微生物学报

Type of sequencing techniques	Advantages	Disadvantages	References
RNA-seq	(1) Quantitative accuracy (2) Wide detection range (3) High accuracy (4) Good repeatability (5) Used to whole genome analysis	rRNA and mitochondrial RNA removal methods are limited and prone to introduce potential errors	[20-22,25-26]
Dual RNA-seq	(1) Detect gene expression of pathogenic bacteria and host cells or tissues simultaneously (2) Can be used for small clinical samples analysis	(1) When sequencing a large number of tissue samples, it is difficult to detect gene expression differences about specific cell type (2) Long term sample processing impairs the integrity of the sample transcriptome	[29-31,33,36]
scRNA-seq	Solved the problem of cellular heterogeneity in organizational samples	(1) There is a loss of spatial information regarding the infection niche during the process of organizational separation (2) High cost	[41-44]

Type of sequencing techniques	Advantages	Disadvantages	References
RNA-seq	(1) Quantitative accuracy (2) Wide detection range (3) High accuracy (4) Good repeatability (5) Used to whole genome analysis	rRNA and mitochondrial RNA removal methods are limited and prone to introduce potential errors	[20-22,25-26]
Dual RNA-seq	(1) Detect gene expression of pathogenic bacteria and host cells or tissues simultaneously (2) Can be used for small clinical samples analysis	(1) When sequencing a large number of tissue samples, it is difficult to detect gene expression differences about specific cell type (2) Long term sample processing impairs the integrity of the sample transcriptome	[29-31,33,36]
scRNA-seq	Solved the problem of cellular heterogeneity in organizational samples	(1) There is a loss of spatial information regarding the infection niche during the process of organizational separation (2) High cost	[41-44]

Predictive tools and links	Theory	Aim	References
RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi)	Dynamic programming-based algorithms, both the classical minimum free energy (MFE) methods and partition function methods	Predict RNA secondary structure	[46-47]
BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)	Base on seeding-and- extending method and Simith-waterman algorithm to generate high scoring segement pairs, finally evaluate the reliability of the comparison	Search similar sequences, structures, and infer the function of unknown sequences	[48-49]
miRanda microRNA target scan (https://www.targetscan.org/vert_72/)	Base on the principle of sequence complementarity, conservative seed match sequence was found that matched to the target 3′UTR, and the target of miRNA was further screened according to thermodynamic stability	Simulate microRNA to obtain potential host targets of sRNA	[50]
IntaRNA 2.0 (http://rna.informatik.uni-freiburg.de/IntaRNA)	Incorporate seed constraints and interaction site accessibility	Predict RNA-RNA hybrids	[51]

Predictive tools and links	Theory	Aim	References
RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi)	Dynamic programming-based algorithms, both the classical minimum free energy (MFE) methods and partition function methods	Predict RNA secondary structure	[46-47]
BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)	Base on seeding-and- extending method and Simith-waterman algorithm to generate high scoring segement pairs, finally evaluate the reliability of the comparison	Search similar sequences, structures, and infer the function of unknown sequences	[48-49]
miRanda microRNA target scan (https://www.targetscan.org/vert_72/)	Base on the principle of sequence complementarity, conservative seed match sequence was found that matched to the target 3′UTR, and the target of miRNA was further screened according to thermodynamic stability	Simulate microRNA to obtain potential host targets of sRNA	[50]
IntaRNA 2.0 (http://rna.informatik.uni-freiburg.de/IntaRNA)	Incorporate seed constraints and interaction site accessibility	Predict RNA-RNA hybrids	[51]

靶向宿主致病菌sRNA的发现及其靶标确定的实验技术进展

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信英蓉 ¹^,² , 杜昕颖 ² , 杨明娟 ² , 宋宏彬 ²^,^* , 王立贵 ²^,^*

微生物学报 | 综述 2024,64(4): 1019-1030

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微生物学报 | 综述 2024, 64(4): 1019-1030

靶向宿主致病菌sRNA的发现及其靶标确定的实验技术进展

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信英蓉¹^,², 杜昕颖², 杨明娟², 宋宏彬²^,^*, 王立贵²^,^*

