Abstract：
Bacterial community collapse due to phage infection is a major risk in cheese making processes. As virulent phages are ubiquitous and diverse in milk fermentation factories, the use of phage-resistant lactic acid bacteria (LAB) is essential to obtain high-quality fermented dairy products. The LAB species Streptococcus thermophilus contains two type II-A CRISPR-Cas systems (CRISPR1 and CRISPR3) that can effectively protect against phage infection. However, virulent streptococcal phages carrying anti-CRISPR proteins (ACR) that block the activity of CRISPR-Cas systems have emerged in yogurt and cheese environments. For example, phages carrying AcrIIA5 can impede both CRISPR1 and CRISPR3 systems, while AcrIIA6 stops only CRISPR1. Here, we explore the activity and diversity of a third streptococcal phage anti-CRISPR protein, namely AcrIIA3. We were able to demonstrate that AcrIIA3 is efficiently active against the CRISPR3-Cas system of S. thermophilus. We used AlphaFold2 to infer the structure of AcrIIA3 and we predicted that this new family of functional ACR in virulent streptococcal phages has a new α-helical fold, with no previously identified structural homologs. Because ACR proteins are being explored as modulators in genome editing applications, we also tested AcrIIA3 against SpCas9. We found that AcrIIA3 could block SpCas9 in bacteria but not in human cells. Understanding the diversity and functioning of anti-defence mechanisms will be of importance in the design of long-term stable starter cultures.
摘要：
由于噬菌体感染导致的细菌群落崩溃是奶酪制作过程中的主要风险。由于有毒噬菌体在牛奶发酵工厂中无处不在且多样化，使用耐噬菌体的乳酸菌（LAB）对于获得高质量的发酵乳制品至关重要。LAB物种嗜热链球菌包含两个II-A型CRISPR-Cas系统（CRISPR1和CRISPR3），可以有效防止噬菌体感染。然而,在酸奶和奶酪环境中出现了携带抗CRISPR蛋白（ACR）的强毒链球菌噬菌体，这些蛋白阻断了CRISPR-Cas系统的活性。例如,携带AcrIIA5的噬菌体可以阻碍CRISPR1和CRISPR3系统，而AcrIIA6只停止CRISPR1。这里,我们探索了第三种链球菌噬菌体抗CRISPR蛋白的活性和多样性，即AcrIIA3。我们能够证明AcrIIA3对嗜热链球菌的CRISPR3-Cas系统具有有效活性。我们使用AlphaFold2来推断AcrIIA3的结构，并且我们预测这一新的功能性ACR家族在毒力链球菌噬菌体中具有新的α螺旋折叠，没有以前鉴定的结构同源物。因为ACR蛋白在基因组编辑应用中被用作调节剂，我们还针对SpCas9测试了AcrIA3。我们发现AcrIA3可以阻断细菌中的SpCas9，但不能阻断人细胞中的SpCas9。了解反防御机制的多样性和功能对于设计长期稳定的发酵剂文化至关重要。
