Metabolism of sialic acid by Bifidobacterium breve UCC2003

Bifidobacteria constitute a specific group of commensal bacteria which inhabit the gastrointestinal tract of humans and other mammals. Bifidobacterium breve UCC2003 has previously been shown to utilise several plant-derived carbohydrates that include cellodextrins, starch and galactan. In the current study, we investigate the ability of this strain to utilise the mucin- and human milk oligosaccharide (HMO)-derived carbohydrate, sialic acid. Using a combination of transcriptomic and functional genomic approaches, we identified a gene cluster dedicated to the uptake and metabolism of sialic acid. Furthermore, we demonstrate that B. breve UCC2003 can cross feed on sialic acid derived from the metabolism of 3’ sialyllactose, a HMO, by Bifidobacterium bifidum PRL2010. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 (O'Connell Motherway et al., 2011) were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.

双歧杆菌（Bifidobacteria）是一类定殖于人类及其他哺乳动物胃肠道的特异性共生菌。此前已有研究证实，短双歧杆菌UCC2003（Bifidobacterium breve UCC2003）可利用多种植物源碳水化合物，包括纤维糊精（cellodextrins）、淀粉及半乳聚糖（galactan）。本研究针对该菌株利用黏蛋白（mucin-）与母乳低聚糖（human milk oligosaccharide, HMO）来源的碳水化合物——唾液酸（sialic acid）的能力展开探究。本研究结合转录组学（transcriptomic）与功能基因组学（functional genomic）手段，鉴定出一个负责唾液酸摄取与代谢的基因簇（gene cluster）。此外，本研究证实，短双歧杆菌UCC2003可利用两歧双歧杆菌PRL2010（Bifidobacterium bifidum PRL2010）代谢母乳低聚糖3’唾液酸乳糖（3’ sialyllactose）所产生的唾液酸，实现交叉喂养。

本研究使用的DNA微阵列（DNA-microarrays）包含针对短双歧杆菌UCC2003全基因组1864个注释基因的寡核苷酸引物（oligonucleotide primers），该微阵列由安捷伦科技公司（Agilent Technologies，美国加利福尼亚州帕洛阿尔托市）设计并提供（O'Connell Motherway等，2011）。细胞裂解、RNA提取（RNA isolation）、RNA质量检测（RNA quality control）、互补DNA（cDNA）合成与标记的实验方法参照此前发表的方案（Pokusaeva等，2009）。标记后的cDNA使用安捷伦基因表达杂交试剂盒（Agilent Gene Expression hybridization kit，货号5188-5242），按照安捷伦双色微阵列基因表达分析手册v4.0（Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual，货号G4140-90050）中的步骤进行杂交。杂交完成后，微阵列按照安捷伦标准流程进行洗涤，并使用安捷伦DNA微阵列扫描仪（Agilent DNA microarray scanner，型号G2565A）进行扫描。扫描得到的图像通过安捷伦特征提取软件（Feature Extraction software，版本9.5）转换为数据文件。DNA微阵列数据的预处理参照此前发表的方案（Garcia De La Nava等，2003）。差异表达分析采用基于t检验变体的Cyber-T分析方法（Long等，2001）。当P值小于0.001且相对于对照组的表达倍数比大于3或小于0.33时，判定该基因存在差异表达。