作者信息

1 郑州大学公共卫生学院, 河南郑州 450001

2 解放军疾病预防控制中心, 北京 100071

Progress in experimental techniques for the discovery and target determination of pathogen sRNAs targeting hosts

Yingrong XIN¹^,², Xinying DU², Mingjuan YANG², Hongbin SONG²^,^*, Ligui WANG²^,^*

Affiliations

1 College of Public Health, Zhengzhou University, Zhengzhou 450001, Henan, China

2 The Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China

出版时间: 2024-04-04 doi: 10.13343/j.cnki.wsxb.20230670

文章导航

摘要

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人类时常暴露于充满各种致病菌的环境中，这些致病菌与人体细胞或组织之间存在多种相互作用。在相互作用的过程中，细菌通过调节自身毒性、侵袭性等致病性，以适应宿主环境并生存下来，同样，宿主细胞也会通过调动自身的免疫系统来抵抗致病菌的入侵。然而，大多数研究者主要聚焦于致病菌sRNA (small RNA, sRNA)自身生理功能的研究，致病菌与宿主相互作用的认识仍然处于起步阶段。因此，如何使用高灵敏性、高分辨率的方法研究致病菌与宿主之间的相互作用成为当前研究面临的一大难题。本文综合国内外相关研究，概述了目前研究致病菌与宿主相互作用常用的技术方法及实验流程，提高对其机制原理的理解，为致病菌sRNA-宿主靶标的相关研究提供技术参考。

关键词

致病菌sRNA / 宿主靶标 / 相互作用 / 实验技术

Abstract

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People are exposed to environments containing various pathogens, which have multiple interactions with human cells or tissues. Pathogens can survive in the host environment by regulating pathogenic conditions such as virulence and invasiveness. At the same time, host cells resist the invasion of pathogens by mobilizing their own immune system. However, researchers mainly focus on the physiological functions of sRNAs in pathogens and have gained limited knowledge about the interactions between pathogens and hosts. How to use highly sensitive and high-resolution methods to study the interactions between pathogens and hosts have become a major challenge in the current research. By reviewing relevant studies, we summarize the commonly used techniques and experimental processes for studying the interactions between pathogens and hosts, aiming to improve the understanding about the mechanisms and principles of these experimental techniques and provide technical references for the research on interactions between pathogen sRNAs and host targets.

Key words

pathogen sRNA / host target / interaction / experimental technique

引用本文

信英蓉, 杜昕颖, 杨明娟, 宋宏彬, 王立贵. 靶向宿主致病菌sRNA的发现及其靶标确定的实验技术进展. 微生物学报, 2024 , 64 (4) : 1019 -1030 . DOI: 10.13343/j.cnki.wsxb.20230670

Yingrong XIN, Xinying DU, Mingjuan YANG, Hongbin SONG, Ligui WANG. Progress in experimental techniques for the discovery and target determination of pathogen sRNAs targeting hosts[J]. Acta Microbiologica Sinica, 2024 , 64 (4) : 1019 -1030 . DOI: 10.13343/j.cnki.wsxb.20230670

正文

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大多数致病菌小RNA (small RNA, sRNA)长度40−500 nt，主要位于基因间区，目前的研究主要集中在致病菌sRNA本身的生理功能，关于革兰氏阴性菌如大肠杆菌、肠道沙门氏菌及志贺氏菌的研究表明，sRNA在调节适应过程中发挥着广泛作用^[1-3]。相比于革兰氏阴性菌，革兰氏阳性菌的sRNA功能研究较少，近些年来才陆续有研究者发现耐甲氧西林金黄色葡萄球菌、单核细胞增生李斯特氏菌和产气荚膜梭菌的sRNA通过调节相关信使RNA (messenger RNA, mRNA)，进而调控其毒力和代谢^[4-6]。总而言之，目前已知的致病菌sRNA发挥调控作用主要方式是通过RNA-RNA碱基直接互补结合、sRNA-RNA结合的强度和持续时间可能取决于互补碱基对的数量；有部分调控方式是通过RNA-蛋白质相互作用，这两种相互作用方式并不是相互独立的，通过RNA-RNA相互作用发挥作用的sRNA也可以结合蛋白质发挥作用^[2]。然而，目前研究者主要关注致病菌sRNA本身的功能研究，或在宿主环境下对自身的调控作用，近年来才逐渐有相关研究探索致病菌sRNA-宿主靶标的作用机制^[7]。

