Small RNAs (sRNAs) and RNA-binding proteins such as Hfq play a critical role in bacterial gene regulation. Few studies in this area are available for Pasteurellaceae species, including Actinobacillus pleuropneumoniae (APP), the causative agent of porcine pleuropneumonia. We previously identified 23 possible sRNAs in APP using bioinformatics and experimental confirmation. One of these, ARRC14, is the subject of the current study.
We performed bioinformatic analyses to determine sequence conservation, and to predict the structure and possible targets of this sRNA. To investigate the function, we constructed single and double mutants arrc14 and hfq in the serovar 8 strain, MIDG2331. The WT and mutant strains were compared for in vitro growth, adhesion, hemolytic activity, stress sensitivity, and virulence in the Galleria mellonella model of infection.
In silico analysis revealed the presence of arrc14 in all 18 APP known serovars (99-100% identity), and in four genera of the Pasteurellaceae family (≈80% identity). Phenotypic analyses showed that Darrc14 appeared to grow more slowly, though the reduction in OD600 was likely due to autoaggregation, as this mutant also and adhered more strongly to both an abiotic surface and porcine epithelial PK15 cells, compared to the WT, Dhfq and Dhfq/Darrc14 strains. Although no significant difference was seen between WT and mutant strains regarding stress resistance (osmotic, temperature, oxidative and antibiotics), Darrc14 appeared more hemolytic and virulent (lower survival of G. mellonella) compared to the WT, Dhfq and Dhfq/Darrc14 strains.
Our results indicate that ARRC14 is a trans-acting sRNA that requires Hfq for binding to its target mRNAs. Using CopraRNA, several putative targets were identified with different predicted functions, indicating a complex gene regulation network by ARRC14. Experimental studies are required to confirm interaction of ARRC14 with these predicted targets and to further determine the role of this sRNA in regulation of virulence of APP.