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Quantitative proteomic analysis of cell envelope preparations under iron starvation stress in Aeromonas hydrophila.

ABSTRACT: Iron homeostasis is an essential process over the entire lives of both hosts and bacterial pathogens, and also plays roles in many other metabolic functions. Currently, knowledge is limited on the iron scavenging mechanism of the cell envelope in the aquatic pathogen, Aeromonas hydrophila. To understand the iron homeostasis mechanism in A. hydrophila, a dimethyl labelling based quantitative proteomics method was used to compare the differential expression of cell envelope proteins under iron starvation.A total of 542 cell envelope proteins were identified by LC-MS/MS, with 66 down-regulated and 104 up-regulated proteins. Bioinformatics analysis showed that outer membrane siderophores, heme and iron receptors, periplasmic iron binding proteins, inner membrane ABC transporters and H(+)-ATP synthase subunits increased in abundance while iron-cluster proteins, electron transport chain and redox proteins were down-regulated. Further q-PCR validation, in vivo addition of exogenous metabolites, and an enzyme inhibition assay revealed that redox, the energy generation process, and ATP synthase elevated the susceptibility of A. hydrophila to iron starvation.Our study demonstrates that the redox and energy generation process, and ATP synthase in A. hydrophila may play critical roles in iron acquisition under conditions of iron-stress. An understanding of the iron scavenging mechanism may be helpful for the development of strategies for preventing and treating A. hydrophila infection.

SUBMITTER: Yao Z

PROVIDER: S-EPMC4957856 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Quantitative proteomic analysis of cell envelope preparations under iron starvation stress in Aeromonas hydrophila.

Yao Zujie Z Wang Zhihong Z Sun Lina L Li Wanxin W Shi Yan Y Lin Ling L Lin Wenxiong W Lin Xiangmin X

BMC microbiology 20160722 1

<h4>Background</h4>Iron homeostasis is an essential process over the entire lives of both hosts and bacterial pathogens, and also plays roles in many other metabolic functions. Currently, knowledge is limited on the iron scavenging mechanism of the cell envelope in the aquatic pathogen, Aeromonas hydrophila. To understand the iron homeostasis mechanism in A. hydrophila, a dimethyl labelling based quantitative proteomics method was used to compare the differential expression of cell envelope prot ...[more]

PMID: 27448791

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Project description:The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of Aeromonas hydrophila is aerolysin related and crucial to the pathogenesis of Aeromonas infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (act), a portion of the act gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (phoA) of Escherichia coli. The act::phoA reporter gene was then introduced into the chromosome of A. hydrophila by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the act promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The act promoter exhibited optimal activity at pH 7.0 and at 37 degrees C, and maximal PhoA activity was noted when the culture was aerated. Using a Vibrio cholerae iron uptake regulator gene (fur) as a probe, a 2.6-kb SalI/HindIII DNA fragment from an A. hydrophila chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the fur gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of V. cholerae. Complementation experiments demonstrated that the A. hydrophila fur gene could restore iron regulation in an E. coli fur-minus mutant. Using the suicide vector pDMS197, we generated a fur isogenic mutant of wild-type A. hydrophila SSU. Northern blot analysis data indicated that the repression in the transcription of the act gene by iron was relieved in the fur isogenic mutant. Further, iron regulation in the fur isogenic mutant of A. hydrophila could be restored by complementation. These results are important in understanding the regulation of the act gene under in vivo conditions.

Project description:Aeromonas is recognized as a human pathogen following ingestion of contaminated food and water. One major problem in Aeromonas identification is that certain species are phenotypically very similar. The antimicrobial resistance is another significant challenge worldwide. We therefore aimed to use mass spectrometry technology for identification and discrimination of Aeromonas species and to screen the antimicrobial resistance of Aeromonas hydrophila (A. hydrophila). A total of 150 chicken meat and water samples were cultured, and then, the isolates were identified biochemically by the Vitek® 2 Compact system. Proteomic identification was performed by MALDI-TOF MS and confirmed by a microchannel fluidics electrophoresis assay. Principal component analysis (PCA) and single-peak analysis created by MALDI were also used to discriminate the Aeromonas species. The antimicrobial resistance of the A. hydrophila isolates was determined by Vitek® 2 AST cards. In total, 43 samples were positive for Aeromonas and comprised 22 A. hydrophila, 12 Aeromonas caviae (A. caviae), and 9 Aeromonas sobria (A. sobria) isolates. Thirty-nine out of 43 (90.69%) Aeromonas isolates were identified by the Vitek® 2 Compact system, whereas 100% of the Aeromonas isolates were correctly identified by MALDI-TOF MS with a score value ≥2.00. PCA successfully separated A. hydrophila, A. caviae and A. sobria isolates into two groups. Single-peak analysis revealed four discriminating peaks that separated A. hydrophila from A. caviae and A. sobria isolates. The resistance of A. hydrophila to antibiotics was 95.46% for ampicillin, 50% for cefotaxime, 45.45% for norfloxacin and pefloxacin, 36.36% for ceftazidime and ciprofloxacin, 31.81% for ofloxacin and 27.27% for nalidixic acid and tobramycin. In conclusion, chicken meat and water were tainted with Aeromonas spp., with a high occurrence of A. hydrophila. MALDI-TOF MS is a powerful technique for characterizing aeromonads at the genus and species levels. Future studies should investigate the resistance of A. hydrophila to various antimicrobial agents.

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Quantitative proteomic analysis of cell envelope preparations under iron starvation stress in Aeromonas hydrophila.

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Quantitative proteomic analysis of cell envelope preparations under iron starvation stress in Aeromonas hydrophila.

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