本综述包括两个主要部分，一部分阐述与宿主相互作用的致病菌sRNA发现的相关技术手段及其演变，另一部分阐述验证sRNA-宿主互作的关键实验，系统地介绍sRNA与宿主相互作用研究的基本流程，以期为sRNA相关研究者提供方法学参考，为致病菌sRNA的发现与致病机制阐明提供参考。

1 与宿主相互作用致病菌sRNA的发现

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1.1 致病菌sRNA发现的技术进展

致病菌sRNA的发现最早始于大肠杆菌，大多数sRNA可调节细菌代谢，因此又被称为调节RNA。聚丙烯酰胺凝胶电泳分析和正磷酸标记总RNA技术是最先用于发现sRNA存在的证据，4.5S和6S RNA就是用该方法最先被鉴定的sRNA^[8]。随后，利用改进的二维凝胶电泳技术发现Spot 42 RNA和10S RNA^[9]，对10S RNA条带进行表征，发现其对应两个大小相似的不同RNA (10Sa和10Sb)，也被称为M2和M1^[10]，上述的sRNAs是在分析邻近蛋白编码基因的转录调控时偶然发现的，随着生物信息学的发展，通过计算机识别候选sRNA成为可能，这是sRNA鉴定的一个重要转折点^[11]。多阵列芯片^[12]可以用于在各种条件下分离的总RNA探测，但该方法只能发现在非常明确条件下表达的sRNA。由于sRNA可以单独编码，也可以在其他基因信息中编码，因此阵列需要全基因组，而不仅仅是开放阅读框(open reading frames, ORFs)或操纵子。总的来说，上述各种方法非常适合于识别已知功能的单一sRNA，具有很强的偶然性，需要大量的重复性实验进行sRNA的筛选，因此，传统发现sRNA的方法逐渐被取代。

目前，靶向宿主的致病菌sRNA的发现通常使用RNA测序(RNA-seq)技术，自从RNA-seq应用于感染生物学领域以来，该技术出现了3个主要阶段。第一阶段是将宿主细胞和细菌物理分离，并分别分析它们的转录组。第二阶段是双RNA测序(dual RNA-seq)，研究者认识到高分辨能力和单核苷酸分辨率对RNA-seq的重要性，使用双RNA测序同时检测和定量来自相互作用的不同生物体的转录本^[13]，提示其在宿主-微生物相互作用中的重要作用^[14-15]。第三个阶段是越来越多的研究者使用单细胞RNA-seq (single-cell RNA-seq, scRNA-seq)来解决致病菌-宿主遇到的细胞异质性问题^[16-17]，目前，这3种RNA测序技术均在应用，如图1所示，本综述对这3种RNA测序方法的工作流程进行简单介绍，研究者根据不同需求可选择不同的测序方式，在国内以RNA-seq应用最为广泛，技术最为成熟，可重复性好，而双RNA-seq和致病菌scRNA-seq国内应用相对较少，主要由于建库及数据分析复杂，现有的方法难以跟上，但是笔者认为，在深入探索致病菌-宿主致病机制过程中，scRNA-seq拥有更好的应用前景。

