铜绿假单胞菌膜囊泡小 RNA PsrR 促进 Rhl 群体感应系统的机制研究

铜绿假单胞菌（Pseudomonas aeruginosa，PA）是一种分布广泛、分泌多种毒力因子、具多重耐药性的条件致病菌。PA的群体感应系统（Quorum sensing, QS）因能调控多种毒力因子而备受关注，其中的一种QS系统Rhl因在病原菌感染中的重要作用而被认为是QS的中央调控中枢。细菌膜囊泡（Membrane vesicles，MV）是近年来发现的细菌组成型分泌的一种纳米囊泡结构，包裹着蛋白质和核酸等多种组分，参与QS等致病过程。非编码小RNA（Small RNA，sRNA）可被包裹进细菌MV中，从而具有潜在靶向结合临近细菌和宿主细胞mRNA的功能、稳定性等多种生理过程，能作为抗菌药物的候选靶点，抑制小RNA的活性能有效的控制致病菌的感染。目前现有研究发现PA能通过QS产生大量MV，保护其内部的小RNA不被降解，但PA MV 富集的小RNA反过来调控QS的工作还很缺乏。

本研究中我们首先通过比较转录组数据挖掘出了PA MV中富集的一个小RNA，命名为PsrR（Pseudomonas aeruginosa sRNA Regulate RhlR）。实验结果表明PsrR可响应PA体内致病部位环境中的低氧信号诱导而高表达，也能通过MV转移到临近生态位的细菌中。PsrR还能与细菌Rhl QS的受体rhlR（C4-HSL receptor）mRNA形成碱基互补配对的相互作用，使鼠李糖脂合成基因rhlABC表达上升，促进RhlR蛋白的表达，最终促进了鼠李糖脂的产量。随后，我们证明了PsrR在提升细菌的群集运动能力的同时，也促进了细菌毒力，导致了更多巨噬细胞的死亡。我们也验证了PsrR在微生物-宿主互作中的作用，发现它还能与宿主人上皮A549细胞中的septin4 mRNA形成碱基互补配对的相互作用，促进Septin4的表达，最终导致细胞的凋亡，具体互作机制有待进一步研究。

综上，本研究全面解析了一个新的在MV中富集的PA小RNA PsrR对细菌自身以及宿主细胞分子靶标的作用机制，为其他研究者们更好地理解铜绿假单胞菌的小RNA以及MV的作用奠定了基础，拓宽了对PA中RNA生物学的认知，并有助于更深入地理解小RNA对于调节铜绿假单胞菌毒力的作用和发现新的抗菌机制。

Pseudomonas aeruginosa (PA) is a widespread, multidrug-resistant, conditionally pathogenic bacterium. PA secretes multiple virulence factors, and its quorum sensing (QS) system plays a key role in regulating multiple virulence factors. One of these QS systems, Rhl, is considered to be the central regulatory hub of QS due to its important role in pathogenic bacterial infections. Bacterial membrane vesicles (MV) are a nanovesicle structures secreted by bacterial constitutive types discovered in recent years that encapsulate a variety of components such as proteins and nucleic acids involved in pathogenic processes such as QS. Small non-coding RNA (sRNA) can be packaged into bacterial MVs and thus have the potential to target binding to adjacent bacterial and host cell mRNAs for a variety of physiological processes such as function, stability, and transcription. Small RNAs can be used as candidate targets for antimicrobial drugs, and inhibition of small RNA activity can effectively control pathogenic bacterial infections. Currently, available studies have shown that PA can produce a large number of MVs via QS to protect its internal small RNAs from degradation, but there is a lack of work on PA MV-enriched small RNAs, which in turn regulate QS.

In this study, we first identified a small RNA enriched in PA MV, named PsrR (Pseudomonas aeruginosa sRNA Regulate RhlR), by comparing transcriptomic data. The results showed that PsrR could be highly expressed in response to the induction of hypoxic signaling in the environment of the PA pathogenic site in vivo, and could also be transferred via MV to bacteria in adjacent ecological niches. PsrR could also form a base-complementary pairing interaction with the mRNA of the bacterial Rhl QS receptor, rhlR (C4-HSL receptor), resulting in the up-regulation of the rhamnolipid synthesizing gene, rhlABC, to promote rhlR protein expression and ultimately rhamnolipid production. Subsequently, we demonstrated that PsrR promoted bacterial virulence while increasing bacterial swarming motility, leading to more macrophage death. We also verified the role of PsrR in microbe-host interactions and found that it also formed base-complementary pairing interactions with SEPTIN4 mRNA in host human epithelial A549 cells, promoting Septin4 expression and ultimately leading to apoptosis, with the exact mechanism of the interactions to be further investigated.

