Dataset Information

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.

SUBMITTER: Qiao Y

PROVIDER: S-EPMC6293093 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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

Qiao Yunqian Y Wang Jiao J Wang He H Chai Baozhong B Rao Chufeng C Chen Xiangdong X Du Shishen S

Applied and environmental microbiology 20181213 1

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 a ...[more]

PMID: 30341077

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Project description:Background Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Despite the identification of several virulence factors the pathogenesis is still poorly understood. We have used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF5054) and T3SS-deficient (isogenic DeltaascV, extremely low-virulent, JF2747) strains in exponential (GP) and stationary (SP) phases of growth. Results Among the different experimental conditions we obtained semi-quantitative values for a total of 2136 A. salmonicida proteins. Proteins of specific A. salmonicida species were proportionally less detected than proteins common to the Aeromonas genus or those shared with other Aeromonas species, suggesting that in vitro growth did not induce the expression of these genes. Four detected proteins which are unidentified in the genome of reference strains of A. salmonicida were homologous to components of the conjugative T4SS of A. hydrophila pRA1 plasmid. Polypeptides of three proteins which are specific to the 01-B526 strain were also discovered. In supernatants (SNs), the number of detected proteins was higher in SP (326 for wt vs 329 for mutant) than in GP (275 for wt vs 263 for mutant). In pellets, the number of identified proteins (a total of 1536) was approximately the same between GP and SP. Numerous highly conserved cytoplasmic proteins were present in A. salmonicida SNs (mainly EF-Tu, EF-G, EF-P, EF-Ts, TypA, AlaS, ribosomal proteins, HtpG, DnaK, peptidyl-prolyl cis-trans isomerases, GAPDH, Enolase, FbaA, TpiA, Pgk, TktA, AckA, AcnB, Mdh, AhpC, Tpx, SodB and PNPase), and several evidences support the theory that their extracellular localization was not the result of cell lysis. According to the Cluster of Orthologous Groups classification, 29% of excreted proteins in A. salmonicida SNs were currently poorly characterized. Conclusions In this part of our work we elucidated the whole in vitro exoproteome of hypervirulent A. salmonicida subsp. salmonicida and showed the secretion of several highly conserved cytoplasmic proteins with putative moonlighting functions and roles in virulence. All together, our results offer new information about the pathogenesis of furunculosis and point out potential candidates for vaccine development.

Project description:Furunculosis, a serious infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida is common in sea-reared rainbow trout production in Denmark. Developing an effective control strategy requires knowledge of the epidemiology, as well as the genomic and virulent variability of the Danish A. salmonicida subsp. salmonicida isolates. To obtain this, the genomes of 101 A. salmonicida subsp. salmonicida, including 99 Danish isolates, one Scottish strain and the type strain NCIMB 1102, were sequenced using the Illumina HiSeq platform. Isolates were de novo assembled, examined for presence of plasmids, virulence and iron acquisition proteins, genomic islands, and antibiotic resistance genes. Single Nucleotide Polymorphisms were aligned and subjected to Bayesian temporal phylogenetic and maximum likelihood tree reconstruction using the published genome of A. salmonicida subsp. salmonicida A449 as reference. Bayesian temporal phylogenetic reconstruction suggests that four major introductions of A. salmonicida subsp. salmonicida into Denmark have occurred. The introductions correlate with the freshwater and subsequent seawater expansion of rainbow trout production. Initial transmission of the bacterium could have been from seawater to freshwater or vice versa, and most minor clades include a mixture of strains from different fresh- and seawater farms. Genomic variation of A. salmonicida subsp. salmonicida mostly appeared to be associated with their plasmids and plasmid encoded virulence factors. Nine A. salmonicida subsp. salmonicida isolates harbored worldwide known antibiotic resistance genes against several antibiotics and there is an indication that 33% of the isolates contained the genomic island AsaGEI1b. These findings not only support the usefulness of whole genome sequencing for genetic studies of homogeneous bacteria in general, but provide novel information about the Danish A. salmonicida subsp. salmonicida population, with implications for vaccine development in efforts to better protect Danish rainbow trout in the future.