1.2 RNA-seq技术在挖掘与宿主互作sRNA的应用

RNA-seq是转录组学研究的重要工具，能够动态检测生物基因组的表达，多维度地反映差异变化。刚兴起的RNA-seq技术通常识别位于ORFs、长度大于100 bp的RNA序列，但是其无法检测出短的ORF (长度 < 100 nt)，以及位于非翻译区(untranslated region, UTRs)或非编码RNA序列，最早用于研究致病菌自身的代谢研究。作为转录组学研究的技术基础，RNA-seq技术在不断地更新换代，目前可用于研究占RNA绝大部分的非编码RNA，研究其结构以及在生命活动中的重要调控作用，同时与蛋白组学相互补充，更全面地呈现细胞水平的变化信息。差异RNA-seq (dRNA-seq)的出现优化了原始的RNA-seq技术，目前应用最为广泛，其通过特异性地降解加工过的转录本来富集初始转录本，通过总体定位转录起始、终止位点测序或末端富集RNA-seq等方式，来确定转录终止位点^[18-19]，该方法可以区分初级转录本和经过加工的RNA，同时可以鉴定出短至8−12 nt的产物RNA，该项技术最早应用于革兰氏阴性菌，如肠道沙门氏菌^[20]，随着研究进步其应用范围逐渐扩大至革兰氏阳性菌，如肺炎链球菌^[21]。

RNA-seq通过物理方法获得感染样本中的致病菌，对获得的胞内致病菌进行转录组测序，即选择性地裂解被感染的真核细胞，通过差分离心法收集胞内细菌^[22]。然而，即使对获得的胞内致病菌进行RNA-seq，由于差分离心难以将致病菌与宿主完全分离，使得收获的致病菌掺杂大量宿主细胞的基因信息，因此采用技术手段减少宿主RNA背景的干扰势在必行。对于细菌转录本样本，目前可以使用商业试剂盒来去除真核核糖体RNA (ribosomal RNA, rRNA)^[23]，这被用于小鼠模型^[24]中霍乱弧菌的体内表达研究，以及小鼠结肠组织感染后恢复的体内表达研究^[25]。另外，也可以使用“杂交选择”直接富集细菌转录本，“杂交选择”是将复杂的cDNA样本与特异性的生物素探针共同孵育，在宿主的cDNA背景下，捕获并富集感兴趣的致病菌cDNA，该方法最近应用于特异富集铜绿假单胞菌或结核分枝杆菌mRNA和sRNA以观察其细胞内表达谱^[26]。细菌转录本的富集是RNA测序技术的关键一环，需要开发新的、高效的方法从复杂、多物种样本中富集目标序列，深入揭示病原菌与宿主间的相互作用。

1.3 双RNA-seq技术在挖掘与宿主互作sRNA的应用

双RNA-seq正被广泛应用于研究跨物种的相互作用，首先是细菌病原体和哺乳动物宿主之间的相互作用^[27]，可以同时检测致病菌(细胞内或细胞外)和被感染宿主中的基因表达，解决了致病菌和被感染细胞在感染过程中持续的信号交换问题。双RNA-seq将未进行物理分离的真核生物和细菌一起处理，有望在致病菌-宿主相互作用中发现新的相互依赖关系，全面分析细菌感染期间宿主和病原体基因的表达。Westermann等^[28]率先利用双RNA-seq方法同时分析血清型鼠伤寒沙门氏菌感染期间病原体和宿主的RNA表达，揭示了细菌核糖体调节因子的分子影响，同时发现phop激活的sRNAPinT在细菌内化后短暂控制入侵相关效应因子和毒力基因的表达。随后，双RNA-seq技术用于挖掘感染过程中病原体和宿主细胞的全局转录组学，揭示了沙门氏菌中与感染相关的RNA结合蛋白ProQ结合的sRNA STnc540通过增加ProQ蛋白活性进一步激活宿主的裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号通路^[29]，并表明在巨噬细胞被感染过程中沙门氏菌处于代谢活性状态，能够重新编程宿主细胞的基因表达^[30]。在树突状细胞(dendritic cells, DCs)^[31]同样发现不同类型的沙门氏菌病原体能重塑宿主细胞的基因表达，同时DCs利用铁处理来防御入侵的沙门氏菌。双RNA-seq最先用于研究专性细胞内病原菌，如结核分枝杆菌^[24,32]和恙虫病东方体^[33]，后来逐渐用于研究兼性细胞内病原菌，如金黄色葡萄球菌^[34]、肺炎链球菌^[35]。

