Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes.
Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes.
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DOI：
10.1128/mbio.02083-21
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发表时间：
2021-12-21
期刊：
影响因子：
6.4
通讯作者：
Novick RP
中科院分区：
文献类型：
--
作者：
Dhasmana N;Ram G;McAllister KN;Chupalova Y;Lopez P;Ross HF;Novick RP
抗菌无人机(ABD)系统的基础是重新定位噬菌体诱导的葡萄球菌致病性岛(SaPI)，用作与抗生素耐药性无关的抗菌剂。通过插入用于四环素抗性(TCR)选择的TetM，将SAPI毒力基因替换为杀菌或抑菌基因，如CRISPR/Cas9/agrA或CRISPR/dCas9/agrP2P3，其表达通过AGRA的双链切割而杀死，或CRISPR/dCas9/agrP2P3，其表达阻断靶生物的毒力。ABD DNA包装在噬菌体颗粒中，在体内和体外攻击他们的葡萄球菌靶标。我们通过转移频率测定ABD滴度，计数存活细胞作为多样性的函数，并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发现，ABD杀死的细胞比转导测量的要多得多。我们对这一现象的研究揭示了ABD系统的几个重要特征：(1)大量进入的ABD DNA分子不能继续建立稳定的转导(即流产)；(2)ABD货物基因在进入后立即表达，即使是流产的ABD；(3)立即电镀在含Tc的琼脂上严重低估了颗粒数量，部分原因是TC抑制蛋白质合成；(4)用CADA(赋予对Cd Cl2的抗性)取代TetM提供了更准确的颗粒计数；(V)表达CRISPR/CAS9/AGRA的ABD可杀死99.99%的感染细胞，并提供最准确的颗粒数量测量以及该系统的原理证明；(Vi)令人惊讶的是，TetM干扰独立于TCR的ABD DNA的稳定建立。
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 tetM for tetracycline resistance (Tcr) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3, whose expression blocks the target organism’s virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets in vivo as well as in vitro. 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 tetM with cadA (conferring resistance to CdCl2) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/cas9/agrA 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 Tcr.