Small RNAs (sRNAs) play important roles in gene regulation and have been frequently connected to the expression of virulence factors in diverse bacteria. Their regulatory activity is often dependent on the interaction with the molecular chaperone Hfq, which facilitates their base-pairing with mRNA targets. Thus far, no sRNA has been described for Actinobacillus pleuropneumoniae (APP), the causative agent of porcine pleuropneumonia, responsible for considerable economic losses in the swine industry. We have used co-immunoprecipitation (coIP) and RNA-Seq to identify specific Hfq-bound RNAs in APP by comparing samples from a wild-type strain (MIDG2331) to those from isogenic mutants (either chromosomal FLAG-tagged hfq, or delta-hfq). Samples were prepared from strains grown under aerobic and anaerobic conditions to early stationary phase. The results were complemented with computational searches for sRNAs using 4 different algorithms (RNAz, INFERNAL, SIPHT and BLASTn over Rfam), and sequences found by at least two of those tools were considered sRNA candidates. The RNASeq results identified at least 15 sRNAs that are Hfq-dependent in APP. While 7 of these are expressed in both aerobic and anaerobic conditions, 5 are exclusively aerobic and 3 exclusively anaerobic. Two sRNAs were highly expressed in all the strains, and are therefore likely Hfq-independent. Among the sRNAs discovered experimentally, 5 were also predicted by the in silico approach, in addition to 18 candidates obtained only computationally. In total, 35 sRNAs were detected in APP. These are widely distributed within the species and include well known global regulators (gcvB, SRP, 6S) as well as novel candidates, many of which are likely involved with virulence, according to their putative targets predicted in silico. Our data indicate a previously unexplored and intricate system of gene regulation through sRNAs and Hfq in APP, and provide a new horizon for further studies.
Financial support: CNPq/ BBSRC (407849/2012_2); FAPEMIG and CAPES.