Introduction

In the search for new potential therapies for pathologies of oxalate, such as kidney stone disease and primary hyperoxaluria, the intestinal microbiome has generated significant interest. Resident oxalate-degrading bacteria inhabit the gastrointestinal tract and reduce absorption of dietary oxalate, thereby potentially lowering the potency of oxalate as a risk factor for kidney stone formation. Although several species of bacteria have been shown to degrade oxalate, select strains of Oxalobacter formigenes (O. formigenes) have thus far demonstrated the unique ability among oxalotrophs to initiate a net intestinal oxalate secretion into the lumen from the bloodstream, allowing them to feed on both dietary and endogenous metabolic oxalate. There is significant interest in this function as a potential therapeutic application for circulating oxalate reduction, although its mechanism of action is still poorly understood. Since this species-exclusive, oxalate-regulating function is reported to be dependent on the use of a currently unidentified secreted bioactive compound, there is much interest in whether O. formigenes produces unique biochemicals that are not expressed by other oxalotrophs which lack the ability to transport oxalate. Hence, this study sought to analyze and compare the metabolomes of O. formigenes and another oxalate degrader, Bifidobacterium animalis subsp. lactis (B. animalis), to determine whether O. formigenes could produce features undetectable in another oxalotroph, thus supporting the theory of a species-exclusive secretagogue compound.

Methods

A comparative metabolomic analysis of O. formigenes strain HC1 (a human isolate) versus B. animalis, another oxalate-degrading human intestinal microbe, was performed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Bacteria were cultured independently in anaerobic conditions, harvested, lysed, and extracted by protein precipitation. Metabolite extracts were chromatographically separated and analyzed by UHPLC-HRMS using reverse phase gradient elution (ACE Excel 2 C18-Pentafluorophenyl column) paired with a Q Exactive™ mass spectrometer.

Objectives

The purpose of this study was to assess whether O. formigenes potentially produces unique biochemicals from other oxalate degraders to better understand its metabolic profile and provide support for the theoretical production of a species-exclusive secretagogue compound responsible for enhancing intestinal oxalate secretion.

Results

We report a panel of metabolites and lipids detected in the O. formigenes metabolome which were undetectable in B. animalis, several of which were identified either by mass-to-charge ratio and retention time matching to our method-specific metabolite library or MS/MS fragmentation. Furthermore, re-examination of data from our previous work showed most of these features were also undetected in the metabolomes of Lactobacillus acidophilus and Lactobacillus gasseri, two other intestinal oxalate degraders.

Conclusions

Our observation of O. formigenes metabolites and lipids which were undetectable in other oxalotrophs suggests that this bacterium likely holds the ability to produce biochemicals not expressed by at least a selection of other oxalate degraders. These findings provide support for the hypothesized biosynthesis of a species-exclusive secretagogue responsible for the stimulation of net intestinal oxalate secretion.

Graphic abstract

中文翻译：

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Oxalobacter formigenes 产生代谢物和脂质在草食性动物双歧杆菌中检测不到

介绍

在寻找针对草酸盐疾病（例如肾结石病和原发性高草酸尿症）的新潜在疗法时，肠道微生物组引起了人们极大的兴趣。常驻的草酸盐降解细菌栖息在胃肠道并减少膳食草酸盐的吸收，从而可能降低草酸盐作为肾结石形成危险因素的效力。尽管细菌的几个种类已显示劣化草酸盐，选择的菌株产甲酸草酸杆菌formigenes（Ò。formigenes) 迄今为止已经证明了草酸营养生物的独特能力，可以启动从血液中将肠道草酸盐净分泌到管腔中，使它们能够以饮食和内源性代谢草酸盐为食。尽管其作用机制仍知之甚少，但人们对该功能作为循环草酸盐还原的潜在治疗应用具有重要意义。由于据报道这种物种独有的草酸盐调节功能依赖于目前未鉴定的分泌性生物活性化合物的使用，因此对O是否存在很大兴趣。formigenes产生独特的生化物质，这些生化物质不被其他缺乏运输草酸盐能力的草酸营养生物所表达。因此，本研究试图分析和比较哦。formigenes和另一种草酸盐降解剂，动物双歧杆菌亚种。球菌（乙。animalis），以确定是否Ò。formigenes可以产生在另一种草酸营养生物中无法检测到的特征，从而支持物种专有的促分泌素化合物的理论。

方法

O 的比较代谢组学分析。formigenes菌株 HC1（人类分离株）与B . Animalis是另一种降解草酸盐的人类肠道微生物，通过超高效液相色谱-高分辨率质谱 (UHPLC-HRMS) 进行分析。细菌在厌氧条件下独立培养，收获、裂解并通过蛋白质沉淀提取。通过 UHPLC-HRMS 使用反相梯度洗脱（ACE Excel 2 C18-五氟苯基柱）与 Q Exactive™ 质谱仪配对，对代谢物提取物进行色谱分离和分析。

目标

本研究的目的是评估O . formigenes可能从其他草酸盐降解剂中产生独特的生化物质，以更好地了解其代谢特征，并为负责增强肠道草酸盐分泌的物种专属促泌剂化合物的理论生产提供支持。

结果

我们报告了一组在O 中检测到的代谢物和脂质。在B中检测不到的formigenes代谢组。Animalis，其中一些是通过质荷比和保留时间与我们的方法特定代谢物库或 MS/MS 碎裂相匹配来鉴定的。此外，从我们以前的工作数据的复检结果显示大部分的这些特点也未被发现中的代谢组嗜酸乳杆菌和乳杆菌，其他两个肠道草酸盐降解菌。

结论

我们对O 的观察。formigenes代谢物和脂质在其他草酸营养生物中检测不到，这表明这种细菌可能具有产生至少一些其他草酸盐降解剂不表达的生化物质的能力。这些发现为负责刺激净肠道草酸分泌的物种专属促泌剂的假设生物合成提供了支持。

图形摘要