PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome.
PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome.
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DOI：
10.1186/s12934-021-01728-7
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发表时间：
2022-01-05
影响因子：
6.4
通讯作者：
Glycoengineering of Veterinary Vaccines consortium (GoVV)
中科院分区：
文献类型：
--
作者：
Terra VS;Mauri M;Sannasiddappa TH;Smith AA;Stevens MP;Grant AJ;Wren BW;Cuccui J;Glycoengineering of Veterinary Vaccines consortium (GoVV)
弯曲杆菌是一种具有全球重要性的动物和人畜共患病原体，迫切需要有效的疫苗，包括利用保守的多糖抗原的疫苗。为此，我们采用蛋白聚糖偶联技术（PGCT）开发了一种多功能大肠杆菌菌株，能够产生多种糖偶联疫苗候选物来对抗空肠弯曲杆菌。我们构建了一株糖工程大肠杆菌菌株，该菌株含有整合在其染色体上的保守的空肠七糖编码区作为模型聚糖。这种方法有三个优点：(i)减少了用于PGCT的质粒和抗生素标记，（ii）快速生成许多聚糖-蛋白组合，并随后快速鉴定大多数抗原蛋白或肽，以及（iii）增加了多糖编码区的遗传稳定性。在本研究中，通过使用模型聚糖表达菌株，我们能够测试空肠梭菌、铜绿假单胞菌（均为革兰氏阴性）和产气荚膜梭菌（革兰氏阳性）的蛋白质作为受体。利用该pgl整合大肠杆菌菌株，可以很容易地生成四种糖缀合物。两个糖缀合物，其中蛋白质和聚糖都来自空肠梭菌（双打疫苗），以及两个糖缀合物，其中聚糖抗原与来自不同病原体的解毒毒素结合（单打疫苗）。由于针对空肠弧菌的减毒活疫苗（LAVS）下游应用于体温高于42℃的家禽，我们研究了温度对大肠杆菌pgl整合菌株蛋白表达和糖基化的影响。我们确定糖基化依赖于温度，并且对于本研究中使用的七糖和载体的组合，可用于糖基化的PglB水平是糖基化反应的一个步骤限制因素。在体外糖基化实验中，我们还证明温度会影响PglB将底物糖基化的能力，而不受其转录水平的影响。在线版本包含补充材料，可在10.1186/s12934-021-01728-7获得。
Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level. The online version contains supplementary material available at 10.1186/s12934-021-01728-7.
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影响因子：
5.5
作者：
Hatz, Christoph F. R.;Bally, Bettina;Fonck, Veronica Gambillara
通讯作者：
Fonck, Veronica Gambillara
影响因子：
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通讯作者：
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