双RNA-seq除了用于细胞作为宿主的研究外，在三维肠道组织模型^[36]通过模仿人类胃肠炎的感染模型中，可以观察到在急性鼠伤寒沙门氏菌感染期间的病原体细胞适应和宿主体的反应^[37]。但在组织或器官宿主中往往存在多种类型的细胞，很难发现不同细胞间特异性的基因变化，因此需要新的测序技术来解决这一问题，深入探讨宿主应对致病菌的防御机制。同样，双RNA-seq也面临着宿主背景基因含量高的难题，除在测序后采用技术手段富集外，在测序前还可以使用绿色荧光蛋白(green fluorescence protein, GFP)标记的沙门氏菌菌株，分离出感染细胞和未被感染细胞，对分选后的宿主细胞进行裂解，随后收集宿主和细菌裂解物，迅速提取总RNA^[28,38]。该方法在测序前降低宿主的基因背景，获得足够数量的细菌读数，然而，长时间的细胞分离和细胞分选不利于转录组的完整性，同样带来了极大的技术挑战。

1.4 单细胞RNA-seq技术在挖掘与宿主互作sRNA的应用

单细胞RNA-seq技术是近年来刚兴起的测序技术，开创了转录组学的新时代。随着人们对病原菌-宿主相互作用研究的深入，发现细胞异质性在宿主-病原体相互作用中或许发挥重要作用，细胞异质性的存在会导致治疗失败和慢性、反复感染^[39]。研究表明鼠伤寒沙门氏菌利用不同细胞类型的固有变异性来逃避宿主的免疫压力，并对抗菌素治疗产生耐药性^[40]，因此，迫切需要新的转录组学方法来挖掘细胞异质性如何影响致病菌-宿主细胞相互作用的结果。真核生物scRNA-seq的检测极限较低，而细菌scRNA-seq必须考虑到致病菌mRNA含量远低于真核细胞，同时，细菌mRNA又不稳定，需要迅速完成细胞裂解、分离和随后的RNA提取，因此，scRNA-seq在微生物领域一直未得到广泛应用。

随着最近改良的细菌scRNA-seq^[41]的出现，单细菌转录组学技术现在已经逐渐应用于致病菌的研究。致病菌在与宿主相互作用时存在不同的异质性，在宿主内致病菌间也存在相互竞争，从而一部分菌株在宿主内竞争存活下来，scRNA-seq的出现使研究优势菌株的形成机制成为可能。microSPLiT是一种最早用于革兰氏菌株的高通量scRNA-seq方法，有研究者分析超过25 000个单一枯草芽孢杆菌细胞的基因表达状态，发现在只有0.1%的个体出现异常状态^[42]，揭示了其在碳利用、胁迫反应、金属吸收和发育决策等调控机制的变化。另外有一项研究使用不依赖polyA的多轮退火和dC尾的定量scRNA- seq (quantitative scRNA-seq, MATQ-seq)，对鼠伤寒沙门氏菌和铜绿假单胞菌分离后进行分析^[43]，可以可靠地捕获生长依赖的基因表达模式。BacDrop是最新的一种高度可扩展的细菌单细胞RNA测序技术，其特点是利用通用的核糖体RNA消耗和组合条形码，实现大规模并行测序，应用于肺炎克雷伯菌的临床分离株，阐明其对抗生素应激的异质性反应^[44]。从感染患者中分离出致病菌，进行细菌scRNA-seq有助于研究其体内活性，并提供关于它们何时、如何以及为什么重新激活相关功能的见解。现对上述介绍的3种RNA测序技术的优缺点进行简单比较(表1)。

