Rose Sasha J, Bermudez Luiz E
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon, USA.
Department of Microbiology, College of Science, Oregon State University, Corvallis, Oregon, USA.
mBio. 2016 Dec 6;7(6):e01597-16. doi: 10.1128/mBio.01597-16.
Extracellular DNA (eDNA) is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM). Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate.
Many bacteria contain extracellular DNA (eDNA) in their biofilm matrix, as it has various biological and physical functions. We recently reported that nontuberculous mycobacteria (NTM) can contain significant quantities of eDNA in their biofilms. In some bacteria, eDNA is derived from dead cells, but that does not appear to be the case for all eDNA-containing organisms, including NTM. In this study, we found that eDNA export in NTM is conditionally dependent on the molecules to which the bacteria are exposed and that bicarbonate positively influences eDNA export. We also identified genes and proteins important for eDNA export, which begins to piece together a description of a mechanism for eDNA. Better understanding of eDNA export can give new targets for the development of antivirulence drugs, which are attractive because resistance to classical antibiotics is currently a significant problem.
细胞外DNA（eDNA）是包括非结核分枝杆菌（NTM）在内的多种病原体生物膜基质的组成部分。细胞裂解是某些细菌中eDNA的来源，但NTM以及其他含eDNA的细菌物种中eDNA的来源仍未明确。在本研究中，研究了影响eDNA输出的条件，并鉴定了参与eDNA输出机制的基因。在建立了一种在未受干扰的生物膜中实时监测eDNA的方法后，研究了影响eDNA的不同条件。碳酸氢盐以不依赖pH的方式对鸟分枝杆菌、脓肿分枝杆菌和龟分枝杆菌中的eDNA输出产生正向影响。含eDNA生物膜中鸟分枝杆菌的表面暴露蛋白质组显示有丰富的碳酸酐酶。用乙氧唑胺对碳酸酐酶进行化学抑制可显著减少eDNA输出。对鸟分枝杆菌中eDNA输出进行的无偏差转座子突变体文库筛选鉴定出许多严重的eDNA输出减弱突变体，包括一个不表达独特的FtsK/SpoIIIE样DNA转运孔的突变体、两个碳酸酐酶失活的突变体、九个属于独特基因组区域的基因失活的突变体，以及许多参与代谢和能量产生的突变体。对包括FtsK/SpoIIIE和碳酸酐酶在内的九个突变体进行互补可显著恢复eDNA输出。有趣的是，一些eDNA输出减弱的突变体在编码通过表面蛋白质组学发现的蛋白质的基因中存在突变，并且更多的突变位于可能编码表面蛋白的操纵子中。总体而言，我们的数据强化了eDNA输出是一种由细菌对碳酸氢盐作出反应而激活的主动机制的证据。
许多细菌在其生物膜基质中含有细胞外DNA（eDNA），因为它具有多种生物学和物理功能。我们最近报道非结核分枝杆菌（NTM）在其生物膜中可含有大量eDNA。在一些细菌中，eDNA来源于死细胞，但对于所有含eDNA的生物体，包括NTM，情况似乎并非如此。在本研究中，我们发现NTM中的eDNA输出有条件地依赖于细菌所接触的分子，并且碳酸氢盐对eDNA输出有正向影响。我们还鉴定了对eDNA输出重要的基因和蛋白质，这开始拼凑出eDNA机制的描述。更好地理解eDNA输出可为抗毒力药物的开发提供新的靶点，由于对传统抗生素的耐药性目前是一个重大问题，抗毒力药物很有吸引力。
