Neisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme.
Neisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme.
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DOI：
10.1371/journal.ppat.1007080
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发表时间：
2018-07
期刊：
影响因子：
6.7
通讯作者：
Criss AK
中科院分区：
文献类型：
--
作者：
Ragland SA;Humbert MV;Christodoulides M;Criss AK
淋病奈瑟菌（Gc）感染富含抗菌蛋白的粘膜部位，包括细菌细胞壁降解酶溶菌酶。某些革兰氏阴性菌产生结合并抑制溶菌酶的蛋白质抑制剂。在这里，我们确定Ng_1063是一种新的Gc溶菌酶抑制剂，我们根据最近发现的第二种溶菌酶抑制剂Ng_1981来定义它的功能。结果表明，Ng_1063序列和结构与mlic型溶菌酶抑制剂具有同源性。重组Ng_1063抑制了溶菌酶介导的Gc敏感突变体和溶菌酶敏感菌黄体微球菌的杀伤。这种抑制活性依赖于Ng_1063的丝氨酸83和赖氨酸103，预测它们与溶菌酶的活性位点残基相互作用。从完整Gc中共免疫沉淀出溶菌酶Ng_1063和Ng_1981。Ng_1063和Ng_1981蛋白水平也在溶菌酶作用下升高。缺乏ng1063和ng1981的Gc对溶菌酶的杀伤敏感性明显高于野生型和单突变菌。当暴露于富含溶菌酶的人眼泪或唾液时，Δ1981Δ1063 Gc的存活率明显低于野生型，加入重组Ng_1981后，存活得以恢复。Δ1981Δ1063突变体Gc存活在产生溶菌酶的人中性粒细胞存在下也降低。我们发现Ng_1063暴露在Gc表面，而Ng_1981同时在胞内池和胞外释放，这表明Gc在多个空间屏障上利用这两种蛋白来完全中和溶菌酶的活性。总之，这些发现确定了Ng_1063和Ng_1981是Gc防御溶菌酶的关键成分。这些蛋白可能是抗微生物治疗的有吸引力的靶点，目的是使Gc对宿主防御敏感和/或用于疫苗开发，这两者都是对抗耐药淋病的迫切需要。粘膜病原体淋病奈瑟菌已经对几乎所有推荐的抗生素产生了耐药性，目前还没有淋病疫苗。有吸引力的治疗发现靶点包括细菌因子，当灭活时，增强细菌对宿主来源的抗菌成分的敏感性。细菌细胞壁降解酶溶菌酶大量存在于粘膜分泌物和先天免疫细胞中。为了抵抗溶菌酶的杀伤，一些细菌产生与溶菌酶结合并直接抑制其活性的蛋白质。在这里，我们证明了淋病奈瑟菌蛋白Ng_1063的溶菌酶抑制活性。我们发现Ng_1063和另一种最近发现的溶菌酶抑制剂Ng_1981都有助于淋病奈瑟菌对溶菌酶的完全耐药，包括对富含溶菌酶的粘膜分泌物和人类中性粒细胞的耐药。虽然Ng_1063和Ng_1981都是溶菌酶的抑制剂，但它们在序列、生物活性和细胞定位上都是不同的。由于Ng_1063和Ng_1981都是细胞外的，我们认为它们可以作为疫苗和药物的靶点，使Gc对人类抗菌防御变得敏感。
The bacterial pathogen Neisseria gonorrhoeae (Gc) infects mucosal sites rich in antimicrobial proteins, including the bacterial cell wall-degrading enzyme lysozyme. Certain Gram-negative bacteria produce protein inhibitors that bind to and inhibit lysozyme. Here, we identify Ng_1063 as a new inhibitor of lysozyme in Gc, and we define its functions in light of a second, recently identified lysozyme inhibitor, Ng_1981. In silico analyses indicated that Ng_1063 bears sequence and structural homology to MliC-type inhibitors of lysozyme. Recombinant Ng_1063 inhibited lysozyme-mediated killing of a susceptible mutant of Gc and the lysozyme-sensitive bacterium Micrococcus luteus. This inhibitory activity was dependent on serine 83 and lysine 103 of Ng_1063, which are predicted to interact with lysozyme’s active site residues. Lysozyme co-immunoprecipitated with Ng_1063 and Ng_1981 from intact Gc. Ng_1063 and Ng_1981 protein levels were also increased in Gc exposed to lysozyme. Gc lacking both ng1063 and ng1981 was significantly more sensitive to killing by lysozyme than wild-type or single mutant bacteria. When exposed to human tears or saliva, in which lysozyme is abundant, survival of Δ1981Δ1063 Gc was significantly reduced compared to wild-type, and survival was restored upon addition of recombinant Ng_1981. Δ1981Δ1063 mutant Gc survival was additionally reduced in the presence of human neutrophils, which produce lysozyme. We found that while Ng_1063 was exposed on the surface of Gc, Ng_1981 was both in an intracellular pool and extracellularly released from the bacteria, suggesting that Gc employs these two proteins at multiple spatial barriers to fully neutralize lysozyme activity. Together, these findings identify Ng_1063 and Ng_1981 as critical components for Gc defense against lysozyme. These proteins may be attractive targets for antimicrobial therapy aimed to render Gc susceptible to host defenses and/or for vaccine development, both of which are urgently needed against drug-resistant gonorrhea. The mucosal pathogen Neisseria gonorrhoeae has acquired resistance to almost all recommended antibiotics, and no gonorrhea vaccine currently exists. Attractive targets for therapeutic discovery include bacterial factors that, when inactivated, enhance bacterial susceptibility to host-derived antimicrobial components. The bacterial cell wall-degrading enzyme lysozyme is abundant in mucosal secretions and innate immune cells. To resist killing by lysozyme, some bacteria produce proteins that bind to and directly inhibit the activity of lysozyme. Here, we demonstrate lysozyme inhibitory activity in the N. gonorrhoeae protein Ng_1063. We found that both Ng_1063 and a second, recently described lysozyme inhibitor, Ng_1981, contribute to full resistance of N. gonorrhoeae to lysozyme, including resistance to lysozyme-rich mucosal secretions and human neutrophils. Although Ng_1063 and Ng_1981 are both inhibitors of lysozyme, they are distinct in their sequences, biological activities, and cellular localizations. Because both Ng_1063 and Ng_1981 are extracellular, we propose they can be targeted for vaccines and drugs that sensitize Gc to human antimicrobial defenses.
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影响因子：
6.7
作者：
Humbert MV;Awanye AM;Lian LY;Derrick JP;Christodoulides M
通讯作者：
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通讯作者：
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