3. Bacteriophages and Cognitive Processing
3.噬菌体与认知处理
To study the interplay between the gut phageome, the gut bacteriome, and executive function, which is one of the six key domains of cognition, Mayneris-Perxachs et al. performed fecal shotgun metagenomics and metabolomics analyses in four human cohorts [128]. They also tested mice that had received fecal microbiota transplantations and Drosophila melanogaster after bacteriophage supplementation. All human participants and animals underwent cognitive tests. As a result of these studies, it was observed that the level of tailed bacteriophages previously classified in the—now abolished—order of Caudovirales (now replaced with the class Caudoviricetes [70,71]) was negatively correlated with trial-making test B time (TMTB) (a neuropsychological test that involves visual scanning and working memory and examines the executive functions of the brain), while the level of Microviridae was positively correlated. In TMTB, participants had to connect numbers and letters in an alternating progressive sequence, which means 1 to A, A to 2, 2 to B, and so on, with precision and time being the main priorities in this test. Higher completion times indicate worse executive functions [129,130]. Sex was also identified as a significant factor when it comes to gut microbiota composition [131]. It was observed that women had lower scores in TMTB, which also correlated with higher Caudovirales levels. Surprisingly, no associations were observed in men. Mayneris-Perxachs et al. also performed the backward digit span test, which measures working memory to assess the participant’s ability to hold information in short-term memory and manipulate that information to produce results [132]. In this case, a higher score indicates better executive performance. This time no association was observed in women. The genomic analysis confirmed that most of the Caudovirales were uncultured and uncharacterized, and from those that could be identified, mostly phages infecting Lactococcus spp., Enterobacteriaceae, Firmicutes (e.g., Eubacterium rectale), or Bacteroidetes (Prevotella copri) were detected. These specific Caudovirales phages belong to the three described families, Siphoviridae, Demerecviridae, and Drexlerviridae, which comprised the former Siphoviridae family. As for the new genome-based Siphoviridae family, a negative association between this family and TMTB results was found. On the contrary, Siphoviridae were associated with better performance in the Stroop test (which measures the ability to inhibit cognitive interference that occurs when the processing of a specific stimulus feature impedes the simultaneous processing of a second stimulus) [133] and both short- and long-term memory [128]. Microviridae phages were associated with significant impairment in executive function in both women and men. Bacteriophages from this family were negatively correlated with Lactobacillus, Streptococcus, and Enterococcus, while they were positively linked to Bacteroides and Prevotella species. Conversely, the Caudovirales were positively associated with lactic acid bacteria. These viruses were supposedly responsible for better executive functions and information processing speed, but also for long- and short-term verbal memory and general cognition, but surprisingly, these correlations were only observed in men [128]. Researchers also found a connection between the specific level of Caudovirales and consumption of dietary products and a fat diet containing medium-chain fatty acids, which is in line with findings that supplementation of medium-chain fatty acids improved synaptic plasticity and cognitive function, both in humans and in mice [134,135]. The studies confirmed that a high sugar diet and high uptake of fructose or exposure to SCFAs increases levels of prophages, which can affect bacterial population by two possible mechanisms: firstly, bacteria are killed as a result of prophage induction and entering the lytic cycle, and secondly, the resulting phage progeny can then kill closely related species of bacteria, thus modulating the bacterial community and levels of metabolites they produce [102,136,137].