2 与致病菌sRNA相互作用宿主靶标的确定

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2.1 生物信息学方法预测sRNA靶标

当获得候选sRNA后，对其靶标的预测显得至关重要。研究者通常采用生物信息学方法进行初步预测^[45]，可以选择的预测工具有多种，如表2所示，对常用的几种sRNA靶标预测工具的原理和目的进行介绍，研究者可以使用RNAfold WebServer^[46-47]对sRNAs的二级结构进行预测，使用动态规划算法预测单个序列的最小自由能二级结构(minimum free energy principle, MFE)，利用质心结构评价RNA结构预测效果，质心和MFE结构之间的高度相似性表明预测可靠。随后可以使用BLAST^[48-49]算法将sRNA的互补序列与NCBI人类参考序列进行比对，以确定是否与人类mRNA完美匹配，利用该方法Koeppen等^[46]在铜绿假单胞菌中发现sRNA52320与人类mRNA的潜在相互作用。由于细菌的sRNAs类似于真核生物的微小RNA (microRNAs, miRNA)，同样可以与mRNA靶点不完全结合来抑制翻译，因此，利用miRanda microRNA靶标扫描^[50]可以预测sRNA可能靶向的宿主mRNA。同时，IntaRNA 2.0^[51]也是强大的预测工具，可以预测microRNA样的相互作用，选取合适的截止点作为相互作用的最小自由能，通常选为−8，该方法认为polyA+被预测为细菌RNA的靶标的次数越多，成为真正靶标的概率越高。截至目前，对物种间RNA通信的研究通常将RNA序列相似性作为唯一的参数，仅限于理论上存在相互作用的可能，其预测的结果往往存在争议，故而需要利用实验手段进行预测结果的验证。

2.2 验证sRNA靶标的实验方法

2.2.1 宿主mRNA靶标的确定

宿主靶标mRNA和宿主靶标蛋白往往是研究者关注的重点。靶标调控通常需要宿主mRNA水平上的变化，通过多种方法进行检测，如在单基因水平上的Northern blotting杂交、引物延伸和RT-PCR^[52]。许多研究发现sRNA可能作用于多个靶点，因此在sRNA表征的初始阶段使用微阵列、RNA-seq或蛋白质组学等全局方法变得必要，这些全局性方法可以将与核蛋白Hfq相关的mRNA作为潜在靶点进行分类^[53]。将受调控的mRNA确定为sRNA的直接靶点，就必须证明存在转录后调控的sRNA-mRNA碱基配对，gfp翻译融合的双质粒系统^[54-55]就是方法之一，该方法主要是检测转录后调控，将两个兼容质粒的sRNA和潜在靶标mRNA在同一细胞内共表达，随后被翻译融合到gfp，通过监测荧光来判断二者是否存在碱基配对结合；该系统使用小质粒，可以很容易在sRNA和靶标中引入点突变来影响调控，该方法在大肠杆菌和沙门氏菌^[54,56]中不同靶点的调控中均有应用。还有一种相似的方法^[57-58]是将5′cDNA末端快速扩增(rapid amplification of cDNA end, RACE)介导的5′UTRs克隆到一个lacZ报告基因上，然后对PBAD启动子下游的融合进行染色体整合，lacZ和上述gfp报告基因可以使用荧光激活细胞分选仪(fluorescence activated cell sorter, FACS)分析，快速筛选潜在的sRNA调控因子和编码辅助因子的基因。代偿性碱基对交换是相对新颖的技术，通过将容易出现PCR错误的染色体sRNA基因进行点突变，然后筛选lacZ融合对靶标基因的调控缺失^[59]。双荧光素酶测定实验目前备受研究者喜爱，基本实验流程如图2所示，构建pmirGLO双荧光素酶miRNA载体，利用酶切位点扩增潜在靶标mRNA的UTR区域，并将其克隆到luc2基因附近，随后将sRNA引入克隆到pmirGLO系统中，通过检测荧光素酶活性来判断sRNA与靶标mRNA是否结合；在嗜肺军团菌^[50]研究中，利用该方法发现sRNARsmY与mRNAddx58的UTR和mRNAcRel结合、tRNA-Phe与mRNAddx58和mRNAirak1结合的证据。

