Abstract
Aeromonas salmonicida subsp. salmonicida (A.s.s) is a major pathogen affecting fisheries worldwide. It is a well-known member of the pigmented Aeromonas species, which produces melanin at ≤ 22 °C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30-35 °C while bacterial growth is not affected. The mechanism and biological significance of this temperature-dependent melanogenesis are not clear. Heterologous expression of an A.s.s. 4-hydroxyphenylpyruvate dioxygenase (HppD), the most crucial enzyme in the HGA-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In the current study, we demonstrated that the extreme thermolability of HppD is responsible for the temperature-dependent melanization of A.s.s. Substitutions in three residues, Ser18, Pro103, or Leu119 of HppD from A.s.s increases the thermolability of this enzyme and results in temperature-independent melanogenesis. Moreover, replacing the corresponding residues of HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A.s.s HppD leads to thermosensitive melanogenesis. Structural analysis suggested that mutations at these sites, especially at position P103, can strengthen the secondary structure of HppD and greatly improve its thermal stability. In addition, we found that HppD sequences of all A.s.s isolates are identical and that two of the three residues are completely conserved within A.s.s isolates, which clearly distinguishes these from other Aeromonas strains. We suggest that this property represents an adaptive strategy to the psychrophilic lifestyle of A.s.s.
Importance Aeromonas salmonicida subsp. salmonicida (A.s.s) is the causative agent of furunculosis, a bacterial septicemia of cold water fish of the Salmonidae family. As it has a well-defined host range, A.s.s has become an ideal model to investigate the co-evolution of host and pathogen. For many pathogens, melanin production is associated with virulence. Although other species of Aeromonas can produce melanin, A.s.s is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here we demonstrate that thermosensitive melanogenesis in A.s.s strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). The strictly conserved hppD sequences among A.s.s and the exclusive thermosensitive pigmentation of these strains might provide insight into the role of melanin in the adaptation to a particular host, and offer a novel molecular marker to readily differentiate A.s.s strains from other A. salmonicida subspecies and Aeromonas species.
