4-Hydroxyphenylpyruvate Dioxygenase Thermolability Is Responsible for Temperature-Dependent Melanogenesis in Aeromonas salmonicida subsp. salmonicida

Abstract

Aeromonas salmonicida subsp. salmonicida is a major pathogen affecting fisheries worldwide and is a well-known pigmented member of the Aeromonas genus. This subspecies produces melanin at ≤22°C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30°C to 35°C, while bacterial growth is unaffected. The mechanism and biological significance of this temperature-dependent melanogenesis remain unclear. Heterologous expression of an A. salmonicida subsp. salmonicida 4-hydroxyphenylpyruvate dioxygenase (HppD), the most critical enzyme in the homogentisic acid (HGA)-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In this study, we demonstrated that the thermolability of HppD is responsible for the temperature-dependent melanization of A. salmonicida subsp. salmonicida. Substitutions of three residues, S18T, P103Q, and L119P, in A. salmonicida subsp. salmonicida HppD increased the thermostability of this enzyme and resulted in temperature-independent melanogenesis. Moreover, the replacement of the corresponding residues in HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A. salmonicida subsp. salmonicida HppD resulted in thermosensitive melanogenesis. A structural analysis suggested that mutations at these sites, especially at position P103, strengthen the secondary structure of HppD and greatly improve its thermal stability. Additionally, we found that the HppD sequences of all A. salmonicida subsp. salmonicida isolates were identical and that two of the three residues were clearly distinct from those of other Aeromonas strains. IMPORTANCE Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a bacterial septicemia of cold-water fish of the Salmonidae family. Although other Aeromonas species can produce melanin, A. salmonicida subsp. salmonicida is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here, we demonstrated that thermosensitive melanogenesis in A. salmonicida subsp. salmonicida strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). Additionally, we confirmed that this thermolabile HppD exhibited higher activity at low temperatures than its mesophilic homologues, suggesting this as an adaptive strategy of this enzyme to the psychrophilic lifestyle of A. salmonicida subsp. salmonicida. The strictly conserved hppD sequences among A. salmonicida subsp. salmonicida isolates and the specific possession of P103 and L119 residues could be used as a reference for the identification of A. salmonicida subsp. salmonicida isolates.

Identificación y caracterización de Aeromonas sp patógenas aisladas de truchas arcoíris (Oncorhynchus mykiss) clínicamente enfermas cultivadas en piscigranjas del Perú

“…Sin embargo, hasta el momento no se habían reportado Aeromonas en truchas de cultivo en Cajamarca. La presentación del pigmento marrón soluble en TSA observado en una de las cuatro cepas A. salmonicida, descrito por Baca (2012) y Buller (2014), varía por el contenido de triptona y la termolabilidad de la hidroxifenilpiruvato dioxigenasa, según lo descrito por Qiao et al (2018).…”

Hiding in Plain Sight: Characterization of Aeromonas Species Isolated from a Recreational Estuary Reveals the Carriage and Putative Dissemination of Resistance Genes

“…Another feature of some species of Aeromonas is the production of melanin and multiple enzymes previously reported in the synthesis pathways of this pigment were found. Melanin plays an important role in the protection against environmental stress, such as radiation, oxidative damage, and heavy metals, among others, and may also contribute to bacterial virulence [35]. For instance, a strain of A. media isolated from a lake in China was able to synthesize high levels of melanin, which was also proposed to be explored from a biotechnological perspective, namely in the photoprotection of bioinsecticides [36].…”

AHL-Based Quorum Sensing Regulates the Biosynthesis of a Variety of Bioactive Molecules in Bacteria

“…225 These compounds contribute to improve the survival of microorganisms by increasing UV resistance, enhancing virulence, and interfering with the immune response of the host. 226,227 In addition, melanin and pyomelanin have antibacterial activities against S. aureus and E. coli and antifungal activity against C. albicans. 228 Acinetobacter baumannii is a human nosocomial pathogen responsible for serious infections such as pneumonia, sepsis, and meningitis.…”

“…In V. anguillarum , VanT, a homologue of V. harveyi LuxR regulator, was shown to be involved in the biosynthesis of melanin and pyomelanin pigments . These compounds contribute to improve the survival of microorganisms by increasing UV resistance, enhancing virulence, and interfering with the immune response of the host. , In addition, melanin and pyomelanin have antibacterial activities against S. aureus and E. coli and antifungal activity against C. albicans …”