Using Tn-seq To Identify Pigmentation-Related Genes of Porphyromonas gingivalis: Characterization of the Role of a Putative Glycosyltransferase
Using Tn-seq To Identify Pigmentation-Related Genes of Porphyromonas gingivalis: Characterization of the Role of a Putative Glycosyltransferase
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DOI：
10.1128/jb.00832-16
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发表时间：
2017-07-01
影响因子：
3.2
通讯作者：
Hu, Linden T.
中科院分区：
文献类型：
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作者：
Klein, Brian A.;Cornacchione, Louis P.;Hu, Linden T.
细胞色素沉着是口腔病原菌牙龈卟啉单胞菌的重要毒力因子。色素沉着与许多细菌功能相关，包括但不限于定植、通过结合氧分子维持局部厌氧环境以及防御免疫细胞产生的活性氧（ROS）。色素相关的基因座确定日期涉及脂多糖，菌毛，血红素收购和加工。我们利用牙龈卟啉单胞菌菌株ATCC 33277的转座子突变体文库，并使用转座子连接的大规模平行测序（Tn-seq）筛选色素缺陷菌落以鉴定参与色素沉着的基因。转座子插入在235个不同的位点，位于67个基因和15个基因间区域，导致改变色素沉着：7个基因先前已被证明参与色素沉着，而75个基因和基因间区域没有。为了进一步确认鉴定，我们在牙龈卟啉单胞菌菌株W83中产生了较小的转座子突变体文库，并鉴定了与ATCC 33277中筛选中鉴定的那些相同的几个基因座中的色素突变，但也鉴定了ATCC 33277筛选中未鉴定的八个基因座中的色素突变。PGN_0361/PG_0264是位于两个tRNA合成酶基因之间并与微型反向重复转座元件相邻的推定糖基转移酶基因，在Tn-seq筛选中鉴定，然后通过靶向缺失和互补进行验证。PGN_0361/PG_0264糖基转移酶中的缺失突变消除色素沉着，调节牙龈菌蛋白酶活性，并改变脂多糖。本研究中确定的其他位点参与色素沉着的机制仍有待确定，但我们的筛选提供了迄今为止最完整的色素沉着基因调查。重要提示牙龈卟啉单胞菌与牙周病的发生和进展有关。一个重要的毒力因子是细菌产生色素的能力。使用转座子文库，我们能够识别参与牙龈卟啉单胞菌色素沉着的已知和新基因。我们确定了一个糖基转移酶，以前没有与色素沉着，这是所需的色素沉着，并确定其参与机制。更好地了解参与色素沉着的基因可能会导致对这一重要毒力特征所涉及的复杂机制的新见解，并可能促进新疗法的开发。
Cellular pigmentation is an important virulence factor of the oral pathogen Porphyromonas gingivalis. Pigmentation has been associated with many bacterial functions, including but not limited to colonization, maintaining a local anaerobic environment by binding oxygen molecules, and defense against reactive oxygen species (ROS) produced by immune cells. Pigmentation-associated loci identified to date have involved lipopolysaccharide, fimbriae, and heme acquisition and processing. We utilized a transposon mutant library of P. gingivalis strain ATCC 33277 and screened for pigmentation-defective colonies using massively parallel sequencing of the transposon junctions (Tn-seq) to identify genes involved in pigmentation. Transposon insertions at 235 separate sites, located in 67 genes and 15 intergenic regions, resulted in altered pigmentation: 7 of the genes had previously been shown to be involved in pigmentation, while 75 genes and intergenic regions had not. To further confirm identification, we generated a smaller transposon mutant library in P. gingivalis strain W83 and identified pigment mutations in several of the same loci as those identified in the screen in ATCC 33277 but also eight that were not identified in the ATCC 33277 screen. PGN_0361/PG_0264, a putative glycosyltransferase gene located between two tRNA synthetase genes and adjacent to a miniature inverted-repeat transposable element, was identified in the Tn-seq screen and then verified through targeted deletion and complementation. Deletion mutations in PGN_0361/PG_0264 glycosyltransferase abolish pigmentation, modulate gingipain protease activity, and alter lipopolysaccharide. The mechanisms of involvement in pigmentation for other loci identified in this study remain to be determined, but our screen provides the most complete survey of genes involved in pigmentation to date.IMPORTANCE P. gingivalis has been implicated in the onset and progression of periodontal disease. One important virulence factor is the bacterium's ability to produce pigment. Using a transposon library, we were able to identify both known and novel genes involved in pigmentation of P. gingivalis. We identified a glycosyltransferase, previously not associated with pigmentation, that is required for pigmentation and determined its mechanism of involvement. A better understanding of the genes involved in pigmentation may lead to new insights into the complex mechanisms involved in this important virulence characteristic and could facilitate development of novel therapeutics.