In conclusion, this study comprehensively analyzed the mechanism of action of PsrR, a novel PA small RNA enriched in MV, on the molecular targets of the bacterium itself as well as the host cells, laying the foundation for other researchers to better understand the roles of P. aeruginosa small RNAs as well as MV, expanding the knowledge of RNA biology in PA, and contributing to a deeper understanding of the role of 小RNAs in regulating P. aeruginosa virulence and the discovery of new interaction mechanisms. P. aeruginosa virulence and the discovery of new antimicrobial mechanisms.

[1] MORADALI M F, GHODS S, REHM B H. Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence [J]. Frontiers in cellular and infection microbiology, 2017, 7(39.
[2] DE OLIVEIRA D M P, FORDE B M, KIDD T J, et al. Antimicrobial Resistance in ESKAPE Pathogens [J]. Clinical microbiology reviews, 2020, 33(3):
[3] BONGIOVANNI M, BARDA B. Pseudomonas aeruginosa Bloodstream Infections in SARS-CoV-2 Infected Patients: A Systematic Review [J]. Journal of clinical medicine, 2023, 12(6):
[4] VILLERET B, SOLHONNE B, STRAUBE M, et al. Influenza A Virus Pre-Infection Exacerbates Pseudomonas aeruginosa-Mediated Lung Damage Through Increased MMP-9 Expression, Decreased Elafin Production and Tissue Resilience [J]. Frontiers in immunology, 2020, 11(117.
[5] DAVIES J C. Pseudomonas aeruginosa in cystic fibrosis: pathogenesis and persistence [J]. Paediatric respiratory reviews, 2002, 3(2): 128-34.
[6] PAPENFORT K, BASSLER B L. Quorum sensing signal-response systems in Gram-negative bacteria [J]. Nature reviews Microbiology, 2016, 14(9): 576-88.
[7] LEE J, ZHANG L. The hierarchy quorum sensing network in Pseudomonas aeruginosa [J]. Protein & cell, 2015, 6(1): 26-41.
[8] DEZIEL E, LEPINE F, MILOT S, et al. Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication [J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(5): 1339-44.
[9] ARMSTRONG D A, LEE M K, HAZLETT H F, et al. Extracellular Vesicles from Pseudomonas aeruginosa Suppress MHC-Related Molecules in Human Lung Macrophages [J]. ImmunoHorizons, 2020, 4(8): 508-19.
[10] MALHOTRA S, HAYES D, JR., WOZNIAK D J. Cystic Fibrosis and Pseudomonas aeruginosa: the Host-Microbe Interface [J]. Clinical microbiology reviews, 2019, 32(3):
[11] REYNOLDS D, KOLLEF M. The Epidemiology and Pathogenesis and Treatment of Pseudomonas aeruginosa Infections: An Update [J]. Drugs, 2021, 81(18): 2117-31.
[12] NAYIR BUYUKSAHIN H, YALCIN E, EMIRALIOGLU N, et al. The effect of Pseudomonas aeruginosa eradication regimens on chronic colonization and clinical outcomes in pediatric patients with cystic fibrosis [J]. Pediatrics international : official journal of the Japan Pediatric Society, 2022, 64(1): e15249.
[13] JURADO-MARTIN I, SAINZ-MEJIAS M, MCCLEAN S. Pseudomonas aeruginosa: An Audacious Pathogen with an Adaptable Arsenal of Virulence Factors [J]. International journal of molecular sciences, 2021, 22(6):
[14] WATERS C M, BASSLER B L. Quorum sensing: cell-to-cell communication in bacteria [J]. Annual review of cell and developmental biology, 2005, 21(319-46.
[15] EBERHARD A, BURLINGAME A L, EBERHARD C, et al. Structural identification of autoinducer of Photobacterium fischeri luciferase [J]. Biochemistry, 1981, 20(9): 2444-9.
[16] BAINTON N J, BYCROFT B W, CHHABRA S R, et al. A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia [J]. Gene, 1992, 116(1): 87-91.
[17] BARR H L, HALLIDAY N, CAMARA M, et al. Pseudomonas aeruginosa quorum sensing molecules correlate with clinical status in cystic fibrosis [J]. The European respiratory journal, 2015, 46(4): 1046-54.
[18] LIU Y C, CHAN K G, CHANG C Y. Modulation of Host Biology by Pseudomonas aeruginosa Quorum Sensing Signal Molecules: Messengers or Traitors [J]. Frontiers in microbiology, 2015, 6(1226.
[19] LEE J, WU J, DENG Y, et al. A cell-cell communication signal integrates quorum sensing and stress response [J]. Nature chemical biology, 2013, 9(5): 339-43.
[20] SEED P C, PASSADOR L, IGLEWSKI B H. Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy [J]. Journal of bacteriology, 1995, 177(3): 654-9.
[21] WINSON M K, CAMARA M, LATIFI A, et al. Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa [J]. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92(20): 9427-31.
[22] VENTRE I, LEDGHAM F, PRIMA V, et al. Dimerization of the quorum sensing regulator RhlR: development of a method using EGFP fluorescence anisotropy [J]. Molecular microbiology, 2003, 48(1): 187-98.
[23] MCKNIGHT S L, IGLEWSKI B H, PESCI E C. The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa [J]. Journal of bacteriology, 2000, 182(10): 2702-8.
[24] CAO H, KRISHNAN G, GOUMNEROV B, et al. A quorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism [J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(25): 14613-8.
[25] REIS R S, PEREIRA A G, NEVES B C, et al. Gene regulation of rhamnolipid production in Pseudomonas aeruginosa – A review [J]. Bioresource Technology, 2011, 102(11): 6377-84.
[26] ABDEL-MAWGOUD A M, LÉPINE F, DÉZIEL E. Rhamnolipids: diversity of structures, microbial origins and roles [J]. Applied Microbiology and Biotechnology, 2010, 86(5): 1323-36.
[27] 段海荣, 黎循航. 铜绿假单胞菌中鼠李糖脂生物合成的研究进展 [J]. 中国生物工程杂志, 2020, 40(09): 43-51.
[28] PEARSON J P, PESCI E C, IGLEWSKI B H. Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes [J]. Journal of bacteriology, 1997, 179(18): 5756-67.
[29] OCHSNER U A, KOCH A K, FIECHTER A, et al. Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa [J]. Journal of bacteriology, 1994, 176(7): 2044-54.
[30] OCHSNER U A, REISER J. Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa [J]. Proceedings of the National Academy of Sciences, 1995, 92(14): 6424-8.

