing early stationary genes and activating carbon overflow pathway. In addition, CodY participates in bacterial virulence in numerous pathogenic bacteria including Bacillus anthracis, the etiological agent of anthrax. As for B. anthracis, CodY positively regulates anthrax toxin expression by enhancing the post-translational accumulation toxin activator AtxA via unknown mechanism. In an endeavor to elucidate the underlying mechanism, this study focuses on the transcriptional profiling of the CodY regulon in B. anthracis during virulence exertion, and on the characterization of a CodY-mediated gene.

Firstly, transcriptional profiles of codY-mutated B. anthracis during late exponential phase in which the virulence expression is maximal were observed. Both overexpression and deletion affected bacterial growth and gene expression: overexpression of CodY significantly inhibited growth and repressed overall gene expression in B. anthracis, whereas deletion mutation delayed the growth rate and repressed gene expression associated with general metabolic regulation and precursor biosynthesis. This provided transcriptional profiles of CodY regulon during growth phase transition and potential positive regulation by CodY in B. anthracis.

Next, transcriptional profiles of codY-deleted B. anthracis of the individual element required for virulence were observed under starvation. Upon iron starvation, codY-deleted B. anthracis overexpressed iron acquisition genes and underexpressed central metabolism genes, whereas the parental strain generally underexpressed carbon overflow pathway-associated genes. Upon glucose starvation, codY-deleted B. anthracis overexpressed nitrogen metabolism genes and underexpressed oxidation-reduction genes, whereas the parental strain showed underexpression of central metabolism genes. Upon carbon dioxide starvation, codY-deleted B. anthracis overexpressed ABC transporter genes and underexpressed virulence related genes, whereas the parental strain showed no significant biological processes changed. As for the integrated gene expression analysis in response to environmental deprivation, genes from the CodY regulon showed complex expression patterns in response to nutrient starvation, reflecting the hierarchical relationship between starvation response and CodY regulation. Transcriptional profiling of codY-deleted B. anthracis also displayed supportive role of CodY in gene regulation. Taken together, this transcriptional profiling demonstrated complex relationship underlying the starvation response and CodY regulation, and suggests that CodY act as a DNA binding pioneer factor to recruit various inhibitors and/or activators.

Lastly, functional characteristics of a putative NlpC/P60 endopeptidase BAS1812 identified from aforementioned transcriptional profiling studies were assessed. Deletion mutant of BAS1812 B. anthracis showed less survivability in late stationary phase under minimal nutrient condition. In addition, BAS1812-deleted B. anthracis showed defective germination and sporulation, and was more susceptible to cell wall destabilizing agent. Loss of BAS1812 also diminished the ability of B. anthracis to degrade peptidoglycan, suggesting that BAS1812 gene product is a peptidoglycan-degrading enzyme that enhances bacterial long-term survival and cell wall maintenance. As previous literatures mentioned its immunogenicity in the infected host serum, it is feasible that BAS1812 in serum represents bacteremia in the host and that BAS1812 serve as a potential biomarker for anthrax treatment and diagnosis.

Collectively, transcriptional profiles associated with B. anthracis CodY in this dissertation suggests that CodY may induce both positive and negative regulation in B. anthracis gene expression, and it acts as a pioneer factor and modulate its mode of action in response to environmental changes. Being a pleiotropic regulator, CodY regulates various genes with potential therapeutic values for anthrax diagnosis and treatment. These findings from this work provide useful information for studying molecular functions of CodY.