Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, an important disease responsible for economic losses to the swine industry worldwide. Its virulence is associated with factors such as capsule, lipopolysaccharide, and proteins including the RTX family toxins. Outer Membrane Vesicles (OMVs) are shed from the surface of many Gram-negative bacteria and can transport different virulence factors, as well as DNA and RNA, including small RNAs (sRNAs). These latter are responsible for regulation of many genes in bacteria. Recently, it has been shown that some bacterial sRNAs can regulate host cell gene expression following delivery by OMVs.
The aim of this study was firstly to determine if OMVs produced by A. pleuropneumoniae contain sRNAs, and secondly to carry out a preliminary identification of sRNAs associated with these OMVs. Hydrostatic filtration was used to concentrate and purify OMVs from A. pleuropneumoniae MIDG2331 (serovar 8). The OMVs were characterized with regards to morphology, protein profile, and RNA profile by TEM, SDS-PAGE, and agarose gel electrophoresis, respectively. An RNAse protection assay was performed to confirm the presence of sRNA inside of OMVs, and specific RT-PCR reactions were done to identify the presence of 16 trans-acting sRNAs previously identified in A. pleuropneumoniae by our group.
Our results indicate that sRNAs are associated with the OMVs produced by A. pleuropneumoniae, and are present both internally and externally. Of the 16 specific sRNAs targeted by RT-PCR amplification, only three were not detected in total RNA from OMVs prior to RNAse protection. Following RNAse treatment, only one OMV-associated sRNA, named ARRC09, was not found inside OMVs. ARRC09 is a novel sRNA from A. pleuropneumoniae, and our group is currently investigating its function. This is the first report of the presence of regulatory sRNAs within OMVs produced by A. pleuropneumoniae.