[31] O’BRIEN K T, NOTO J G, NICHOLS-O’NEILL L, et al. Potent Irreversible Inhibitors of LasR Quorum Sensing in Pseudomonas aeruginosa [J]. ACS Medicinal Chemistry Letters, 2014, 6(2): 162-7.

[32] MCINNIS C E, BLACKWELL H E. Thiolactone modulators of quorum sensing revealed through library design and screening [J]. Bioorganic & Medicinal Chemistry, 2011, 19(16): 4820-8.

[33] NADAL JIMENEZ P, KOCH G, THOMPSON J A, et al. The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa [J]. Microbiology and Molecular Biology Reviews, 2012, 76(1): 46-65.

[34] HOFFMAN L R, KULASEKARA H D, EMERSON J, et al. Pseudomonas aeruginosa lasR mutants are associated with cystic fibrosis lung disease progression [J]. Journal of Cystic Fibrosis, 2009, 8(1): 66-70.

[35] LEE J, WU J, DENG Y, et al. A cell-cell communication signal integrates quorum sensing and stress response [J]. Nature chemical biology, 2013, 9(5): 339-43.

[36] LEE J, ZHANG L. The hierarchy quorum sensing network in Pseudomonas aeruginosa [J]. Protein & cell, 2014, 6(1): 26-41.

[37] KREINDLER J L, BJARNSHOLT T, JENSEN P, et al. Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients [J]. PloS one, 2010, 5(4): e10115.

[38] CHEN R, DÉZIEL E, GROLEAU M-C, et al. Social cheating in a Pseudomonas aeruginosa quorum-sensing variant [J]. Proceedings of the National Academy of Sciences, 2019, 116(14): 7021-6.

[39] CRUZ R L, ASFAHL K L, VAN DEN BOSSCHE S, et al. RhlR-Regulated Acyl-Homoserine Lactone Quorum Sensing in a Cystic Fibrosis Isolate of Pseudomonas aeruginosa [J]. mBio, 2020, 11(2):

[40] FELTNER J B, WOLTER D J, POPE C E, et al. LasR Variant Cystic Fibrosis Isolates Reveal an Adaptable Quorum-Sensing Hierarchy in Pseudomonas aeruginosa [J]. mBio, 2016, 7(5):

[41] KING J D, KOCÍNCOVÁ D, WESTMAN E L, et al. Review: Lipopolysaccharide biosynthesis inPseudomonas aeruginosa [J]. Innate Immunity, 2009, 15(5): 261-312.

[42] ALSHALCHI S A, ANDERSON G G. Expression of the lipopolysaccharide biosynthesis gene lpxD affects biofilm formation of Pseudomonas aeruginosa [J]. Archives of Microbiology, 2014, 197(2): 135-45.

[43] HUSZCZYNSKI S M, LAM J S, KHURSIGARA C M. The Role of Pseudomonas aeruginosa Lipopolysaccharide in Bacterial Pathogenesis and Physiology [J]. Pathogens, 2019, 9(1): 6.

