Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis.

中文翻译：

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使用随机转座子突变文库对人早幼粒细胞系中吞噬无形体生长所必需的基因进行生物统计学预测

无形体吞噬细胞 (Ap) 是人类无形体病的病原体，是一种细胞内细菌，可导致北美第二常见的蜱传疾病。为了解决这些病原体缺乏遗传系统的问题，我们使用随机 Himar1 转座子诱变来生成能够在人类早幼粒细胞（HL-60 细胞）中复制的 Ap 突变体库。Illumina 测序确定了 1195 个非随机分布的插入。由于突变体的密度是非饱和的，没有插入的基因要么对 Ap 至关重要，要么随机保留。为了解决这个问题，我们应用了一种生物统计学方法来预测必需基因。由于转座子插入基因组 TA 二核苷酸位点的机会对于所有位点应该是相同的，我们使用马尔可夫链蒙特卡罗模型来估计非突变基因对 Ap 必不可少的概率。预测的必需基因包括那些编码结构核糖体蛋白、参与代谢的酶、IV 型分泌系统成分、抗氧化防御分子和假设蛋白的基因。我们使用计算机后基因组方法来预测对 HL-60 细胞中的 Ap 复制至关重要的基因。这些结果将有助于靶向基因研究它们在人类无形体病发病机制中的作用。我们使用计算机后基因组方法来预测对 HL-60 细胞中的 Ap 复制至关重要的基因。这些结果将有助于靶向基因研究它们在人类无形体病发病机制中的作用。我们使用计算机后基因组方法来预测对 HL-60 细胞中的 Ap 复制至关重要的基因。这些结果将有助于靶向基因研究它们在人类无形体病发病机制中的作用。