Klein Brian A, Cornacchione Louis P, Collins Marisha, Malamy Michael H, Duncan Margaret J, Hu Linden T
Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts, USA.
Department of Microbiology, The Forsyth Institute, Cambridge, Massachusetts, USA.
J Bacteriol. 2017 Jun 27;199(14). doi: 10.1128/JB.00832-16. Print 2017 Jul 15.
Cellular pigmentation is an important virulence factor of the oral pathogen 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 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 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. 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 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.
细胞色素沉着是口腔病原体的一种重要毒力因子。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抵御免疫细胞产生的活性氧（ROS）。迄今为止确定的与色素沉着相关的基因座涉及脂多糖、菌毛以及血红素的获取和加工。我们利用牙龈卟啉单胞菌菌株ATCC 33277的转座子突变体文库，并通过对转座子连接点进行大规模平行测序（Tn-seq）来筛选色素沉着缺陷菌落，以鉴定参与色素沉着的基因。位于67个基因和15个基因间区域的235个不同位点的转座子插入导致色素沉着改变：其中7个基因先前已被证明与色素沉着有关，而75个基因和基因间区域此前未知。为进一步确认鉴定结果，我们在牙龈卟啉单胞菌菌株W83中构建了一个较小的转座子突变体文库，并在与ATCC 33277筛选中鉴定出的几个相同基因座中发现了色素突变，但也有8个在ATCC 33277筛选中未被鉴定出来。PGN_0361/PG_0264是一个假定的糖基转移酶基因，位于两个tRNA合成酶基因之间，且与一个微型反向重复转座元件相邻，在Tn-seq筛选中被鉴定出来，随后通过靶向缺失和互补进行了验证。PGN_0361/PG_0264糖基转移酶的缺失突变消除了色素沉着，调节了牙龈蛋白酶的活性，并改变了脂多糖。本研究中鉴定出的其他基因座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对参与色素沉着基因最全面的调查。牙龈卟啉单胞菌与牙周病的发生和发展有关。一个重要的毒力因子是该细菌产生色素的能力。通过使用转座子文库，我们能够鉴定出参与牙龈卟啉单胞菌色素沉着已知和新的基因。我们鉴定出一种以前与色素沉着无关的糖基转移酶，它是色素沉着所必需的，并确定了其参与机制。更好地理解参与色素沉着的基因可能会为这一重要毒力特征所涉及的复杂机制带来新的见解，并可能促进新型治疗方法的开发。