[44] SAMPEDRO I, PARALES R E, KRELL T, et al. Pseudomona schemotaxis [J]. FEMS microbiology reviews, 2014, n/a-n/a.

[45] HAIKO J, WESTERLUND-WIKSTRÖM B. The Role of the Bacterial Flagellum in Adhesion and Virulence [J]. Biology, 2013, 2(4): 1242-67.

[46] BURROWS L L. Pseudomonas aeruginosa Twitching Motility: Type IV Pili in Action [J]. Annual review of microbiology, 2012, 66(1): 493-520.

[47] YAN S, WU G. Can Biofilm Be Reversed Through Quorum Sensing in Pseudomonas aeruginosa? [J]. Frontiers in microbiology, 2019, 10(

[48] PENA R T, BLASCO L, AMBROA A, et al. Relationship Between Quorum Sensing and Secretion Systems [J]. Frontiers in microbiology, 2019, 10(

[49] ANANTHARAJAH A, MINGEOT-LECLERCQ M-P, VAN BAMBEKE F. Targeting the Type Three Secretion System in Pseudomonas aeruginosa [J]. Trends in Pharmacological Sciences, 2016, 37(9): 734-49.

[50] STRATEVA T, MITOV I. Contribution of an arsenal of virulence factors to pathogenesis of Pseudomonas aeruginosa infections [J]. Annals of Microbiology, 2011, 61(4): 717-32.

[51] HALLDORSSON S, GUDJONSSON T, GOTTFREDSSON M, et al. Azithromycin Maintains Airway Epithelial Integrity during Pseudomonas aeruginosa Infection [J]. American Journal of Respiratory Cell and Molecular Biology, 2010, 42(1): 62-8.

[52] ZULIANELLO L, CANARD C, KÖHLER T, et al. Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia byPseudomonas aeruginosa [J]. Infection and Immunity, 2006, 74(6): 3134-47.

[53] DAUNER M, SKERRA A. Scavenging Bacterial Siderophores with Engineered Lipocalin Proteins as an Alternative Antimicrobial Strategy [J]. ChemBioChem, 2019, 21(5): 601-6.

[54] GELLATLY S L, HANCOCK R E W. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses [J]. Pathogens and Disease, 2013, 67(3): 159-73.

[55] MOURA-ALVES P, PUYSKENS A, STINN A, et al. Host monitoring of quorum sensing during Pseudomonas aeruginosa infection [J]. Science, 2019, 366(6472):

[56] LIN C K, KAZMIERCZAK B I. Inflammation: A Double-Edged Sword in the Response to Pseudomonas aeruginosa Infection [J]. Journal of innate immunity, 2017, 9(3): 250-61.

[57] LOVEWELL R R, PATANKAR Y R, BERWIN B. Mechanisms of phagocytosis and host clearance ofPseudomonas aeruginosa [J]. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2014, 306(7): L591-L603.

[58] CAREVIC M, Z H, FUCHS K, et al. CXCR1 Regulates Pulmonary Anti-Pseudomonas Host Defense [J]. Journal of innate immunity, 2016, 8(4): 362-73.

[59] LAVOIE E G, WANGDI T, KAZMIERCZAK B I. Innate immune responses to Pseudomonas aeruginosa infection [J]. Microbes and Infection, 2011, 13(14-15): 1133-45.

[60] MAUCH R M, JENSEN P, MOSER C, et al. Mechanisms of humoral immune response against Pseudomonas aeruginosa biofilm infection in cystic fibrosis [J]. Journal of Cystic Fibrosis, 2018, 17(2): 143-52.

[61] LI Y, JIN L, CHEN T. The Effects of Secretory IgA in the Mucosal Immune System [J]. BioMed Research International, 2020, 2020(1-6.

[62] HOEKSTRA D, VAN DER LAAN J W, DE LEIJ L, et al. Release of outer membrane fragments from normally growing Escherichia coli [J]. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1976, 455(3): 889-99.

[63] BROWN L, WOLF J M, PRADOS-ROSALES R, et al. Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi [J]. Nature reviews Microbiology, 2015, 13(10): 620-30.

[64] PEREZ-CRUZ C, CARRION O, DELGADO L, et al. New type of outer membrane vesicle produced by the Gram-negative bacterium Shewanella vesiculosa M7T: implications for DNA content [J]. Applied and environmental microbiology, 2013, 79(6): 1874-81.

[65] KUEHN M J, KESTY N C. Bacterial outer membrane vesicles and the host-pathogen interaction [J]. Genes & development, 2005, 19(22): 2645-55.

[66] RENELLI M, MATIAS V, LO R Y, et al. DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential [J]. Microbiology, 2004, 150(Pt 7): 2161-9.