为了研究肠道噬菌体组、肠道细菌组和执行功能（认知的六个关键领域之一）之间的相互作用，Mayneris-Perxachs 等人对四个人类队列进行了粪便枪元基因组学和代谢组学分析[ 128]。他们还测试了接受粪便微生物群移植的小鼠和补充噬菌体后的黑腹果蝇。所有人类参与者和动物都接受了认知测试。这些研究的结果表明，以前被归入噬尾病毒纲（现已被噬尾病毒目取代[70, 71]）的噬尾细菌的水平与试作测试 B 时间（TMTB）（一种神经心理学测试，涉及视觉扫描和工作记忆，考察大脑的执行功能）呈负相关，而微小病毒科的水平则呈正相关。在 TMTB 中，受试者必须以交替渐进的顺序将数字和字母连接起来，即 1 到 A、A 到 2、2 到 B，依此类推。完成时间越长，说明执行功能越差[129, 130]。性别也被认为是影响肠道微生物群组成的一个重要因素[131]。据观察，女性的 TMTB 分数较低，这也与 Caudovirales 水平较高有关。令人惊讶的是，在男性中没有观察到相关性。Mayneris-Perxachs 等人还进行了后向数字跨度测试，该测试测量工作记忆，以评估受试者将信息保存在短时记忆中并操作该信息以产生结果的能力[132]。在这种情况下，得分越高表明执行能力越强。这次在女性中没有观察到任何关联。基因组分析证实，大多数 Caudovirales 都是未培养和未定性的，而在那些可以确定的噬菌体中，大部分都检测到了感染乳球菌属、肠杆菌科、固执菌属（如直肠杆菌）或类杆菌科（Prevotella copri）的噬菌体。这些特异的 Caudovirales 噬菌体属于三个已描述的科，即 Siphoviridae、Demerecviridae 和 Drexlerviridae，它们组成了前 Siphoviridae 科。至于基于基因组的新 Siphoviridae 科，发现该科与 TMTB 结果呈负相关。相反，Siphoviridae 与在 Stroop 测试（该测试测量抑制认知干扰的能力，当处理特定刺激特征时会阻碍同时处理第二个刺激）[133] 以及短期和长期记忆[128] 中表现较好有关。 微小病毒科噬菌体与女性和男性的执行功能明显受损有关。该家族的噬菌体与乳酸杆菌、链球菌和肠球菌呈负相关，而与乳酸杆菌和普雷沃特氏菌呈正相关。相反，Caudovirales 与乳酸菌呈正相关。据称，这些病毒不仅能提高执行功能和信息处理速度，还能提高长期和短期口头记忆力和一般认知能力，但令人惊讶的是，这些相关性只在男性中观察到[128]。研究人员还发现，Caudovirales 的特定水平与食用含有中链脂肪酸的膳食产品和脂肪饮食之间存在联系，这与补充中链脂肪酸可改善突触可塑性和认知功能的研究结果一致，无论是在人类还是在小鼠身上都是如此[ 134, 135]。这些研究证实，高糖饮食、果糖摄入量高或接触 SCFAs 会增加噬菌体的水平，而噬菌体可通过两种可能的机制影响细菌数量：首先，噬菌体诱导细菌进入溶菌循环而被杀死；其次，由此产生的噬菌体后代可杀死密切相关的细菌种类，从而调节细菌群落及其产生的代谢物水平[102, 136, 137]。
To study the interplay between the gut phageome, the gut bacteriome, and executive function, which is one of the six key domains of cognition, Mayneris-Perxachs et al. performed fecal shotgun metagenomics and metabolomics analyses in four human cohorts [128]. They also tested mice that had received fecal microbiota transplantations and Drosophila melanogaster after bacteriophage supplementation. All human participants and animals underwent cognitive tests. As a result of these studies, it was observed that the level of tailed bacteriophages previously classified in the—now abolished—order of Caudovirales (now replaced with the class Caudoviricetes [70,71]) was negatively correlated with trial-making test B time (TMTB) (a neuropsychological test that involves visual scanning and working memory and examines the executive functions of the brain), while the level of Microviridae was positively correlated. In TMTB, participants had to connect numbers and letters in an alternating progressive sequence, which means 1 to A, A to 2, 2 to B, and so on, with precision and time being the main priorities in this test. Higher completion times indicate worse executive functions [129,130]. Sex was also identified as a significant factor when it comes to gut microbiota composition [131]. It was observed that women had lower scores in TMTB, which also correlated with higher Caudovirales levels. Surprisingly, no associations were observed in men. Mayneris-Perxachs et al. also performed the backward digit span test, which measures working memory to assess the participant’s ability to hold information in short-term memory and manipulate that information to produce results [132]. In this case, a higher score indicates better executive performance. This time no association was observed in women. The genomic