Project description:Furunculosis, which is caused by Aeromonas salmonicida subsp. salmonicida, is a major salmonid disease in fish farms worldwide. Several plasmids found in this bacterium confer phenotypes such drug resistance and virulence. Small plasmids (pAsa1, pAsa2, pAsa3, and pAsal1) related to ColE1- and ColE2-type replicons are usually present in its normal plasmidome. In the present study, with the objective to investigate if these plasmids display particularities related to the origin of the isolates bearing them, a total of 153 isolates, including 78 new and 75 previously described, were analyzed for the presence of small plasmids by PCR and DNA restriction fragment profiling. A geographical dichotomy between Canadian and European isolates for their propensity to do not have pAsa3 or pAsal1 was found. In addition, the genotyping analysis led to the identification of two European isolates harboring an unusual pAsal1. An investigation by next-generation sequencing (NGS) of these two isolates shed light on two pAsal1 variants (pAsal1C and pAsal1D). As with pAsal1B, another pAsal1 variant previously described, these two new variants bore a second insertion sequence (ISAS5) in addition to the usual ISAS11. The characterization of these variants suggested that they could predominate over the wild-type pAsal1 in stressful conditions such as growth at temperatures of 25°C and above. To obtain a comprehensive portrait of the mutational pressure on small plasmids, 26 isolates whose DNA had been sequenced by NGS were investigated. pAsa3 and pAsal1 were more prone to mutations than pAsa1 and pAsa2, especially in the mobA gene, which encodes a relaxase and a primase. Lastly, the average copy number of each plasmid per cell was assessed using raw sequencing data. A clear trend with respect to the relative proportion per cell of each plasmid was identified. Our large-scale study revealed a geographical dichotomy in small plasmid repertoire in addition to a clear trend for pAsa3 and pAsal1 to be more frequently altered. Moreover, we present the discovery of two new variants of pAsal1: pAsal1C and pAsal1D.

Project description:BACKGROUND: Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish. RESULTS: The nucleotide sequences of the A. salmonicida subsp. salmonicida A449 chromosome and two large plasmids are characterized. The chromosome is 4,702,402 bp and encodes 4388 genes, while the two large plasmids are 166,749 and 155,098 bp with 178 and 164 genes, respectively. Notable features are a large inversion in the chromosome and, in one of the large plasmids, the presence of a Tn21 composite transposon containing mercury resistance genes and an In2 integron encoding genes for resistance to streptomycin/spectinomycin, quaternary ammonia compounds, sulphonamides and chloramphenicol. A large number of genes encoding potential virulence factors were identified; however, many appear to be pseudogenes since they contain insertion sequences, frameshifts or in-frame stop codons. A total of 170 pseudogenes and 88 insertion sequences (of ten different types) are found in the A. salmonicida genome. Comparison with the A. hydrophila ATCC 7966T genome reveals multiple large inversions in the chromosome as well as an approximately 9% difference in gene content indicating instances of single gene or operon loss or gain.A limited number of the pseudogenes found in A. salmonicida A449 were investigated in other Aeromonas strains and species. While nearly all the pseudogenes tested are present in A. salmonicida subsp. salmonicida strains, only about 25% were found in other A. salmonicida subspecies and none were detected in other Aeromonas species. CONCLUSION: Relative to the A. hydrophila ATCC 7966T genome, the A. salmonicida subsp. salmonicida genome has acquired multiple mobile genetic elements, undergone substantial rearrangement and developed a significant number of pseudogenes. These changes appear to be a consequence of adaptation to a specific host, salmonid fish, and provide insights into the mechanisms used by the bacterium for infection and avoidance of host defence systems.

Dataset Information

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

Publications

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

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