[67] PATTON J G, FRANKLIN J L, WEAVER A M, et al. Biogenesis, delivery, and function of extracellular RNA [J]. Journal of extracellular vesicles, 2015, 4(27494.

[68] MACDONALD I A, KUEHN M J. Offense and defense: microbial membrane vesicles play both ways [J]. Research in microbiology, 2012, 163(9-10): 607-18.

[69] COOKE A C, FLOREZ C, DUNSHEE E B, et al. Pseudomonas Quinolone Signal-Induced Outer Membrane Vesicles Enhance Biofilm Dispersion in Pseudomonas aeruginosa [J]. mSphere, 2020, 5(6):

[70] LIN J, ZHANG W, CHENG J, et al. A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition [J]. Nature communications, 2017, 8(1):

[71] NAZIK H, SASS G, ANSARI S R, et al. Novel intermicrobial molecular interaction: Pseudomonas aeruginosa Quinolone Signal (PQS) modulates Aspergillus fumigatus response to iron [J]. Microbiology, 2020, 166(1): 44-55.

[72] ELLIS T N, KUEHN M J. Virulence and immunomodulatory roles of bacterial outer membrane vesicles [J]. Microbiology and molecular biology reviews : MMBR, 2010, 74(1): 81-94.

[73] MASHBURN L M, WHITELEY M. Membrane vesicles traffic signals and facilitate group activities in a prokaryote [J]. Nature, 2005, 437(7057): 422-5.

[74] KADURUGAMUWA JL B T. Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion [J]. J Bacteriol 1995; 177: 3998–4008 PMID: 7608073, 1995,

[75] BOMBERGER J M, MACEACHRAN D P, COUTERMARSH B A, et al. Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles [J]. PLoS pathogens, 2009, 5(4): e1000382.

[76] BAUMAN S J, KUEHN M J. Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells [J]. BMC microbiology, 2009, 9(26.

[77] ELHENAWY W, DEBELYY M O, FELDMAN M F. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles [J]. mBio, 2014, 5(2): e00909-14.

[78] STENTZ R, OSBORNE S, HORN N, et al. A bacterial homolog of a eukaryotic inositol phosphate signaling enzyme mediates cross-kingdom dialog in the mammalian gut [J]. Cell reports, 2014, 6(4): 646-56.

[79] RAKOFF-NAHOUM S, COYNE M J, COMSTOCK L E. An ecological network of polysaccharide utilization among human intestinal symbionts [J]. Current biology : CB, 2014, 24(1): 40-9.

[80] LAPPANN M, OTTO A, BECHER D, et al. Comparative proteome analysis of spontaneous outer membrane vesicles and purified outer membranes of Neisseria meningitidis [J]. Journal of bacteriology, 2013, 195(19): 4425-35.

[81] PRADOS-ROSALES R, WEINRICK B C, PIQUE D G, et al. Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition [J]. Journal of bacteriology, 2014, 196(6): 1250-6.

[82] LIN J, ZHANG W, CHENG J, et al. A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition [J]. Nature communications, 2017, 8(14888.

[83] COOKE A C, FLOREZ C, DUNSHEE E B, et al. Pseudomonas Quinolone Signal-Induced Outer Membrane Vesicles Enhance Biofilm Dispersion in Pseudomonas aeruginosa [J]. mSphere, 2020, 5(6):

[84] YUN S H, PARK E C, LEE S Y, et al. Antibiotic treatment modulates protein components of cytotoxic outer membrane vesicles of multidrug-resistant clinical strain, Acinetobacter baumannii DU202 [J]. Clinical proteomics, 2018, 15(28.

[85] DEVOS S, VAN OUDENHOVE L, STREMERSCH S, et al. The effect of imipenem and diffusible signaling factors on the secretion of outer membrane vesicles and associated Ax21 proteins in Stenotrophomonas maltophilia [J]. Frontiers in microbiology, 2015, 6(298.

[86] STENTZ R, HORN N, CROSS K, et al. Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against beta-lactam antibiotics [J]. The Journal of antimicrobial chemotherapy, 2015, 70(3): 701-9.

[87] JAIN S, PILLAI J. Bacterial membrane vesicles as novel nanosystems for drug delivery [J]. International journal of nanomedicine, 2017, 12(6329-41.

[88] SAHR T, ESCOLL P, RUSNIOK C, et al. Translocated Legionella pneumophila small RNAs mimic eukaryotic microRNAs targeting the host immune response [J]. Nature communications, 2022, 13(1):

[89] WHITELEY M, KOEPPEN K, HAMPTON T H, et al. A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles [J]. PLoS pathogens, 2016, 12(6): e1005672.

