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Immunization with alkyl hydroperoxide reductase subunit C reduces Fusobacterium nucleatum load in the intestinal tract.

ABSTRACT: Fusobacterium nucleatum (Fn) is an important tumour-associated bacterium in colorectal cancer (CRC). The antioxidant protein alkyl hydroperoxide reductase subunit C (AhpC) can induce strong antibacterial immune response during various pathogen infections. Our study aimed to evaluate the efficacy of Fn-AhpC as a candidate vaccine. In this work, by western blot analysis, we showed that Fn-AhpC recombinant protein could be recognized specifically by antibodies present in the sera of CRC patients; using the mouse Fn-infection model, we observed that systemic prophylactic immunization with AhpC/alum conferred significant protection against infection in 77.3% of mice. In addition, we measured the anti-AhpC antibody level in the sera of CRC patients and found that there was no obvious increase of anti-AhpC antibodies in the early-stage CRC group. Furthermore, we treated Fn with the sera from both immunized mice and CRC patients and found that sera with high anti-AhpC antibodies titre could inhibit Fn growth. In conclusion, our findings support the use of AhpC as a potential vaccine candidate against inhabitation or infection of Fn in the intestinal tract, which could provide a practical strategy for the prevention of CRC associated with Fn infection.

SUBMITTER: Guo SH

PROVIDER: S-EPMC5585165 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Immunization with alkyl hydroperoxide reductase subunit C reduces Fusobacterium nucleatum load in the intestinal tract.

Guo Song-He SH Wang Hai-Fang HF Nian Zhi-Gang ZG Wang Yi-Dan YD Zeng Qiu-Yao QY Zhang Ge G

Scientific reports 20170905 1

Fusobacterium nucleatum (Fn) is an important tumour-associated bacterium in colorectal cancer (CRC). The antioxidant protein alkyl hydroperoxide reductase subunit C (AhpC) can induce strong antibacterial immune response during various pathogen infections. Our study aimed to evaluate the efficacy of Fn-AhpC as a candidate vaccine. In this work, by western blot analysis, we showed that Fn-AhpC recombinant protein could be recognized specifically by antibodies present in the sera of CRC patients; u ...[more]

PMID: 28874771

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Project description:AIM:To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria. METHODS:Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined. RESULTS:Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION:AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.

Project description:Fusobacterium nucleatum, an anaerobic Gram-negative bacterium frequently found in the human oral cavity and some extra-oral sites, is implicated in several important diseases: periodontitis, adverse pregnancy outcomes, and colorectal cancer. To date, how this obligate anaerobe copes with oxidative stress and host immunity within multiple human tissues remains unknown. Here, we uncovered a critical role in this process of a multigene locus encoding a single, fused methionine sulfoxide reductase (MsrAB), a two-component signal transduction system (ModRS), and thioredoxin (Trx)- and cytochrome c (CcdA)-like proteins, which are induced when fusobacterial cells are exposed to hydrogen peroxide. Comparative transcriptome analysis revealed that the response regulator ModR regulates a large regulon that includes trx, ccdA, and many metabolic genes. Significantly, specific mutants of the msrAB locus, including msrAB, are sensitive to reactive oxygen species and defective in adherence/invasion of colorectal epithelial cells. Strikingly, the msrAB mutant is also defective in survival in macrophages, and it is severely attenuated in virulence in a mouse model of preterm birth, consistent with its failure to spread to the amniotic fluid and colonize the placenta. Clearly, the MsrAB system regulated by the two-component system ModRS represents a major oxidative stress defense pathway that protects fusobacteria against oxidative damage in immune cells and confers virulence by enabling attachment and invasion of multiple target tissues. IMPORTANCE F. nucleatum colonizes various human tissues, including oral cavity, placenta, and colon. How this obligate anaerobe withstands oxidative stress in host immune cells has not been described. We report here that F. nucleatum possesses a five-gene locus encoding a fused methionine sulfoxide reductase (MsrAB), a two-component signal transduction system (ModRS), and thioredoxin- and cytochrome c-like proteins. Regulated by ModRS, MsrAB is essential for resistance to reactive oxygen species, adherence/invasion of colorectal epithelial cells, and survival in macrophage. Unable to colonize placenta and spread to amniotic fluid, the msrAB mutant failed to induce preterm birth in a murine model.

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Immunization with alkyl hydroperoxide reductase subunit C reduces Fusobacterium nucleatum load in the intestinal tract.

Publications

Immunization with alkyl hydroperoxide reductase subunit C reduces Fusobacterium nucleatum load in the intestinal tract.

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