analysis confirmed that most of the Caudovirales were uncultured and uncharacterized, and from those that could be identified, mostly phages infecting Lactococcus spp., Enterobacteriaceae, Firmicutes (e.g., Eubacterium rectale), or Bacteroidetes (Prevotella copri) were detected. These specific Caudovirales phages belong to the three described families, Siphoviridae, Demerecviridae, and Drexlerviridae, which comprised the former Siphoviridae family. As for the new genome-based Siphoviridae family, a negative association between this family and TMTB results was found. On the contrary, Siphoviridae were associated with better performance in the Stroop test (which measures the ability to inhibit cognitive interference that occurs when the processing of a specific stimulus feature impedes the simultaneous processing of a second stimulus) [133] and both short- and long-term memory [128]. Microviridae phages were associated with significant impairment in executive function in both women and men. Bacteriophages from this family were negatively correlated with Lactobacillus, Streptococcus, and Enterococcus, while they were positively linked to Bacteroides and Prevotella species. Conversely, the Caudovirales were positively associated with lactic acid bacteria. These viruses were supposedly responsible for better executive functions and information processing speed, but also for long- and short-term verbal memory and general cognition, but surprisingly, these correlations were only observed in men [128]. Researchers also found a connection between the specific level of Caudovirales and consumption of dietary products and a fat diet containing medium-chain fatty acids, which is in line with findings that supplementation of medium-chain fatty acids improved synaptic plasticity and cognitive function, both in humans and in mice [134,135]. The studies confirmed that a high sugar diet and high uptake of fructose or exposure to SCFAs increases levels of prophages, which can affect bacterial population by two possible mechanisms: firstly, bacteria are killed as a result of prophage induction and entering the lytic cycle, and secondly, the resulting phage progeny can then kill closely related species of bacteria, thus modulating the bacterial community and levels of metabolites they produce [102,136,137].
为了研究肠道噬菌体组、肠道细菌组和执行功能（认知的六个关键领域之一）之间的相互作用，Mayneris-Perxachs 等人对四个人类队列进行了粪便枪元基因组学和代谢组学分析[ 128]。他们还测试了接受粪便微生物群移植的小鼠和补充噬菌体后的黑腹果蝇。所有人类参与者和动物都接受了认知测试。这些研究的结果表明，以前被归入噬尾病毒纲（现已被噬尾病毒目取代[70, 71]）的噬尾细菌的水平与试作测试 B 时间（TMTB）（一种神经心理学测试，涉及视觉扫描和工作记忆，考察大脑的执行功能）呈负相关，而微小病毒科的水平则呈正相关。在 TMTB 中，受试者必须以交替渐进的顺序将数字和字母连接起来，即 1 到 A、A 到 2、2 到 B，依此类推。完成时间越长，说明执行功能越差[129, 130]。性别也被认为是影响肠道微生物群组成的一个重要因素[131]。据观察，女性的 TMTB 分数较低，这也与 Caudovirales 水平较高有关。令人惊讶的是，在男性中没有观察到相关性。Mayneris-Perxachs 等人还进行了后向数字跨度测试，该测试测量工作记忆，以评估受试者将信息保存在短时记忆中并操作该信息以产生结果的能力[132]。在这种情况下，得分越高表明执行能力越强。这次在女性中没有观察到任何关联。基因组分析证实，大多数 Caudovirales 都是未培养和未定性的，而在那些可以确定的噬菌体中，大部分都检测到了感染乳球菌属、肠杆菌科、固执菌属（如直肠杆菌）或类杆菌科（Prevotella copri）的噬菌体。这些特异的 Caudovirales 噬菌体属于三个已描述的科，即 Siphoviridae、Demerecviridae 和 Drexlerviridae，它们组成了前 Siphoviridae 科。至于基于基因组的新 Siphoviridae 科，发现该科与 TMTB 结果呈负相关。