The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response.IMPORTANCEClostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen's growth.

中文翻译：

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免疫蛋白钙卫蛋白通过限制锌影响艰难梭菌代谢。


肠道内有多种微生物群，它们必须竞争营养物质才能生存，但控制病原体定植的具体限制营养物质尚未明确定义。艰难梭菌定植通常需要事先破坏微生物群，这表明在资源竞争中战胜共生菌对于建立艰难梭菌感染 (CDI) 至关重要。在 CDI 过程中，免疫蛋白钙卫蛋白 (CP) 被释放到肠腔中，以螯合锌 (Zn) 和其他必需营养金属。然而，锌限制对艰难梭菌定植的影响尚不清楚。为了确定艰难梭菌对锌限制的反应，我们对暴露于 CP 的艰难梭菌进行了 RNA 测序。在含有 CP 的培养基中，艰难梭菌上调涉及金属稳态和氨基酸代谢的基因。为了确定感染期间重要的 CP 响应基因，我们测量了小鼠 CDI 模型中选定的艰难梭菌转录本相对于体外表达的丰度。参与硒 (Se) 依赖性脯氨酸发酵的基因转录本在感染期间和对 CP 的反应中增加。脯氨酸发酵基因转录的增加取决于 CP Zn 结合和脯氨酸可用性，但脯氨酸发酵只有在补充 Se 时才会增强。 CP缺陷小鼠不能抑制艰难梭菌脯氨酸发酵依赖性生长，表明CP介导的锌螯合以及有限的硒限制了艰难梭菌脯氨酸发酵。总体而言，这些结果强调了艰难梭菌定植如何依赖于多种营养素的可用性，而多种营养素的丰度受宿主反应的动态影响。重要性艰难梭菌感染（CDI）是抗生素后院内感染的主要原因。 抗生素治疗可能会成功，但多达三分之一的人会反复感染。了解控制艰难梭菌定植的机制对于设计原发性和复发性 CDI 的新疗法至关重要。在这里，我们发现限制营养物质可以控制 CDI 期间艰难梭菌的代谢，并影响病原体的整体适应性。具体来说，免疫蛋白 CP 限制了锌的利用率并增加了脯氨酸发酵所需的艰难梭菌基因的转录。矛盾的是，这导致艰难梭菌脯氨酸发酵减少。这种发酵减少是由于脯氨酸发酵所需的另一种营养物质硒的可用性有限。因此，CP介导的锌限制与低硒水平相结合总体上降低了艰难梭菌在肠道中的适应性。这些结果强调了营养物质可用性如何影响艰难梭菌定植的复杂性，并提供了对驱动病原体生长的关键代谢过程的深入了解。