[90] CHOI J W, KIM S C, HONG S H, et al. Secretable Small RNAs via Outer Membrane Vesicles in Periodontal Pathogens [J]. Journal of dental research, 2017, 96(4): 458-66.

[91] WESTERMANN A J, FÖRSTNER K U, AMMAN F, et al. Dual RNA-seq unveils noncoding RNA functions in host–pathogen interactions [J]. Nature, 2016, 529(7587): 496-501.

[92] KOEPPEN K, NYMON A, BARNABY R, et al. Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa [J]. Proceedings of the National Academy of Sciences, 2021, 118(28):

[93] DUTTA T, SRIVASTAVA S. Small RNA-mediated regulation in bacteria: A growing palette of diverse mechanisms [J]. Gene, 2018, 656(60-72.

[94] SUTHERLAND C, MURAKAMI K S, LOVETT S T, et al. An Introduction to the Structure and Function of the Catalytic Core Enzyme of Escherichia coli RNA Polymerase [J]. EcoSal Plus, 2018, 8(1):

[95] KLEIN G, RAINA S. Small regulatory bacterial RNAs regulating the envelope stress response [J]. Biochemical Society transactions, 2017, 45(2): 417-25.

[96] MIKULIK K. Structure and functional properties of prokaryotic small noncoding RNAs [J]. Folia microbiologica, 2003, 48(4): 443-68.

[97] MICHAUX C, VERNEUIL N, HARTKE A, et al. Physiological roles of small RNA molecules [J]. Microbiology, 2014, 160(Pt 6): 1007-19.

[98] DELIHAS N, FORST S. MicF: an antisense RNA gene involved in response of Escherichia coli to global stress factors [J]. Journal of molecular biology, 2001, 313(1): 1-12.

[99] STORZ G, VOGEL J, WASSARMAN K M. Regulation by small RNAs in bacteria: expanding frontiers [J]. Molecular cell, 2011, 43(6): 880-91.

[100] CHEN Y, XUE D, SUN W, et al. sRNA OsiA Stabilizes Catalase mRNA during Oxidative Stress Response of Deincoccus radiodurans R1 [J]. Microorganisms, 2019, 7(10):

[101] GOTTESMAN S, STORZ G. Bacterial small RNA regulators: versatile roles and rapidly evolving variations [J]. Cold Spring Harbor perspectives in biology, 2011, 3(12):

[102] VOGEL J, LUISI B F. Hfq and its constellation of RNA [J]. Nature reviews Microbiology, 2011, 9(8): 578-89.

[103] STORZ G, ALTUVIA S, WASSARMAN K M. An abundance of RNA regulators [J]. Annual review of biochemistry, 2005, 74(199-217.

[104] MA L, WANG J, WANG S, et al. Synthesis of multiple Pseudomonas aeruginosa biofilm matrix exopolysaccharides is post‐transcriptionally regulated [J]. Environmental microbiology, 2012, 14(8): 1995-2005.

[105] COYNE M J, RUSSELL K S, COYLE C L, et al. The Pseudomonas aeruginosa algC gene encodes phosphoglucomutase, required for the synthesis of a complete lipopolysaccharide core [J]. Journal of bacteriology, 1994, 176(12): 3500-7.

[106] OLVERA C, GOLDBERG J B, S ¡NCHEZ R, et al. ThePseudomonas aeruginosa algCgene product participates in rhamnolipid biosynthesis [J]. FEMS Microbiology Letters, 1999, 179(1): 85-90.

[107] WILDERMAN P J, SOWA N A, FITZGERALD D J, et al. Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis [J]. Proceedings of the National Academy of Sciences, 2004, 101(26): 9792-7.

[108] NELSON C E, HUANG W, BREWER L K, et al. Proteomic Analysis of the Pseudomonas aeruginosa Iron Starvation Response Reveals PrrF Small Regulatory RNA-Dependent Iron Regulation of Twitching Motility, Amino Acid Metabolism, and Zinc Homeostasis Proteins [J]. Journal of bacteriology, 2019, 201(12):

[109] THOMASON M K, VOICHEK M, DAR D, et al. A rhlI 5′ UTR-Derived sRNA Regulates RhlR-Dependent Quorum Sensing in Pseudomonas aeruginosa [J]. mBio, 2019, 10(5):

[110] MALGAONKAR A, NAIR M. Quorum sensing in Pseudomonas aeruginosa mediated by RhlR is regulated by a small RNA PhrD [J]. Scientific reports, 2019, 9(1): 432.

[111] KOEPPEN K, HAMPTON T H, JAREK M, et al. A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles [J]. PLoS pathogens, 2016, 12(6): e1005672.

[112] TSATSARONIS J A, FRANCH-ARROYO S, RESCH U, et al. Extracellular Vesicle RNA: A Universal Mediator of Microbial Communication? [J]. Trends in microbiology, 2018, 26(5): 401-10.

