Department of Microbiology, New York University School of Medicinegrid.201076.2, New York, New York, USA.
Cytometry and Cell Sorting Laboratory, New York University School of Medicinegrid.201076.2, New York, New York, USA.
mBio. 2021 Dec 21;12(6):e0208321. doi: 10.1128/mBio.02083-21. Epub 2021 Nov 16.
The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting for tetracycline resistance (Tc) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/, whose expression kills by double-strand cleavage of , or CRISPR/, whose expression blocks the target organism's virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets as well as . We determine ABD titers by transfer frequency, enumerate surviving cells as a function of multiplicity, and analyze the fate of ABD DNA with green fluorescent protein. An initial study revealed surprisingly that many more cells were killed by the ABD than were measured by transduction. Our study of this phenomenon has revealed several important features of the ABD system: (i) a significant number of entering ABD DNA molecules do not go on to establish stable transductants (i.e., are abortive); (ii) ABD cargo genes are expressed immediately following entry, even by the abortive ABDs; (iii) immediate plating on Tc-containing agar seriously underestimates particle numbers, partly owing to Tc inhibition of protein synthesis; (iv) replacement of with (conferring resistance to CdCl) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/ kill ∼99.99% of infected cells and provide the most accurate measurement of particle numbers as well as proof of principle for the system; and (vi) surprisingly, TetM interferes with stable establishment of ABD DNA independently of Tc. For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert. We have long used for these purposes but show here that tetM not only underestimates particle titers, by over 20-fold in some experiments, but also seriously impedes stable establishment of the therapeutic particle DNA. Given that is a very convenient and widely used selective marker, publication of these findings is of considerable importance to the microbiological community as well as an interesting illustration of the unpredictable biological effects of genes taken out of their native context.
抗菌无人机 (ABD) 系统基于重新利用噬菌体诱导的葡萄球菌致病性岛 (SaPI) 作为对抗生素耐药性不敏感的抗菌剂。ABD 通过插入四环素抗性 (Tc) 选择的 ，用杀菌或抑菌基因（如 CRISPR/ ）替换 SaPI 毒力基因，其表达通过双链断裂杀死 ，或 CRISPR/ ，其表达阻止靶生物体的毒力。ABD DNA 被包装在噬菌体样颗粒中，这些颗粒攻击其葡萄球菌靶标 以及 。我们通过转移频率确定 ABD 滴度，根据倍数计数存活细胞，并使用绿色荧光蛋白分析 ABD DNA 的命运。一项初步研究令人惊讶地表明，被 ABD 杀死的细胞数量远远超过通过转导测量的数量。我们对这一现象的研究揭示了 ABD 系统的几个重要特征：（i）进入 ABD 的大量 DNA 分子不会继续建立稳定的转导子（即，是流产的）；（ii）ABD 货物基因在进入后立即表达，即使是流产的 ABD 也是如此；（iii）立即在含有 Tc 的琼脂平板上进行平板计数严重低估了颗粒数量，部分原因是 Tc 抑制蛋白质合成；（iv）用 （赋予对 CdCl 的抗性）替换 提供了更准确的颗粒计数；（v）表达 CRISPR/的 ABD 杀死 ∼99.99%的感染细胞，并提供了对颗粒数量最准确的测量以及对该系统的原理证明；（vi）令人惊讶的是，TetM 独立于 Tc 干扰 ABD DNA 的稳定建立。对于颗粒状治疗剂，如 ABD，准确计数颗粒对于评估制备程序和计算治疗剂量至关重要。同样重要的是，用于这两个目的的选择性标记应该是生物惰性的。我们长期以来一直使用 用于这些目的，但这里表明 tetM 不仅严重低估了颗粒滴度，在某些实验中超过 20 倍，而且严重阻碍了治疗性颗粒 DNA 的稳定建立。鉴于 是一种非常方便且广泛使用的选择性标记，这些发现的发表不仅对微生物学社区具有重要意义，而且对从其天然环境中取出的基因的不可预测的生物学效应也是一个有趣的说明。
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