Israeli Ma'ayan, Elia Uri, Rotem Shahar, Cohen Hila, Tidhar Avital, Bercovich-Kinori Adi, Cohen Ofer, Chitlaru Theodor
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel.
Front Microbiol. 2019 Feb 18;10:255. doi: 10.3389/fmicb.2019.00255. eCollection 2019.
Anthrax is a lethal disease caused by the Gram-positive spore-producing bacterium . We previously demonstrated that disruption of gene, encoding the chaperone/protease HtrA (High Temperature Requirement A of ) results in significant virulence attenuation, despite unaffected ability of Δ strains (in which the gene was deleted) to synthesize the key anthrax virulence factors: the exotoxins and capsule. Δ strains exhibited increased sensitivity to stress regimens as well as silencing of the secreted starvation-associated Neutral Protease A (NprA) and down-modulation of the bacterial S-layer. The virulence attenuation associated with disruption of the gene was suggested to reflect the susceptibility of Δ mutated strains to stress insults encountered in the host indicating that HtrA represents an important pathogenesis determinant. As all HtrA serine proteases, HtrA exhibits a protease catalytic domain and a PDZ domain. In the present study we interrogated the relative impact of the proteolytic activity (mediated by the protease domain) and the PDZ domain (presumably necessary for the chaperone activity and/or interaction with substrates) on manifestation of phenotypic characteristics mediated by HtrA. By inspecting the phenotype exhibited by Δ strains -complemented with either a wild-type, truncated (ΔPDZ), or non-proteolytic form (mutated in the catalytic serine residue) of HtrA, as well as strains exhibiting modified chromosomal alleles, it is shown that (i) the proteolytic activity of HtrA is essential for its N-terminal autolysis and subsequent release into the extracellular , while the PDZ domain was dispensable for this process, (ii) the PDZ domain appeared to be dispensable for most of the functions related to stress resilience as well as involvement of HtrA in assembly of the bacterial S-layer, (iii) conversely, the proteolytic activity but not the PDZ domain, appeared to be dispensable for the role of HtrA in mediating up-regulation of the extracellular protease NprA under starvation stress, and finally (iv) in a murine model of anthrax, the HtrA PDZ domain, was dispensable for manifestation of virulence. The unexpected dispensability of the PDZ domain may represent a unique characteristic of HtrA amongst bacterial serine proteases of the HtrA family.
炭疽病是由革兰氏阳性产芽孢细菌引起的一种致命疾病。我们之前证明，编码伴侣蛋白/蛋白酶HtrA（的高温需求A）的基因被破坏会导致毒力显著减弱，尽管Δ菌株（其中基因被删除）合成关键炭疽病毒力因子（外毒素和荚膜）的能力未受影响。Δ菌株对应激方案的敏感性增加，同时分泌的饥饿相关中性蛋白酶A（NprA）沉默，细菌S层下调。与基因破坏相关的毒力减弱被认为反映了Δ突变菌株对宿主中遇到的应激损伤的易感性，这表明HtrA是一个重要的致病决定因素。与所有HtrA丝氨酸蛋白酶一样，HtrA具有一个蛋白酶催化结构域和一个PDZ结构域。在本研究中，我们探究了蛋白水解活性（由蛋白酶结构域介导）和PDZ结构域（可能是伴侣蛋白活性和/或与底物相互作用所必需的）对HtrA介导的表型特征表现的相对影响。通过检查用野生型、截短型（ΔPDZ）或非蛋白水解形式（催化丝氨酸残基发生突变）的HtrA互补的Δ菌株以及表现出修饰染色体等位基因的菌株所呈现的表型，结果表明：（i）HtrA的蛋白水解活性对其N端自溶以及随后释放到细胞外是必不可少的，而PDZ结构域对这一过程是可有可无的；（ii）PDZ结构域对于大多数与应激恢复力相关的功能以及HtrA参与细菌S层的组装似乎是可有可无的；（iii）相反，蛋白水解活性而非PDZ结构域，对于HtrA在饥饿应激下介导细胞外蛋白酶NprA上调的作用似乎是可有可无的；最后（iv）在炭疽病小鼠模型中，HtrA的PDZ结构域对于毒力的表现是可有可无的。PDZ结构域出人意料的可有可无性可能代表了HtrA在HtrA家族细菌丝氨酸蛋白酶中独有的特征。
