Identification and functional characterization of two heme uptake systems required for virulence of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the phylum Bacteroidetes, causes bacterial cold-water disease in salmonids worldwide. Infected fish exhibit tissue damages and develop hemorrhagic septicemia. The molecular factors involved in host colonization are mostly unknown. In this study, we identified two TonB-dependent receptors that are required for iron homeostasis and virulence in rainbow trout (Oncorhynchus mykiss). Transcriptional analyses revealed that their expression is tightly controlled and upregulated under specific iron sources and concentrations. Characterization of single and double deletion mutants showed that the two receptors act without redundancy: BfpR is required for optimal growth in the presence of high hemoglobin level, while HfpR confers the capacity to acquire nutrient iron from heme or hemoglobin under iron scarcity. The gene hfpY, co-transcribed with hfpR, encodes a protein belonging to the HmuY family but with low sequence conservation compared to other members of this family. We demonstrated that HfpY is able to bind heme in vitro and contributes significantly to host colonization and disease severity in experimentally infected rainbow trout. Overall, these results are consistent with a model in which both BfpR and Hfp systems promote heme uptake and respond to distinct signals to adapt iron acquisition to the different stages of pathogenesis. Our findings give insight into the molecular basis of F. psychrophilum pathogenicity and point to the newly identified heme outer membrane receptors as promising targets for antibacterial development. Author summary: Infectious diseases are a major threat for human and animal health. Bacterial pathogens impact aquaculture, a sector that accounts for half of the human fish consumption, and mechanistic insights into pathogenesis allow for improved control strategies. Pathogens have to retrieve essential nutrients to multiply. Host-derived heme constitutes the largest iron source during infection and pathogens have evolved fine-tuned iron acquisition systems to overcome host scavenging mechanisms while preventing iron toxicity. These mechanisms are poorly understood in pathogens of the family Flavobacteriaceae that have a critical impact on the fish farming, animal welfare and environment. Here, we report the identification and characterization of two heme uptake systems important for adaptation under distinct iron sources conditions encountered during infection and for virulence and host colonization in Flavobacterium psychrophilum, a serious pathogen of salmonids. ### Competing Interest Statement The authors have declared no competing interest.