[113] LEE H J. Microbe-Host Communication by Small RNAs in Extracellular Vesicles: Vehicles for Transkingdom RNA Transportation [J]. International journal of molecular sciences, 2019, 20(6):

[114] FURUSE Y, FINETHY R, SAKA H A, et al. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells [J]. PloS one, 2014, 9(9): e106434.

[115] WEIBERG A, WANG M, LIN F M, et al. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways [J]. Science, 2013, 342(6154): 118-23.

[116] WESTERMANN A J, FORSTNER K U, AMMAN F, et al. Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions [J]. Nature, 2016, 529(7587): 496-501.

[117] CHOI J W, KIM S C, HONG S H, et al. Secretable Small RNAs via Outer Membrane Vesicles in Periodontal Pathogens [J]. Journal of dental research, 2017, 96(4): 458-66.

[118] AYTAR ELIK P, DERKUŞ B, ERDOĞAN K, et al. Bacterial membrane vesicle functions, laboratory methods, and applications [J]. Biotechnology Advances, 2022, 54(107869.

[119] KULP A, KUEHN M J. Biological Functions and Biogenesis of Secreted Bacterial Outer Membrane Vesicles [J]. Annual review of microbiology, 2010, 64(1): 163-84.

[120] TOYOFUKU M, MORINAGA K, HASHIMOTO Y, et al. Membrane vesicle-mediated bacterial communication [J]. The ISME Journal, 2017, 11(6): 1504-9.

[121] DONG Z, CHEN Y. Transcriptomics: Advances and approaches [J]. Science China Life Sciences, 2013, 56(10): 960-7.

[122] SRIVASTAVA A, GEORGE J, KARUTURI R K M. Transcriptome Analysis [J]. 2019, 792-805.

[123] COPPENS L, LAVIGNE R. SAPPHIRE: a neural network based classifier for σ70 promoter prediction in Pseudomonas [J]. BMC Bioinformatics, 2020, 21(1):

[124] ANDERSSON R, SANDELIN A. Determinants of enhancer and promoter activities of regulatory elements [J]. Nature Reviews Genetics, 2019, 21(2): 71-87.

[125] SILVA I J, BARAHONA S, EYRAUD A, et al. SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA [J]. Proceedings of the National Academy of Sciences, 2019, 116(8): 3042-51.

[126] OTAKA H, ISHIKAWA H, MORITA T, et al. PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action [J]. Proceedings of the National Academy of Sciences, 2011, 108(32): 13059-64.

[127] LIVNY J, WALDOR M K. Identification of small RNAs in diverse bacterial species [J]. Current opinion in microbiology, 2007, 10(2): 96-101.

[128] ADHYA S, GOTTESMAN M. Control of Transcription Termination [J]. Annual review of biochemistry, 1978, 47(1): 967-96.

[129] RAY-SONI A, BELLECOURT M J, LANDICK R. Mechanisms of Bacterial Transcription Termination: All Good Things Must End [J]. Annual review of biochemistry, 2016, 85(1): 319-47.

[130] MELAMED S, PEER A, FAIGENBAUM-ROMM R, et al. Global Mapping of Small RNA-Target Interactions in Bacteria [J]. Molecular cell, 2016, 63(5): 884-97.

[131] CAO P, FLEMING D, MOUSTAFA D A, et al. A Pseudomonas aeruginosa small RNA regulates chronic and acute infection [J]. Nature, 2023, 618(7964): 358-64.

[132] WANG X-W, LIU C-X, CHEN L-L, et al. RNA structure probing uncovers RNA structure-dependent biological functions [J]. Nature chemical biology, 2021, 17(7): 755-66.

[133] DENMAN R B. Using RNAFOLD to predict the activity of small catalytic RNAs [J]. BioTechniques, 1993, 15(6): 1090-5.

[134] CAIAZZA N C, SHANKS R M, O'TOOLE G A. Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa [J]. Journal of bacteriology, 2005, 187(21): 7351-61.

[135] ZUKER M. Mfold web server for nucleic acid folding and hybridization prediction [J]. Nucleic acids research, 2003, 31(13): 3406-15.

[136] HELLMAN L M, FRIED M G. Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions [J]. Nature Protocols, 2007, 2(8): 1849-61.

[137] JIA T, WU P, LIU B, et al. The phosphate-induced small RNA EsrL promotes E. coli virulence, biofilm formation, and intestinal colonization [J]. Science Signaling, 2023, 16(767):

[138] SORGER-DOMENIGG T, SONNLEITNER E, KABERDIN V R, et al. Distinct and overlapping binding sites of Pseudomonas aeruginosa Hfq and RsmA proteins on the non-coding RNA RsmY [J]. Biochemical and Biophysical Research Communications, 2007, 352(3): 769-73.

