Barbieri Giorgia, Stefano Alessia, Pietrantoni Agostina, Fratini Federica, Cavallero Serena, Tozzoli Rosangela, Chiani Paola, Boni Arianna, Morabito Stefano, Michelacci Valeria
Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, Italy.
Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy.
Front Cell Infect Microbiol. 2025 Aug 8;15:1621341. doi: 10.3389/fcimb.2025.1621341. eCollection 2025.
Shiga toxin (Stx)-producing (STEC) harboring virulence determinants of Extraintestinal pathogenic (ExPEC) are currently emerging in Europe as a cause of severe disease. Among the ExPEC features identified in STEC, the gene is associated with an augmented production of Outer Membrane Vesicles (OMV). OMVs produced by strains have been shown to deliver toxins and small RNAs, but information on the latter genetic component is still scanty. We investigated the small RNAs contained in the OMVs produced by two -positive STEC strains producing Stx2 belonging to O26:H11 and O80:H2 serotypes, isolated from a human case of Hemolytic Uraemic Syndrome (HUS) and from a milk sample, respectively. The OMVs were purified from overnight cultures using two sequential ultracentrifugation steps at 100.000 and 200.000 x of 2h each. The OMVs collected were analyzed by Nanoparticle Tracking Analysis (NTA) and Electron Microscopy (EM) to define their morphology, size and concentration. The whole genome sequences of the two test strains and three additional -positive STEC strains were analyzed to identify sequences of small RNAs. Specific Real Time PCR assays were deployed to detect the small RNAs within OMVs. The EM and NTA characterization confirmed the presence of spherical particles, and showed that a cleaner preparation was obtained after the second step of ultracentrifugation (200.000 x ). We identified the sequences of 27 putative small RNAs present in the genome of -positive STEC strains and absent in that of a non-pathogenic . Nine of them were identified and quantified in the OMVs produced by the two test strains. A regulatory role in bacterial DNA replication, integration and in stress-response was hypothesized for the identified small RNAs, some of which encompassing all the domains of life. STnc_100 was the only small RNA identified in the OMVs produced by both strains. Interestingly, a copy of STnc_100 sequence is harbored downstream the operon carried by Stx2-encoding phages in both the strains and has a complementarity region for gene, suggesting a modulation in Stx production or release. Our results indicate that OMVs release may exert regulatory functions, putatively influencing the crosstalk with the host and the gut microbiota during the infection process.
携带肠外致病性大肠杆菌（ExPEC）毒力决定因素的产志贺毒素（Stx）大肠杆菌（STEC）目前在欧洲作为严重疾病的病因正在出现。在STEC中鉴定出的ExPEC特征中，该基因与外膜囊泡（OMV）产量增加有关。已证明由该菌株产生的OMV可递送毒素和小RNA，但关于后一种遗传成分的信息仍然很少。我们研究了从一名溶血尿毒综合征（HUS）患者和一份牛奶样本中分别分离出的属于O26:H11和O80:H2血清型、产生Stx2的两株STEC阳性菌株所产生的OMV中所含的小RNA。使用两个连续的超速离心步骤（每次2小时，分别为100,000和200,000×g）从过夜培养物中纯化OMV。对收集的OMV进行纳米颗粒跟踪分析（NTA）和电子显微镜（EM）分析，以确定其形态、大小和浓度。分析了两株测试菌株和另外三株STEC阳性菌株的全基因组序列，以鉴定小RNA的序列。采用特异性实时PCR检测法检测OMV中的小RNA。EM和NTA表征证实了球形颗粒的存在，并表明在超速离心的第二步（200,000×g）后获得了更纯净的制剂。我们鉴定出了STEC阳性菌株基因组中存在、而非致病性菌株基因组中不存在的27个假定小RNA的序列。其中9个在两株测试菌株产生的OMV中被鉴定和定量。推测所鉴定的小RNA在细菌DNA复制、整合和应激反应中具有调节作用，其中一些涵盖了生命的所有领域。STnc_100是两株菌株产生的OMV中唯一鉴定出的小RNA。有趣的是，两株菌株中编码Stx2的噬菌体携带的操纵子下游都含有一份STnc_100序列的拷贝，并且与该基因有一个互补区域，表明其对Stx产生或释放有调节作用。我们的结果表明，OMV释放可能发挥调节功能，可能在感染过程中影响与宿主和肠道微生物群的相互作用。
