Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USA.
Division of Oral Biology and Medicine, School of Dentistry, University of California, Los Angeles, California, USA.
Mol Oral Microbiol. 2022 Oct;37(5):206-217. doi: 10.1111/omi.12365. Epub 2022 Mar 25.
Actinomyces oris plays an important role in oral biofilm development. Like many gram-positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA-a genetic suppressor of srtA deletion lethality-not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild-type cells, under elevated concentrations of mono- and divalent cations the formation of mono- and multi-species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, compared to the wild-type strain. Altogether, the results support the notion that the cell wall-anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris.
口腔放线菌在口腔生物膜的形成中起着重要作用。与许多革兰氏阳性菌一样，口腔放线菌产生大量表面蛋白，这些蛋白通过一种名为管家天冬酰胺转肽酶 SrtA 的保守转肽酶锚定在细菌肽聚糖上；然而，许多口腔放线菌表面蛋白在生物膜形成中的生物学作用在很大程度上是未知的。在这里，我们报告了糖蛋白 GspA-一种 srtA 缺失致死性的遗传抑制剂-不仅促进生物膜的形成，而且在阳离子胁迫下维持细胞膜的完整性。与野生型细胞相比，突变细胞在缺乏 gspA 的情况下形成单种和多种生物膜的能力在单种和二价阳离子浓度升高时显著降低，尽管这两种细胞类型在存在阳离子的情况下浮游生长是无法区分的。由于 gspA 的过表达对缺乏 gspA 和 srtA 的细胞是致命的，我们进行了遗传筛选以鉴定涉及细胞活力的 GspA 决定因素。对有活力克隆的 DNA 测序和生化特性分析表明，两个关键半胱氨酸残基和一个丝氨酸残基的突变严重影响了 GspA 的糖基化和生物膜的形成。此外，缺乏 gspA 的突变细胞对十二烷基硫酸钠（一种溶解细胞质膜的去污剂）明显敏感，这表明 gspA 突变细胞的细胞包膜发生了改变。与这一观察结果一致的是，与野生型菌株相比，gspA 突变体表现出增加的膜通透性，而与 GspA 糖基化无关。总的来说，这些结果支持了这样一种观点，即细胞壁锚定的糖蛋白 GspA 为口腔放线菌促进的生物膜发展中抵抗阳离子胁迫提供了一种防御机制。
