Biologically active flavins from riboflavin (vitamin B2) are essential for intracellular redox reactions and extracellular bacterial physiology. Most bacterial pathogens can either synthesize riboflavin de novo or scavenge riboflavin from the host tissues through high-affinity transporters.
Aeromonas salmonicida is a Gram-negative pathogen of fresh and marine water fish and the etiological agent of furunculosis and its riboflavin supply pathways have not been studied. Here, we characterized the riboflavin provision pathways in A. salmonicida using in-silico and experimental approaches. We found that A. salmonicida possesses a riboflavin biosynthetic pathway (RBP) and a transporter ribN. Transcriptional orchestration analysis of riboflavin supply genes by RT-PCR revealed that the main RBP operon consists of ribD, ribE1, ribBA, and ribH genes, together with nrdR and nusB genes. ribE1 and bifunctional ribBA from the main RBP operon have duplicated paralogs outside the main operon; ribE2 (ribE1), ribA, and ribB (ribBA). Regulation analysis using RNA-Seq, and qPCR showed that the ribB transcriptional unit, conserving a putative FMN riboswitch, is negatively regulated by riboflavin. To study the role of paralogs found in Riboflavin Biosynthetic Pathway (RBP) and riboflavin transporter (ribN) in A. salmonicida pathogenesis and physiology, mutants of ribE1, ribE2, ribBA, ribA, ribB, ribN, and ribA-ribE1 were constructed, characterized, and evaluated in the marine teleost, lumpfish (Cyclopterus lumpus). Groups of 60 fish were intraperitoneally injected with 0.1 ml (104 CFU/dose) of the respective A. salmonicida wild type and mutant strains. Tissue samples were collected at different time points to determine bacterial colonization. Mortality was recorded until 30 days post-infection (dpi). All fish died within 10 dpi from the ΔribE2, ΔribBA, ribN, and wild type infected groups, whereas 100% of the fish infected with ΔribE1, ΔribA, ΔribB, and ribA-ribE1 survived. Surviving fish were challenged with 103 CFU/dose (10 LD50) of A. salmonicida wild type to determine the mutants’ utility as live attenuated vaccine. The attenuated mutants (ΔribE1, ΔribA, ΔribB, and ribA-ribE1) conferred protection with a low relative percentage of survival (10-20%). In summary, ribE1, ribA, and ribB of RBP play an essential role in riboflavin provision to A. salmonicida during host colonization and impacting bacterial virulence.