[139] SOBRERO P, VALVERDE C. The bacterial protein Hfq: much more than a mere RNA-binding factor [J]. Critical Reviews in Microbiology, 2012, 38(4): 276-99.

[140] GEBHARDT M J, FARLAND E A, BASU P, et al. Hfq-licensed RNA–RNA interactome in Pseudomonas aeruginosa reveals a keystone sRNA [J]. Proceedings of the National Academy of Sciences, 2023, 120(21):

[141] MOSTOWY S, COSSART P. Septins: the fourth component of the cytoskeleton [J]. Nature reviews Molecular cell biology, 2012, 13(3): 183-94.

[142] BRIDGES A A, GLADFELTER A S. Fungal pathogens are platforms for discovering novel and conserved septin properties [J]. Current opinion in microbiology, 2014, 20(42-8.

[143] THAVARAJAH R, VIDYA K, JOSHUA E, et al. Potential role of septins in oral carcinogenesis: An update and avenues for future research [J]. Journal of oral and maxillofacial pathology : JOMFP, 2012, 16(1): 73-8.

[144] EDISON N, ZURI D, MANIV I, et al. The IAP-antagonist ARTS initiates caspase activation upstream of cytochrome C and SMAC/Diablo [J]. Cell death and differentiation, 2012, 19(2): 356-68.

[145] ABBAS R, LARISCH S. Targeting XIAP for Promoting Cancer Cell Death-The Story of ARTS and SMAC [J]. Cells, 2020, 9(3):

[146] ZHAO X, FENG H, WANG Y, et al. Septin4 promotes cell death in human colon cancer cells by interacting with BAX [J]. International journal of biological sciences, 2020, 16(11): 1917-28.

[147] MAMRIEV D, ABBAS R, KLINGLER F M, et al. A small-molecule ARTS mimetic promotes apoptosis through degradation of both XIAP and Bcl-2 [J]. Cell death & disease, 2020, 11(6): 483.

[148] HAO Q, CHEN J, LIAO J, et al. p53 induces ARTS to promote mitochondrial apoptosis [J]. Cell death & disease, 2021, 12(2): 204.

[149] SHEN S, LIU M, WU Y, et al. Involvement of SEPT4_i1 in hepatocellular carcinoma: SEPT4_i1 regulates susceptibility to apoptosis in hepatocellular carcinoma cells [J]. Molecular biology reports, 2012, 39(4): 4519-26.

[150] JEON T W, YANG H, LEE C G, et al. Electro-hyperthermia up-regulates tumour suppressor Septin 4 to induce apoptotic cell death in hepatocellular carcinoma [J]. International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group, 2016, 32(6): 648-56.

[151] EDISON N, CURTZ Y, PALAND N, et al. Degradation of Bcl-2 by XIAP and ARTS Promotes Apoptosis [J]. Cell reports, 2017, 21(2): 442-54.

[152] FU R H, HUANG L C, LIN C Y, et al. Modulation of ARTS and XIAP by Parkin Is Associated with Carnosic Acid Protects SH-SY5Y Cells against 6-Hydroxydopamine-Induced Apoptosis [J]. Molecular neurobiology, 2018, 55(2): 1786-94.

[153] EDISON N, REINGEWERTZ T H, GOTTFRIED Y, et al. Peptides mimicking the unique ARTS-XIAP binding site promote apoptotic cell death in cultured cancer cells [J]. Clinical cancer research : an official journal of the American Association for Cancer Research, 2012, 18(9): 2569-78.

[154] WU S, ZHANG Y, YOU S, et al. Septin4 promotes cardiomyocytes apoptosis by enhancing the VHL-mediated degradation of HIF-1α [J]. Cell Death Discovery, 2021, 7(1):

[155] ZHU D, SONG K, CHEN J, et al. Expression of Septin4 in Schistosoma japonicum-infected mouse livers after praziquantel treatment [J]. Parasites & vectors, 2015, 8(19.

[156] ZHANG N, ZHANG Y, ZHAO S, et al. Septin4 as a novel binding partner of PARP1 contributes to oxidative stress induced human umbilical vein endothelial cells injure [J]. Biochem Biophys Res Commun, 2018, 496(2): 621-7.

[157] VILLARROYA-BELTRI C, GUTIÉRREZ-VÁZQUEZ C, SÁNCHEZ-CABO F, et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs [J]. Nature communications, 2013, 4(1):

[158] SANTANGELO L, GIURATO G, CICCHINI C, et al. The RNA-Binding Protein SYNCRIP Is a Component of the Hepatocyte Exosomal Machinery Controlling MicroRNA Sorting [J]. Cell reports, 2016, 17(3): 799-808.