相反，Siphoviridae 与在 Stroop 测试（该测试测量抑制认知干扰的能力，当处理特定刺激特征时会阻碍同时处理第二个刺激）[133] 以及短期和长期记忆[128] 中表现较好有关。 微小病毒科噬菌体与女性和男性的执行功能明显受损有关。该家族的噬菌体与乳酸杆菌、链球菌和肠球菌呈负相关，而与乳酸杆菌和普雷沃特氏菌呈正相关。相反，Caudovirales 与乳酸菌呈正相关。据称，这些病毒不仅能提高执行功能和信息处理速度，还能提高长期和短期口头记忆力和一般认知能力，但令人惊讶的是，这些相关性只在男性中观察到[128]。研究人员还发现，Caudovirales 的特定水平与食用含有中链脂肪酸的膳食产品和脂肪饮食之间存在联系，这与补充中链脂肪酸可改善突触可塑性和认知功能的研究结果一致，无论是在人类还是在小鼠身上都是如此[ 134, 135]。这些研究证实，高糖饮食、果糖摄入量高或接触 SCFAs 会增加噬菌体的水平，而噬菌体可通过两种可能的机制影响细菌数量：首先，噬菌体诱导细菌进入溶菌循环而被杀死；其次，由此产生的噬菌体后代可杀死密切相关的细菌种类，从而调节细菌群落及其产生的代谢物水平[102, 136, 137]。
4. Possible Application of Bacteriophages in the Treatment of CNS-Related Diseases
4.噬菌体在治疗中枢神经系统相关疾病中的可能应用
4.1. CNS Infections 4.1.中枢神经系统感染
Bacteria, viruses, fungi, and parasites have different strategies for invading the CNS. Among bacteria infecting the CNS, the most common are Mycobacterium spp., Brucella spp., Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, Borrelia burgdorferi, Staphylococcus aureus, and E. coli [138,139,140]. The spectrum of bacterial infections is wide, ranging from brain abscesses and septicemia to meningoencephalitis, and they can result from hematogenous spread from another site of the patient’s body, contiguous spread from the upper airways, injury, or surgery. Some bacteria, such as M. tuberculosis, can enter the CNS via lymphatic nodes [141]. For newborns, the most threatening bacteria are Streptococcus agalactiae and E. coli, followed by S. pneumoniae and N. meningitidis in the first weeks of life [142]. As an effect of bacterial infection, a specific cascade of events is initiated. In particular, events in the brain significantly modulate the feedback effect on the gut microbiota [19,143,144].
细菌、病毒、真菌和寄生虫入侵中枢神经系统的策略各不相同。在感染中枢神经系统的细菌中，最常见的是分枝杆菌属、布鲁氏菌属、单核细胞增生李斯特菌、脑膜炎奈瑟菌、肺炎链球菌、流感嗜血杆菌、布氏杆菌、金黄色葡萄球菌和大肠杆菌[138, 139, 140]。细菌感染的范围很广，从脑脓肿和败血症到脑膜脑炎都有可能，可由患者身体其他部位的血源性传播、上呼吸道的毗连传播、外伤或手术引起。有些细菌，如结核杆菌，可通过淋巴结进入中枢神经系统[141]。对新生儿来说，最具威胁性的细菌是无乳链球菌和大肠杆菌，其次是肺炎双球菌和脑膜炎双球菌[142]。细菌感染会引发一系列特定事件。特别是，大脑中的事件会显著调节肠道微生物群的反馈效应[19, 143, 144]。
There are three main strategies for neurotropic pathogens to cross the BBB: (i) transcellular migration; (ii) paracellular migration; or (iii) a Trojan horse mechanism [145]. The mechanism of the transcellular entrance is based on the pathogen’s ability to bind to BMECs. The pathogen is then endocytosed, transported within the vacuole through BMECs, and finally released to the brain tissue. In the paracellular mechanism, the pathogen disrupts tight junctions and/or induces the production of pro-inflammatory cytokines, e.g., TNFα, IL-6, or IL-1β. Bacterial infection induces an inflammatory response via glial mediators, which are crucial in the communication between the host’s immune system and the brain, but also between the gut microbiota and the brain [146,147]. In the Trojan horse mechanism, pathogens infect phagocytes and—hidden inside them—cross the BBB and infect the CNS. Several bacteria, including E. coli, group B streptococci, S. pneumoniae, N. meningitidis, L. monocytogenes, and M. tuberculosis, cross the BBB transcellularly, but L. monocytogenes and M. tuberculosis can also cross the BBB via the Trojan horse mechanism [148,149].
