Characterization of the small RNAs carried by outer membrane vesicles produced by hlyF-positive Shiga toxin-producing Escherichia coli
Shiga toxin (Stx)–producing Escherichia coli (STEC) harboring virulence determinants of Extraintestinal pathogenic E. coli (ExPEC) are currently emerging in Europe as a cause of severe disease. Among the ExPEC features identified in STEC, the gene hlyF is associated with an augmented production of O...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-08-01
|
| Series: | Frontiers in Cellular and Infection Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2025.1621341/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849392114357501952 |
|---|---|
| author | Giorgia Barbieri Giorgia Barbieri Alessia Stefano Agostina Pietrantoni Federica Fratini Serena Cavallero Rosangela Tozzoli Paola Chiani Arianna Boni Stefano Morabito Valeria Michelacci |
| author_facet | Giorgia Barbieri Giorgia Barbieri Alessia Stefano Agostina Pietrantoni Federica Fratini Serena Cavallero Rosangela Tozzoli Paola Chiani Arianna Boni Stefano Morabito Valeria Michelacci |
| author_sort | Giorgia Barbieri |
| collection | DOAJ |
| description | Shiga toxin (Stx)–producing Escherichia coli (STEC) harboring virulence determinants of Extraintestinal pathogenic E. coli (ExPEC) are currently emerging in Europe as a cause of severe disease. Among the ExPEC features identified in STEC, the gene hlyF is associated with an augmented production of Outer Membrane Vesicles (OMV). OMVs produced by E. coli 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 hlyF-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 g 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 hlyF-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 g). We identified the sequences of 27 putative small RNAs present in the genome of hlyF-positive STEC strains and absent in that of a non-pathogenic E. coli. 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 stx2 operon carried by Stx2-encoding phages in both the strains and has a complementarity region for stxB 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. |
| format | Article |
| id | doaj-art-ad1a31beb1a34b9d86466ffe97c16ca5 |
| institution | Kabale University |
| issn | 2235-2988 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj-art-ad1a31beb1a34b9d86466ffe97c16ca52025-08-20T03:40:50ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882025-08-011510.3389/fcimb.2025.16213411621341Characterization of the small RNAs carried by outer membrane vesicles produced by hlyF-positive Shiga toxin-producing Escherichia coliGiorgia Barbieri0Giorgia Barbieri1Alessia Stefano2Agostina Pietrantoni3Federica Fratini4Serena Cavallero5Rosangela Tozzoli6Paola Chiani7Arianna Boni8Stefano Morabito9Valeria Michelacci10Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, ItalyDepartment of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, ItalyDepartment of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, ItalyIstituto Superiore di Sanità, Core Facilities Technical-Scientific Service, Rome, ItalyIstituto Superiore di Sanità, Core Facilities Technical-Scientific Service, Rome, ItalyDepartment of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, ItalyDepartment of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, ItalyDepartment of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, ItalyDepartment of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, ItalyDepartment of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, ItalyDepartment of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, ItalyShiga toxin (Stx)–producing Escherichia coli (STEC) harboring virulence determinants of Extraintestinal pathogenic E. coli (ExPEC) are currently emerging in Europe as a cause of severe disease. Among the ExPEC features identified in STEC, the gene hlyF is associated with an augmented production of Outer Membrane Vesicles (OMV). OMVs produced by E. coli 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 hlyF-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 g 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 hlyF-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 g). We identified the sequences of 27 putative small RNAs present in the genome of hlyF-positive STEC strains and absent in that of a non-pathogenic E. coli. 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 stx2 operon carried by Stx2-encoding phages in both the strains and has a complementarity region for stxB 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.https://www.frontiersin.org/articles/10.3389/fcimb.2025.1621341/fullouter membrane vesiclesOMVssmall RNAShiga toxin-producing Escherichia coliextraintestinal pathogenic Escherichia coliinfection |
| spellingShingle | Giorgia Barbieri Giorgia Barbieri Alessia Stefano Agostina Pietrantoni Federica Fratini Serena Cavallero Rosangela Tozzoli Paola Chiani Arianna Boni Stefano Morabito Valeria Michelacci Characterization of the small RNAs carried by outer membrane vesicles produced by hlyF-positive Shiga toxin-producing Escherichia coli Frontiers in Cellular and Infection Microbiology outer membrane vesicles OMVs small RNA Shiga toxin-producing Escherichia coli extraintestinal pathogenic Escherichia coli infection |
| title | Characterization of the small RNAs carried by outer membrane vesicles produced by hlyF-positive Shiga toxin-producing Escherichia coli |
| title_full | Characterization of the small RNAs carried by outer membrane vesicles produced by hlyF-positive Shiga toxin-producing Escherichia coli |
| title_fullStr | Characterization of the small RNAs carried by outer membrane vesicles produced by hlyF-positive Shiga toxin-producing Escherichia coli |
| title_full_unstemmed | Characterization of the small RNAs carried by outer membrane vesicles produced by hlyF-positive Shiga toxin-producing Escherichia coli |
| title_short | Characterization of the small RNAs carried by outer membrane vesicles produced by hlyF-positive Shiga toxin-producing Escherichia coli |
| title_sort | characterization of the small rnas carried by outer membrane vesicles produced by hlyf positive shiga toxin producing escherichia coli |
| topic | outer membrane vesicles OMVs small RNA Shiga toxin-producing Escherichia coli extraintestinal pathogenic Escherichia coli infection |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2025.1621341/full |
| work_keys_str_mv | AT giorgiabarbieri characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT giorgiabarbieri characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT alessiastefano characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT agostinapietrantoni characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT federicafratini characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT serenacavallero characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT rosangelatozzoli characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT paolachiani characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT ariannaboni characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT stefanomorabito characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli AT valeriamichelacci characterizationofthesmallrnascarriedbyoutermembranevesiclesproducedbyhlyfpositiveshigatoxinproducingescherichiacoli |