BACKGROUND Moraxella catarrhalis is a leading cause of otitis media (OM) and chronic obstructive pulmonary disease (COPD). M. catarrhalis contains a Type III DNA adenine methyltransferase (ModM) that is phase-variably expressed (i.e., its expression is subject to random, reversible ON/OFF switching). ModM has six target recognition domain alleles (modM1-6), and we have previously shown that modM2 is the predominant allele, while modM3 is associated with OM. Phase-variable DNA methyltransferases mediate epigenetic regulation and modulate pathogenesis in several bacteria. ModM2 of M. catarrhalis regulates the expression of a phasevarion containing genes important for colonization and infection. Here we describe the phase-variable expression of modM3, the ModM3 methylation site and the suite of genes regulated within the ModM3 phasevarion. RESULTS Phase-variable expression of modM3, mediated by variation in length of a 5'-(CAAC)n-3' tetranucleotide repeat tract in the open reading frame was demonstrated in M. catarrhalis strain CCRI-195ME with GeneScan fragment length analysis and western immunoblot. We determined that ModM3 is an active N6-adenine methyltransferase that methylates the sequence 5'-ACm6ATC-3'. Methylation was detected at all 4446 5'-ACATC-3' sites in the genome when ModM3 is expressed. RNASeq analysis identified 31 genes that are differentially expressed between modM3 ON and OFF variants, including five genes that are involved in the response to oxidative and nitrosative stress, with potential roles in biofilm formation and survival in anaerobic environments. An in vivo chinchilla (Chinchilla lanigera) model of otitis media demonstrated that transbullar challenge with the modM3 OFF variant resulted in an increased middle ear bacterial load compared to a modM3 ON variant. In addition, co-infection experiments with NTHi and M. catarrhalis modM3 ON or modM3 OFF variants revealed that phase variation of modM3 altered survival of NTHi in the middle ear during early and late stage infection. CONCLUSIONS Phase variation of ModM3 epigenetically regulates the expression of a phasevarion containing multiple genes that are potentially important in the progression of otitis media.

中文翻译：

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卡他莫拉氏菌相变DNA甲基转移酶ModM3是一种表观遗传调节剂，可影响中耳炎体内模型中细菌的存活。

背景技术卡他莫拉菌是中耳炎（OM）和慢性阻塞性肺疾病（COPD）的主要原因。卡他莫拉氏菌包含III型DNA腺嘌呤甲基转移酶（ModM），其相位可变地表达（即其表达受到随机，可逆的ON / OFF转换的影响）。ModM具有六个目标识别域等位基因（modM1-6），并且我们先前已经证明modM2是主要等位基因，而modM3与OM相关。相变DNA甲基转移酶介导表观遗传调控，并调节几种细菌的发病机理。卡他莫拉氏菌的ModM2调节一个含有对定殖和感染很重要的基因的相变蛋白的表达。在这里，我们描述了modM3的相变表达，ModM3甲基化位点以及在ModM3相变内调节的基因组。结果通过GeneScan片段长度分析和Western分析证实了卡他莫拉氏菌CCRI-195ME菌株在开放阅读框中通过5'-（CAAC）n-3'四核苷酸重复序列的长度变化介导的modM3的相变表达。免疫印迹。我们确定ModM3是一种活性N6-腺嘌呤甲基转移酶，可甲基化序列5'-ACm6ATC-3'。表达ModM3时，在基因组的所有4446个5'-ACATC-3'位点都检测到了甲基化。RNASeq分析确定了在modM3 ON和OFF变体之间差异表达的31个基因，包括与氧化应激和亚硝化应激有关的5个基因，它们在厌氧环境中生物膜形成和存活中具有潜在作用。中耳炎的体内黄鼠（Chinchilla lanigera）模型表明，与modM3 ON变体相比，modM3 OFF变种的经小脑挑战导致中耳细菌负荷增加。此外，使用NTHi和粘膜炎莫德氏菌modM3 ON或modM3 OFF变体进行的共感染实验表明，modM3的相变会改变早期和晚期感染期间中耳NTHi的存活率。结论ModM3的相变在表观遗传上调节了包含多个基因的相变的表达，这些基因在中耳炎的发展中可能是重要的。卡他莫拉modM3 ON或modM3 OFF变体表明，在早期和晚期感染期间，modM3的相变改变了中耳NTHi的存活。结论ModM3的相变在表观遗传上调节了包含多个基因的相变的表达，这些基因在中耳炎的发展中可能是重要的。卡他性菌modM3 ON或modM3 OFF变体表明，在早期和晚期感染期间，modM3的相变改变了中耳NTHi的存活。结论ModM3的相变在表观遗传上调节了包含多个基因的相变的表达，这些基因在中耳炎的发展中可能是重要的。