Aeromonas salmonicida is a Gram-negative pathogen of fresh and marine water fish and the etiological agent of furunculosis. Most bacterial pathogens can either synthesize riboflavin de novo or scavenge riboflavin from the host tissues through high-affinity transporters. Biologically active flavins from riboflavin (vitamin B2) are essential for intracellular redox reactions and extracellular bacterial physiology. Riboflavin supply pathways of A. salmonicida have not been studied.
First , we in-silico and experimentally characterized the riboflavin provision pathways in A. salmonicida . In this pathogen, the riboflavin biosynthetic pathway (RBP) coexists with the transporter ribN . Transcriptional orchestration of riboflavin supply genes by RT-PCR revealed that the main RBP operon consists of ribD , ribE1 , ribBA, and ribH together with genes nrdR and nusB , which are involved in functions that are not directly related to riboflavin biosynthesis. 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 qPCR showed that the ribB transcriptional unit, conserving a putative FMN riboswitch, is negatively regulated by riboflavin.
Secondly, to study the role of paralogs found in Riboflavin Biosynthetic Pathway (RBP) and riboflavin transporter (ribN ) in A. salmonicida in lumpfish (Cyclopterus lumpus) , mutants of ribE1 , ribE2 , ribBA , ribA , ribB , ribN , and ribA-ribE1 were constructed and characterized. 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). Surviving fish were challenged with 103 CFU/dose (10 LD50) of A. salmonicida wild type. 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 s urvived. After the challenge , fully 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 play an essential role in A. salmonicida virulence during host colonization . This study was funded by Canada-First, Ocean Frontier Institute, and NSERC-Discovery (RGPIN-2018-05942).