2.2.2 宿主蛋白靶标的确定

无论靶标蛋白是被激活还是被抑制，通过聚丙烯酰胺凝胶电泳或Western blotting方法可用于研究sRNA在蛋白水平上的调控，但该方法需要已知蛋白的特异性抗体；如果没有特异性抗体，染色体表位标记方法^[60]可以检测调控蛋白，该方法使用血凝素(hemagglutinin, HA)标记的反向自转运体入侵蛋白(invAHA)作为鼠伤寒沙门氏菌三型分泌系统1 (T1)表达的转录报告基因，使用基于GFP和InvAHA的双报告方法用于T1基因表达分析，发现SPI-1启动子PprgH和PsicA分别对GFP和InvAHA的驱动作用。蛋白质与mRNA的共纯化联合质谱分析也是一个可行的方法，目前已知的与细菌sRNA相互作用的蛋白除了Hfq以外，还可能与sRNA引导mRNA降解复合物中的RNase E^[61]相互作用，有项研究发现色氨酸酶是ColE1质粒编码的Rcd RNA的靶点，利用Rcd与琼脂糖交联，与大肠杆菌裂解液孵育，将总蛋白进行质谱鉴定。通过sTarPicker方法和蛋白质组学分析，发现热休克蛋白(DnaK)可能是福氏志贺菌sRNASsr1的直接靶点，调控其在酸性环境的耐受性^[58]，还有在体外用链霉素载体标记大肠杆菌RNA，并通过亲和层析发现几种相互作用的蛋白，包括聚合酶亚基、Hfq和S1^[62]。上述各种方法提供了有价值的参考，以寻找与sRNA相关或靶向的蛋白质。

sRNA的靶标蛋白发现主要集中在致病菌自身蛋白，尚无关于sRNA直接靶向宿主蛋白的研究，但是方法学都是通用的，可以借鉴上述方法进行相关研究，这也反映了对mRNA靶点研究的偏向性和缺乏生物计算算法来全局预测蛋白质靶点，笔者认为，将RNA-seq技术与蛋白组学结果联合分析，再附加以上实验方法进行验证，或许可以用于致病菌sRNA与宿主靶标蛋白相互作用研究。

3 展望

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目前，致病菌sRNA-宿主靶标相关研究方法处于起步阶段，这些方法仍然有待发展完善，比如双RNA-seq难以区分致病菌是处于宿主细胞质、细胞核还是细胞膜，scRNA-seq的出现解决了具有相同基因组的宿主细胞异质性的问题，但其花费较大且所需要的样本量也较大，对致病菌的应用也刚刚兴起还未成熟，以上各种技术方法并非独立的，还可以联合使用，比如单细胞双RNA-seq在未来或许有很好的应用前景。同时，研究者们也在寻找新的发现和功能表征的技术方法及实验策略，不断优化测序深度，以期发现不稳定存在以及短的sRNA，未来sRNA和基因表达的分析方式依然会发生重大转变。

本文关注的宿主靶点侧重于mRNA和蛋白质，因为这一主题自致病菌-宿主相互作用研究起始未曾有过改变，众多mRNA介导的sRNA活性调控的相关研究也印证该观点。目前宿主mRNA作为致病菌sRNA靶标是研究者的重心，因为相关的全局测序、预测、实验手段都相对完善，笔者认为，由于缺乏可靠的蛋白靶点全局预测方法，sRNA对蛋白质活性的调节目前仍然被低估了。此外，关于CRISPR介导的免疫调节，DNA作为调节RNA活性的靶点尚未得到完全解决，这也提示我们宿主DNA或许也可能是致病菌sRNA潜在调节靶标。在感染沙门氏菌的人类宿主细胞中，长链非编码RNA (long noncoding RNA, lncRNA)表达谱的改变较mRNA更快，而在致病菌中，sRNA通常作为重要调控因子，反映致病菌所处何种宿主环境^[28]，这提示lncRNA未来或许能成为致病菌-宿主相互作用研究的重点。

基金

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国家重点研发计划(2022YFC2602304)

参考文献

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文献

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参考文献引证文献

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2024年第64卷第4期

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doi: 10.13343/j.cnki.wsxb.20230670

