Recombinant enolase of Candida albicans could induce the formation of neutrophil extracellular traps

doi:10.3877/cma.j.issn.1674-1366.2018.02.002

Abstract

Abstract:

Objective

To investigate the effect of enolase of Candida albicans (C.albicans) on human neutrophils.

Methods

Molecular cloning method was applied to plasmids construcion, cloning, expression and recombinant enolase purification, and density gradient centrifugation was used to isolate the human neutrophils. The effects of recombinant enolase on human neutrophils were observed by DCFH-DA method, SYTOX green and immunofluorescence. The positive control (SC5314 group) was neutrophils stimulated by C.albicans SC5314 (neitrophil∶C.albicans = 5∶1) and the negative control was neutrophils without stimulation. One-Way ANOVA and Tukey′s test were applied for statistical analysis.

Results

Recombinant enolase, 46 kDa, was successfully cloned and purified. Recombinant enolase could stimulate neutrophils to produce the intracellular ROS, which has no significant difference compared with those induced by C.albicans SC5314 group at 30, 60 and 120 min. Sytox green quantitative and immunoflu-orescence observation of extracellular DNA showed that recombinant enolase also could induce human neutrophils to form NETs. NETs quantitative results showed that there was no significant difference between SC5314 group[ (12.41 ± 2.00) %] and enolase group[ (11.0 ± 2.5) %] at 2 h (F = 6.13, P= 0.0886) , but enolase group[ (22.2 ± 2.0) %) ]was significant lower than SC5314 group [ (31.4 ± 3.1) %] at 4 h (F = 9.745, P = 0.0370) .

Conclusion

Recombinant enolase of C.albicans could induce human neu-trophils to form NETs.

Key words: Enolase, Candida albicans, Neutrophils, Extracellular traps

Luping Sun, Xiaohuan Zhang, Jinhong Yang, Yanglan Liu, Yuxue Ou, Yan Hu. Recombinant enolase of Candida albicans could induce the formation of neutrophil extracellular traps[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(02): 76-82.

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Figures/Tables 5

References 24

[1]	Qin Y，Zhang L，Xu Z，et al. Innate immune cell response upon Candida albicans infection[J]. Virulence，2016，7（5）：512-526.
[2]	Brown GD. Innate antifungal immunity：the key role of phagocytes[J]. Annu Rev Immunol，2011（29）：1-21.
[3]	Kumar V，Sharma A. Neutrophils：Cinderella of innate immune system[J]. Int Immunopharmacol，2010，10（11）：1325-1334.
[4]	Segal AW. How neutrophils kill microbes[J]. Annu Rev Immunol，2005（23）：197-223.
[5]	Wright HL，Moots RJ，Bucknall RC，et al. Neutrophil function in inflammation and inflammatory diseases[J]. Rheumatology（Oxford），2010，49（9）：1618-1631.
[6]	Brinkmann V，Reichard U，Goosmann C，et al. Neutrophil extracellular traps kill bacteria[J]. Science，2004，303（5663）：1532-1535.
[7]	Brinkmann V，Zychlinsky A. Neutrophil extracellular traps：is immunity the second function of chromatin？[J]. J Cell Biol，2012，198（5）：773-783.
[8]	Urban CF，Reichard U，Brinkmann V，et al. Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms[J]. Cell Microbiol，2006，8（4）：668-676.
[9]	Gow NA，Hube B. Importance of the Candida albicans cell wall during commensalism and infection[J]. Curr Opin Microbiol，2012，15（4）：406-412.
[10]	Hall RA，Gow NA. Mannosylation in Candida albicans：role in cell wall function and immune recognition[J]. Mol Microbiol，2013，90（6）：1147-1161.
[11]	Kustrzeba-Wojcicka I，Golczak M. Enolase from Candida albicans--purification and characterization[J]. Comp Biochem Physiol B Biochem Mol Biol，2000，126（1）：109-120.
[12]	He ZX，Chen J，Li W，et al. Serological response and diagnostic value of recombinant candida cell wall protein enolase，phospho-glycerate kinase，and β-glucosidase[J]. Front Microbiol，2015（6）：920.
[13]	Angiolella L，Facchin M，Stringaro A，et al. Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics[J]. J Infect Dis，1996，173（3）：684-690.
[14]	Pitarch A，Díez-Orejas R，Molero G，et al. Analysis of the serologic response to systemic Candida albicans infection in a murine model[J]. Proteomics，2001，1（4）：550-559.
[15]	Li Wq，Hu Xc，Zhang X，et al. Immunisation with the glycolytic enzyme enolase confers effective protection against Candida albicans infection in mice[J]. Vaccine，2011，29（33）：5526-5533.
[16]	Zhang X，Zhao S，Sun L，et al. Different virulence of candida albicans is attributed to the ability of escape from neutrophil extracellular traps by secretion of DNase[J]. Am J Transl Res，2017，9（1）：50-62.
[17]	Montagnoli C，Sandini S，Bacci A，et al. Immunogenicity and protective effect of recombinant enolase of Candida albicans in a murine model of systemic candidiasis[J]. Med Mycol，2004，42（4）：319-324.
[18]	Mori Y，Yamaguchi M，Terao Y，et al. α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps[J]. J Biol Chem，2012，287（13）：10472-10481.
[19]	Bergmann S，Wild D，Diekmann O，et al. Identification of a novel plasmin（ogen）-binding motif in surface displayed alpha-enolase of Streptococcus pneumoniae[J]. Mol Microbiol，2003，49（2）：411-423.
[20]	von Köckritz-Blickwede M，Goldmann O，Thulin P，et al. Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation[J]. Blood，2008，111（6）：3070-3080.
[21]	Bianchi M，Hakkim A，Brinkmann V，et al. Restoration of NET formation by gene therapy in CGD controls aspergillosis[J]. Blood，2009，114（13）：2619-2622.
[22]	Behnen M，Leschczyk C，Möller S，et al. Immobilized immune complexes induce neutrophil extracellular trap release by human neutrophil granulocytes via FcγRⅢB and Mac-1[J]. J Immunol，2014，193（4）：1954-1965.
[23]	Keshari RS，Verma A，Barthwal MK，et al. Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced NETs release from human neutrophils[J]. J Cell Biochem，2013，114（3）：532-540.
[24]	Papayannopoulos V，Metzler KD，Hakkim A，et al. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps[J]. J Cell Biol，2010，191（3）：677-691.

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