神经病原体穿过 BBB 的主要策略有三种：(i) 跨细胞迁移；(ii) 细胞旁迁移；或 (iii) 木马机制[ 145]。跨细胞入口机制基于病原体与 BMECs 结合的能力。然后病原体被内吞，通过 BMECs 在液泡内运输，最后释放到脑组织。在细胞旁机制中，病原体会破坏紧密连接和/或诱导产生促炎细胞因子，如 TNFα、IL-6 或 IL-1β。细菌感染通过神经胶质介质诱导炎症反应，神经胶质介质在宿主免疫系统与大脑之间以及肠道微生物群与大脑之间的交流中至关重要[146, 147]。在特洛伊木马机制中，病原体感染吞噬细胞，并隐藏在吞噬细胞内穿过 BBB 感染中枢神经系统。包括大肠杆菌、B 组链球菌、肺炎双球菌、脑膜炎双球菌、单核细胞增多症和结核杆菌在内的几种细菌可经细胞穿过 BBB，但单核细胞增多症和结核杆菌也可通过特洛伊木马机制穿过 BBB [ 148, 149]。
Viruses can infect the CNS either by directly traversing the BBB through one of the three routes described above or by taking nonhematogenous routes. Figure 2 presents possible routes of penetrating the BBB and entering the CNS by bacteriophages. This can be achieved by retrograde axonal transport from peripheral nerves to the CNS, the nasal olfactory epithelium, and neurons. These viruses can stimulate the production of pro-inflammatory cytokines just like bacteria, but they were also reported to stimulate the production of metalloproteases in BMECs, astrocytes, and pericytes. As an effect of the activity of these enzymes, tight junctions can be destabilized by initiating the RhoA kinase pathway and enhancing the permeability of BMEC monolayers in vitro [150,151].
病毒既可以通过上述三种途径之一直接穿越 BBB 感染中枢神经系统，也可以通过非血源性途径感染中枢神经系统。图 2 列出了噬菌体穿透 BBB 进入中枢神经系统的可能途径。噬菌体可通过轴突逆行运输从外周神经进入中枢神经系统、鼻嗅上皮细胞和神经元。与细菌一样，这些病毒也能刺激促炎细胞因子的产生，但据报道，它们还能刺激BMECs、星形胶质细胞和周细胞产生金属蛋白酶。由于这些酶的活性，可通过启动 RhoA 激酶途径破坏紧密连接的稳定性，并增强体外 BMEC 单层的通透性[150, 151]。
Figure 2. Possible routes of entering the CNS by bacteriophages. (A) Intranasal administration; (a) transcellular pathway; (b) intracellular pathway. (B) Entering from circulation through the BBB by either transcellular pathway or by the Trojan horse mechanism.
图 2.噬菌体进入中枢神经系统的可能途径。(A) 经鼻给药；(a) 经细胞途径；(b) 细胞内途径。(B) 通过跨细胞途径或特洛伊木马机制从血液循环中通过 BBB 进入中枢神经系统。