프로테옴 분석을 통한 탄저 병원성 관련 단백질의 동정

Abstract

Bacillus anthracis the causative agent of anthrax has been subjected for the extensive study to understand the cellular and physiological characteristics that enhance to elucidate the underlying mechanism of the disease. Toxin gene regulation in B. anthracis is controlled by a complex mechanism and that is not fully understood. Coordination of several plasmid and chromosome encoded regulators with the host related signals is essential for the positive regulation of the virulence factors. In addition, the anthrax toxin activator AtxA serves as a key regulatory element by positive regulation of the toxin and the antiphagocytic poly-γ-D-glutamic acid capsule genes are essential to B. anthracis virulence. The activity of AtxA is also regulated by multiple regulatory circuits. Transcription of atxA is dependent on a dual promoter and repressed by the transition state regulator AbrB, which, in turn, is regulated by components of the sporulation phosphorelay. The activity of AtxA protein is posttranslationally regulated through phosphorylation and dephosphorylation of two histidine residues. Transcriptional repression of the AtxA regulatory protein by two small c-type cytochromes results in down-regulation of toxin genes expression. CodY a global regulator of stationary phase gene also controls the toxin gene expression by regulating AtxA accumulation posttranslationally. Expression of B. anthracis virulence factors is stimulated by conditions suggestive of the host environment. Temperature and CO2 levels mimic the host related signals affecting the transcription of AtxA and toxin genes. Optimal toxin expression levels occur at 37 ° C in media supplemented with bicarbonate, in vitro conditions thought to mimic those of the mammalian host. The proteome components (both cellular and secretome) of B. anthracis have been subjected for recent study in order to identify functionally active genes, subcellular proteins, disease-specific proteins, and immunoreactive proteins. To identify virulence associated proteins, we have employed two B. anthracis strains in the presence or absence of plasmid pXO1 which carries the anthrax toxin genes and the master transcriptional regulator AtxA. Additionally, low (28 ° C) temperatures and host mimic temperatures (37 ° C) were introduced during growth that affects both AtxA and toxin expression. In our first series of experiments, strain and temperature regulated secretome proteins were analyzed. Differential expression of several plasmid and chromosomal proteins enabled us to identify a poll of proteins documented as a virulence factor and potential vaccine candidate of B. anthracis and of other bacteria. Cellular proteins of two B. anthracis strains (Sterne and Δ Sterne) were subjected for our second series of study while bacterial cells were grown in host mimic conditions or induced toxin expression conditions. Under the same growing conditions, differentially expressed proteins in two strains were identified through two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF MS) proposed to be regulated by the plasmid encoded regulator including AtxA. A large proportion of the identified proteins were functionally related with cellular metabolism, DNA binding activity, ion binding proteins, amino acid biosynthetic process, and energy production. Several stress response proteins and the proteins documented as virulence factors either in B. anthracis or in other pathogenic bacteria species were also identified in our study in addition to the secretome proteins. Local DNA topology such as intrinsic curvature is associated with the regulatory region of three anthrax classical toxin genes and appears to be a common trait of most bacterial toxins or virulence gene promoters. In the third and final series of our study we have employed several B. anthracis genes encoding as a putative virulence factor identified by our first and second series of experiments. Both in silico and in vitro analyses suggest the presence of intrinsic DNA curvature associated with these genes promoting factors. No evidence shown regarding AtxA dependent regulation of these genes suggests that such DNA topology might be associated with temperature dependent regulation of these genes. Taken together these results suggest that a large number of cellular and extracellular proteins including immunogenic and putative virulence factors in B. anthracis are regulated by the plasmid encoded regulators such as AtxA and environmental signals such as temperature or synergistically both of them. Additionally, the presence of similar DNA topology in promoter region of these genes evoked by the temperature resembles the classical toxin genes, and consequently might play a role in recognition of the promoters by the transcription machinery and / or trans-acting regulators such as AtxA.