接收时间：2023-11-01
首发时间：2026-03-19
出版时间：2024-04-04

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收稿日期：2023-11-01
录用日期：2024-01-29

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National Key Research and Development Program of China(2022YFC2602304)

国家重点研发计划(2022YFC2602304)

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1 郑州大学公共卫生学院, 河南郑州 450001

2 解放军疾病预防控制中心, 北京 100071

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Type of sequencing techniques	Advantages	Disadvantages	References
RNA-seq	(1) Quantitative accuracy (2) Wide detection range (3) High accuracy (4) Good repeatability (5) Used to whole genome analysis	rRNA and mitochondrial RNA removal methods are limited and prone to introduce potential errors	[20-22,25-26]
Dual RNA-seq	(1) Detect gene expression of pathogenic bacteria and host cells or tissues simultaneously (2) Can be used for small clinical samples analysis	(1) When sequencing a large number of tissue samples, it is difficult to detect gene expression differences about specific cell type (2) Long term sample processing impairs the integrity of the sample transcriptome	[29-31,33,36]
scRNA-seq	Solved the problem of cellular heterogeneity in organizational samples	(1) There is a loss of spatial information regarding the infection niche during the process of organizational separation (2) High cost	[41-44]

Type of sequencing techniques

Advantages

Disadvantages

References

RNA-seq

(1) Quantitative accuracy
(2) Wide detection range
(3) High accuracy
(4) Good repeatability
(5) Used to whole genome analysis

rRNA and mitochondrial RNA removal methods are limited and prone to introduce potential errors

[20-22,25-26]

Dual RNA-seq

(1) Detect gene expression of pathogenic bacteria and host cells or tissues simultaneously
(2) Can be used for small clinical samples analysis

(1) When sequencing a large number of tissue samples, it is difficult to detect gene expression differences about specific cell type
(2) Long term sample processing impairs the integrity of the sample transcriptome

[29-31,33,36]

scRNA-seq

Solved the problem of cellular heterogeneity in organizational samples

(1) There is a loss of spatial information regarding the infection niche during the process of organizational separation
(2) High cost

[41-44]

Predictive tools and links	Theory	Aim	References
RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi)	Dynamic programming-based algorithms, both the classical minimum free energy (MFE) methods and partition function methods	Predict RNA secondary structure	[46-47]
BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)	Base on seeding-and- extending method and Simith-waterman algorithm to generate high scoring segement pairs, finally evaluate the reliability of the comparison	Search similar sequences, structures, and infer the function of unknown sequences	[48-49]
miRanda microRNA target scan (https://www.targetscan.org/vert_72/)	Base on the principle of sequence complementarity, conservative seed match sequence was found that matched to the target 3′UTR, and the target of miRNA was further screened according to thermodynamic stability	Simulate microRNA to obtain potential host targets of sRNA	[50]
IntaRNA 2.0 (http://rna.informatik.uni-freiburg.de/IntaRNA)	Incorporate seed constraints and interaction site accessibility	Predict RNA-RNA hybrids	[51]

Predictive tools and links

Theory

Aim

References

RNAfold WebServer
(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi)

Dynamic programming-based algorithms, both the classical minimum free energy (MFE) methods and partition function methods

Predict RNA secondary structure

[46-47]

BLAST
(https://blast.ncbi.nlm.nih.gov/Blast.cgi)

Base on seeding-and- extending method and Simith-waterman algorithm to generate high scoring segement pairs, finally evaluate the reliability of the comparison

Search similar sequences, structures, and infer the function of unknown sequences

[48-49]

miRanda microRNA target scan
(https://www.targetscan.org/vert_72/)

Base on the principle of sequence complementarity, conservative seed match sequence was found that matched to the target 3′UTR, and the target of miRNA was further screened according to thermodynamic stability

Simulate microRNA to obtain potential host targets of sRNA

[50]

IntaRNA 2.0
(http://rna.informatik.uni-freiburg.de/IntaRNA)

Incorporate seed constraints and interaction site accessibility

Predict RNA-RNA hybrids

[51]