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Membrane vesicle-mediated release of bacterial RNA.

Sjöström AE, Sandblad L, Uhlin BE, Wai SN - Sci Rep (2015)

Bottom Line: Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs.The results showed that RNAs originating from intergenic regions were the most abundant.Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.

ABSTRACT
Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.

No MeSH data available.


Related in: MedlinePlus

Analysis of OMV-associated RNA.RNA extracted from overnight cultures of wt and HTRR mutant Vibrio cholerae O1 El Tor A1552 whole cells (wc) and vesicles (v) using the Total RNA Norgen kit. RNA (3.5 μg) was run on a 12% polyacrylamide denaturing gel and transferred to a membrane. (A) Gel stained with GelRed. Positions of bands missing in case of mutant strains are indicated by asterisks (*). (B) Northern blot membrane probed with probe vc2478,5. (C) Northern blot membrane re-probed with probe vc0190,5. (D) Northern blot membrane re-probed with probe vca0526,5.
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f2: Analysis of OMV-associated RNA.RNA extracted from overnight cultures of wt and HTRR mutant Vibrio cholerae O1 El Tor A1552 whole cells (wc) and vesicles (v) using the Total RNA Norgen kit. RNA (3.5 μg) was run on a 12% polyacrylamide denaturing gel and transferred to a membrane. (A) Gel stained with GelRed. Positions of bands missing in case of mutant strains are indicated by asterisks (*). (B) Northern blot membrane probed with probe vc2478,5. (C) Northern blot membrane re-probed with probe vc0190,5. (D) Northern blot membrane re-probed with probe vca0526,5.

Mentions: To verify that the vesicle RNA sequences aligned to the chromosomal V. cholerae DNA— corresponding to the HTRR regions — in fact give rise to the vesicle transcripts, we constructed three mutants (AES206, AES207, and AES208) in which the chromosomal DNA corresponding to each of these HTRRs was deleted and replaced by Km-cassettes. RNA from vesicle preparations and whole cell extracts of wild type A1552 and the three mutants were subjected to a 12% polyacrylamide denaturing gel electrophoresis and stained with GelRed (Fig. 2A). All vesicle-isolated RNA (v) samples exhibited a similar but unique band pattern, different from the likewise similar but unique whole cell extracted RNA (wc) sample pattern. Asterisks (*) in the AES207 and AES208 mutant lanes show where bands are missing in the samples from mutants. The gel-separated RNA transcripts were transferred to a membrane for Northern blotting and hybridized with probes vc2478,5 (Supplementary Fig. S4B), vc0190,5 (Supplementary Fig. S4C), and vca0526,5 (Supplementary Fig. S4D). Probing verified that these RNAs from HTRRs correspond to the identified DNA sequences on the chromosome (Fig. 2B–D). Interestingly, the size of the bands missing in the AES207 and AES208 RNA wc lanes are the same as that of the transcripts recognized by the vc2478,5 and vca0526,5 probes (Fig. 2A,B and D). Thus, the vc2478,5 and vca0526,5 region transcripts are apparently abundant enough to be visible by GelRed staining.


Membrane vesicle-mediated release of bacterial RNA.

Sjöström AE, Sandblad L, Uhlin BE, Wai SN - Sci Rep (2015)

Analysis of OMV-associated RNA.RNA extracted from overnight cultures of wt and HTRR mutant Vibrio cholerae O1 El Tor A1552 whole cells (wc) and vesicles (v) using the Total RNA Norgen kit. RNA (3.5 μg) was run on a 12% polyacrylamide denaturing gel and transferred to a membrane. (A) Gel stained with GelRed. Positions of bands missing in case of mutant strains are indicated by asterisks (*). (B) Northern blot membrane probed with probe vc2478,5. (C) Northern blot membrane re-probed with probe vc0190,5. (D) Northern blot membrane re-probed with probe vca0526,5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4612299&req=5

f2: Analysis of OMV-associated RNA.RNA extracted from overnight cultures of wt and HTRR mutant Vibrio cholerae O1 El Tor A1552 whole cells (wc) and vesicles (v) using the Total RNA Norgen kit. RNA (3.5 μg) was run on a 12% polyacrylamide denaturing gel and transferred to a membrane. (A) Gel stained with GelRed. Positions of bands missing in case of mutant strains are indicated by asterisks (*). (B) Northern blot membrane probed with probe vc2478,5. (C) Northern blot membrane re-probed with probe vc0190,5. (D) Northern blot membrane re-probed with probe vca0526,5.
Mentions: To verify that the vesicle RNA sequences aligned to the chromosomal V. cholerae DNA— corresponding to the HTRR regions — in fact give rise to the vesicle transcripts, we constructed three mutants (AES206, AES207, and AES208) in which the chromosomal DNA corresponding to each of these HTRRs was deleted and replaced by Km-cassettes. RNA from vesicle preparations and whole cell extracts of wild type A1552 and the three mutants were subjected to a 12% polyacrylamide denaturing gel electrophoresis and stained with GelRed (Fig. 2A). All vesicle-isolated RNA (v) samples exhibited a similar but unique band pattern, different from the likewise similar but unique whole cell extracted RNA (wc) sample pattern. Asterisks (*) in the AES207 and AES208 mutant lanes show where bands are missing in the samples from mutants. The gel-separated RNA transcripts were transferred to a membrane for Northern blotting and hybridized with probes vc2478,5 (Supplementary Fig. S4B), vc0190,5 (Supplementary Fig. S4C), and vca0526,5 (Supplementary Fig. S4D). Probing verified that these RNAs from HTRRs correspond to the identified DNA sequences on the chromosome (Fig. 2B–D). Interestingly, the size of the bands missing in the AES207 and AES208 RNA wc lanes are the same as that of the transcripts recognized by the vc2478,5 and vca0526,5 probes (Fig. 2A,B and D). Thus, the vc2478,5 and vca0526,5 region transcripts are apparently abundant enough to be visible by GelRed staining.

Bottom Line: Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs.The results showed that RNAs originating from intergenic regions were the most abundant.Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.

ABSTRACT
Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.

No MeSH data available.


Related in: MedlinePlus