Deacetylation of sialic acid by esterases potentiates pneumococcal neuraminidase activity for mucin utilization, colonization and virulence.
Deacetylation of sialic acid by esterases potentiates pneumococcal neuraminidase activity for mucin utilization, colonization and virulence.
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DOI：
10.1371/journal.ppat.1006263
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发表时间：
2017-03
期刊：
影响因子：
6.7
通讯作者：
Yesilkaya H
中科院分区：
文献类型：
--
作者：
Kahya HF;Andrew PW;Yesilkaya H
肺炎球菌神经氨酸酶是一种关键酶，用于宿主聚糖的顺序去糖基化，并且在宿主存活、定殖和肺炎链球菌引起的感染的发病机制中起重要作用。可以影响神经氨酸酶活性的因素之一是其底物唾液酸中存在的乙酰化的量和位置。我们假设肺炎球菌酯酶通过去除唾液酸的乙酰化作用增强神经氨酸酶活性，这将对肺炎球菌在粘蛋白、定植和毒力上的存活产生重大影响。这些假设进行了测试，使用同基因突变体和重组酯酶在微生物，生物化学和体内试验。我们发现肺炎球菌酯酶活性至少由四个基因编码，SPD_0534（EstA）被发现负责主要的酯酶活性，并且肺炎球菌酯酶对短酰基链具有特异性。使用天然底物的酯酶活性测定表明，Axe和EstA酯酶都可以使用乙酰化木聚糖和牛颌下粘蛋白（BSM），一种高度乙酰化的底物，但只有EstA对三丁酸甘油酯（甘油三酯）有活性。用Axe或EstA孵育BSM导致乙酸盐以时间和浓度依赖性方式释放，用任一酶预处理BSM增加随后暴露于神经氨酸酶A时的唾液酸释放。qRT-PCR结果显示，estA和axe在BSM染毒后和呼吸道组织中的表达水平均升高。estA单独突变或与nanA（神经氨酸酶A的编码）组合，或将其推定的丝氨酸活性位点替换为丙氨酸，降低了肺炎球菌在肺炎球菌肺炎小鼠模型中利用BSM作为唯一碳源的能力、唾液酸释放、定殖和毒力。神经氨酸酶活性对于肺炎球菌定殖和毒力至关重要，因为它是有效切割宿主聚糖以满足营养需求、附着和微生物通过生物膜移位所必需的。末端唾液酸的修饰，如O-乙酰化，会影响神经氨酸酶的效力。在这项研究中，我们调查是否肺炎球菌酯酶可以增强神经氨酸酶的活性，通过去乙酰化唾液酸。我们发现，肺炎球菌酯酶活性编码的至少四个基因，具体的短酰基链酯，和乙酰化的酯酶的去除增强肺炎球菌神经氨酸酶活性的粘蛋白利用，定植和毒力。因此，本研究阐明了宿主去糖基化对肺炎球菌定植和毒力的复杂性和重要性，并揭示了治疗干预的潜在靶点。
Pneumococcal neuraminidase is a key enzyme for sequential deglycosylation of host glycans, and plays an important role in host survival, colonization, and pathogenesis of infections caused by Streptococcus pneumoniae. One of the factors that can affect the activity of neuraminidase is the amount and position of acetylation present in its substrate sialic acid. We hypothesised that pneumococcal esterases potentiate neuraminidase activity by removing acetylation from sialic acid, and that will have a major effect on pneumococcal survival on mucin, colonization, and virulence. These hypotheses were tested using isogenic mutants and recombinant esterases in microbiological, biochemical and in vivo assays. We found that pneumococcal esterase activity is encoded by at least four genes, SPD_0534 (EstA) was found to be responsible for the main esterase activity, and the pneumococcal esterases are specific for short acyl chains. Assay of esterase activity by using natural substrates showed that both the Axe and EstA esterases could use acetylated xylan and Bovine Sub-maxillary Mucin (BSM), a highly acetylated substrate, but only EstA was active against tributyrin (triglyceride). Incubation of BSM with either Axe or EstA led to the acetate release in a time and concentration dependent manner, and pre-treatment of BSM with either enzyme increased sialic acid release on subsequent exposure to neuraminidase A. qRT-PCR results showed that the expression level of estA and axe increased when exposed to BSM and in respiratory tissues. Mutation of estA alone or in combination with nanA (codes for neuraminidase A), or the replacement of its putative serine active site to alanine, reduced the pneumococcal ability to utilise BSM as a sole carbon source, sialic acid release, colonization, and virulence in a mouse model of pneumococcal pneumonia. Neuraminidase activity is critical for pneumococcal colonization and virulence as it is required for efficient cleavage of host glycans for nutritional requirements, attachment, and translocation of the microbe through biological membranes. Modifications, such as O-acetylation, in terminal sialic acid can affect the potency of neuraminidase. In this study we investigated whether pneumococcal esterases could potentiate neuraminidase activity by de-acetylating sialic acid. We found that the pneumococcal esterase activity is coded by at least four genes, specific for short acyl chain esters, and the removal of acetylation by esterases potentiates pneumococcal neuraminidase activity for mucin utilisation, colonization and virulence. Hence, this study elucidates the complexity and importance of host de-glycosylation for pneumococcal colonization and virulence, and reveals a potential target for therapeutic intervention.