Genetic Manipulation of Corynebacterium mastitidis to Better Understand the Ocular Microbiome.
Genetic Manipulation of Corynebacterium mastitidis to Better Understand the Ocular Microbiome.
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乳腺棒状杆菌的基因操作以更好地了解眼部微生物组。
DOI：
10.1167/iovs.64.2.19
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发表时间：
2023-02-01
影响因子：
4.4
通讯作者：
中科院分区：
文献类型：
--
作者：
棒状杆菌属是通常与眼表面相关的革兰氏阳性细菌。从小鼠眼睛中分离出乳房棒状杆菌，并证明其诱导有益的免疫应答，可以保护眼睛免受病原性感染。因为眼睛相关的棒状杆菌属。没有得到很好的描述，我们生成了一个C。肥大转座子（Tn）突变体库，以更好地了解眼睛定植细菌的性质。用定制的基于Tn5的转座子进行Tn诱变，所述转座子掺入荧光蛋白mCherry的无启动子基因。我们使用流式细胞术和酶测定筛选我们的文库，以鉴定证明我们方法的实用性的有用突变体。mCherry+细菌的荧光激活细胞分选（FACS）使我们能够鉴定出使用显微镜在鼠眼表面上可检测到的高度荧光突变体。我们还鉴定了不能水解尿素的功能性敲除，UreaseKO。虽然尿酸是泪液中产生的抗微生物因子，但UreaseKO细菌保持了定殖眼睛的能力，表明定殖不需要尿素水解。在体外和体内，与野生型C相比，两种突变体都保持了刺激保护性免疫的潜力。桅杆总之，我们描述了一种方法，基因改造的眼睛殖民微生物，C。桅杆此外，本文概述的程序将允许继续开发用于修饰眼棒状杆菌属的遗传工具，这将导致更全面地了解眼表面微生物组和宿主免疫之间的相互作用。
Corynebacterium spp. are Gram-positive bacteria commonly associated with the ocular surface. Corynebacterium mastitidis was isolated from mouse eyes and was demonstrated to induce a beneficial immune response that can protect the eye from pathogenic infection. Because eye-relevant Corynebacterium spp. are not well described, we generated a C. mast transposon (Tn) mutant library to gain a better understanding of the nature of eye-colonizing bacteria. Tn mutagenesis was performed with a custom Tn5-based transposon that incorporated a promoterless gene for the fluorescent protein mCherry. We screened our library using flow cytometry and enzymatic assays to identify useful mutants that demonstrate the utility of our approach. Fluorescence-activated cell sorting (FACS) of mCherry+ bacteria allowed us to identify a highly fluorescent mutant that was detectable on the murine ocular surface using microscopy. We also identified a functional knockout that was unable to hydrolyze urea, UreaseKO. Although uric acid is an antimicrobial factor produced in tears, UreaseKO bacterium maintained an ability to colonize the eye, suggesting that urea hydrolysis is not required for colonization. In vitro and in vivo, both mutants maintained the potential to stimulate protective immunity as compared to wild-type C. mast. In sum, we describe a method to genetically modify an eye-colonizing microbe, C. mast. Furthermore, the procedures outlined here will allow for the continued development of genetic tools for modifying ocular Corynebacterium spp., which will lead to a more complete understanding of the interactions between the microbiome and host immunity at the ocular surface.
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微生物群对铜绿假单胞菌诱导的角膜炎的抗性。
影响因子：
6.7
作者：
Kugadas A;Christiansen SH;Sankaranarayanan S;Surana NK;Gauguet S;Kunz R;Fichorova R;Vorup-Jensen T;Gadjeva M
通讯作者：
Gadjeva M
DOI：
10.1016/j.jtos.2016.04.004
发表时间：
2016-07
期刊：
The ocular surface
影响因子：
--
作者：
Kugadas A;Gadjeva M
通讯作者：
Gadjeva M
影响因子：
32.4
作者：
Sakamoto K;Jin SP;Goel S;Jo JH;Voisin B;Kim D;Nadella V;Liang H;Kobayashi T;Huang X;Deming C;Horiuchi K;Segre JA;Kong HH;Nagao K
通讯作者：
Nagao K
影响因子：
2.1
作者：
Nolden, L;Beckers, G;Burkovski, A
通讯作者：
Burkovski, A
影响因子：
3.4
作者：
Ozkan, Jerome;Majzoub, Marwan E.;Willcox, Mark
通讯作者：
Willcox, Mark