嗜麦芽窄食单胞菌表达IV型蛋白质分泌系统(T4SS),该系统可促进其他细菌的接触依赖性杀伤,并部分通过分泌效应子TfcB来实现。这里,我们报告了TfcB的结构,包含类似于糖基水解酶(GH-19)几丁质酶的催化结构域的N-末端结构域和用于被T4SS识别和转运的C-末端结构域。利用双杂交测定法来测量与T4SS偶联蛋白VirD4的效应子相互作用,我们记录了另外五个T4SS底物的存在。其中之一是蛋白质20845,一种带注释的核酸酶。缺乏20845基因的嗜麦芽嗜血杆菌突变体因杀死大肠杆菌而受损,肺炎克雷伯菌,还有铜绿假单胞菌.此外,克隆的20845基因赋予了强大的毒性,当20845与其同源免疫蛋白共表达时,重组大肠杆菌被拯救。20845效应是一种899个氨基酸的蛋白质,在其N端由GHH核酸酶结构域组成,不确定函数的一个大的中心区域,和一个用于分泌的C末端。在预测的催化位点具有突变的20845基因的工程化变体不会阻碍大肠杆菌,表明20845的抗菌作用涉及其核酸酶活性。使用流式细胞术与DNA染色,我们确定20845,但不是它的突变变体,导致目标细菌DNA含量的损失。数据库搜索显示,20845的未表征同源物存在于一系列细菌中。这些数据表明嗜麦芽窄食链球菌T4SS通过多种毒性效应物的作用促进细菌间竞争,包括一种强效的,新型DNase。嗜麦芽窄食单胞菌是一种多重耐药菌,革兰氏阴性细菌,是一种新兴的人类病原体。囊性纤维化患者特别容易感染嗜麦芽窄食链球菌。在医院供水系统和各种类型的感染中,嗜麦芽菌与其他细菌共存,包括铜绿假单胞菌等其他病原体。我们先前证明了嗜麦芽窄食链球菌具有功能性VirB/D4VI型蛋白质分泌系统(T4SS),可促进其他细菌的接触依赖性杀伤。由于大多数关于抗菌系统的工作涉及VI型分泌系统,这一观察仍然值得注意。此外,嗜麦芽窄食链球菌目前单独作为表达抗细菌T4SS的人病原体的模型。利用生化,遗传,和细胞生物学方法,我们现在报道了一种新型抗菌核酸酶(TfdA)的发现和杀菌T4SS效应子(TfcB)的首次结构测定。
Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).