http / /cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology

ISSN 1007-7626 CN 11-3870 / Q http / /cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology 2016 1 32 1 93 ~ 98 DOI 10. 13865 /j. cnki. cjbmb. 2016. 01. 14 HdeA 1 3 2 3 3 3 * 1 422000 2 422000 3 421001 KpHdeA KpHdeA KpHdeA KpHdeA KpHdeA KpHdeA KpHdeA KpHdeA Q936 Characterization of the Klebsiella pneumoniae HdeA Protein under Acidic Condition 1 LIU Yan 3 LIU Dan 2 YOU Xiao-Xing 3 Jiang Chuan-Hao 3 LI Ran-Hui 3 * 1 Shaoyang Medical College Shaoyang 422000 Hunan China 2 Shaoyang Municipal Center for Disease Control and Prevention Shaoyang 422000 Hunan China 3 Insititute of Pathogenic Biology University of South China Hengyang 421001 Hunan China Abstract Klebsiella pneumoniae is an important conditional pathogen in hospital infection which frequently colonizes the respiratory and intestinal tracts of human. How K. pneumoniae escapes the acid stress of gastric acid and then enters the intestinal remains unclear. The expression of KpHdeA was upregulated in K. pneumoniae under acid stress. The coding sequence of KpHdeA was cloned from K. pneumoniae and overexpressed in Escherichia coli. Soluble KpHdeA was purified by Ni 2 + -affinity chromatography with apparent homogeneity. The light scattering assay showed that the acid-induced alcohol dehydrogenase ADH aggregation was attenuated by KpHdeA. The fluorescence emission spectra indicated that the hydrophobic surfaces of KpHdeA protein were exposed under acidic condition. CD spectra also indicated that KpHdeA protein could form a disordered conformation when treated with acid. Additionally over-expression of KpHdeA could confer anti-acid capability on E. coli. It was concluded that KpHdeA was a molecular chaperone and was crucial for acid resistance of K. pneumoniae. 2015-02-07 2015-05-11 2014 5 No. 31000091 No. 14B164 * Tel 0734-8282907 E-mail ranhui81@ 163. com Received February 7 2015 Accepted May 11 2015 Supported by Key Laboratory of Hunan Province for Special Pathogens Prevention and Control Foundation under Grant No. 2014-5 National Natural Science Foundation of China No. 31000091 and Outstanding Youth Project of Scientific Research Fund of Hunan Provincial Education Department No. 14B164 * Corresponding author Tel 0734-8282907 E-mail ranhui81@ 163. com

94 32 Key words Klebsiella pneumonia molecular chaperone anti-acid capability secondary structure ph 1 ~ 3 TaKaRa NheⅠ BamHⅠ T4 DNA IPTG Fermentas Trizol Invitrogen PCR Invitrogen 1 2 Ni 2 + GE 3 1. 2 KpHdeA LB 37 200 r /min 50 μl 5 ml LB ph7. 0 6 h 8 000 r /min ph 4 5 1 min ph 2 4 LB 1 h 2 h 8 000 r /min 6 1 min TaKaRa RNA HdeA RNA cdna HdeA KpHdeA 7-9 HdeA 16 S RNA PCR 10 KpHdeA KHF 5'-GGCGCACTGTT ph HdeA TTTAACCTCTGC-3' KHR 5'- HdeA HdeA ACGTTGAGAACCGCATCC TCCG-3' 16S RNA 16F 5'-ATGACCAGCCACACTGGAAC-3' 16R 5'-CTTCCTCCCCGCTGAAAGTA-3' 11 HdeA HdeA 14 PCR 2 SYBR Premix Ex TM TaqII 12. 5 μl 50 ROX Reference Dye 0. 5 μl 10 12 1 μl 10 μmol /L cdna Klebsiella pneumoniae 0. 5 μl RNase-free 25 μl 95 30 s 95 5 s 60 31 s 40 13 14 KpHdeA 16S 15 RNA Ct KpHdeA 16 17 1. 3 KpHdeA HdeA GenBank KpHdeA KpHdeA F 5' -GGGCTAGCATGAAAGT TAAATCATTGG-3' NheⅠ 1 1. 1 NH12 30 s 72 60 s 30 72 10 min PCR Top10 BL21 Star DE3 pet28a HpHdeA PCR DNA ExTaq GenBank KpHdeA R 5'-TTGGATCCTTAATAGATTTCCTTTTTC AGATT -3' BamHⅠ DNA PCR KpHdeA PCR 94 5 min 94 30 s 55 1. 4 pet28a-kphdea DNA pet28a KpHdeA

1 HdeA 95 NheⅠ BamHⅠ 190 ~ 250 nm CD pet28a KpHdeA T4 DNA 25 0. 5 nm 1. 0 nm 16 12 h Top100. 5 s 3 LB 37 16 h 1. 9 PCR BL /pet28a BL /pet28a- Invitrogen 1. 5 KpHdeA pet28a-kphdea A 600 0. 5 0. 5 BL21 Star DE3 BL / pet28a-kphdea LB 37 200 r /min A 600 0. 1 1 200 LB ph 1 100 LB 3. 0 0. 5 mmol /L IPTG 37 A 600 0. 5 A 600 0. 5 mmol /L IPTG 28 6 h 3 PBS 12 000 r /min 1. 10 10 min 0. 45 μm Ni 2 + 50 mmol /L NaH 2 PO 4 0. 3 mol /L NaCl 40 mmol /L ph 8. 0 50 mmol /L NaH 2 PO 4 0. 3 mol /L NaCl 250 mmol /L 2 ph 8. 0 2. 1 KpHdeA SDS-PAGE Bradford KpHdeA 1. 6 KpHdeA ADH ph 4. 0 1 h KpHdeA ADH Sigma KpHdeA 8 ph 2. 0 1h 1 0 1 1 1 2 1 3 ADH KpHdeA 10 Fig. 1 10 μmol /L 150 mmol /L KH 2 PO 4 150 mmol /L NaCl 150 mmol /L NH 4 2 SO 4 ph 2. 0 4. 0 30 min F-4500 400 nm 25 3 3 1. 7 KpHdeA ANS 1-Anilinonaphthalene-8-sulphonate ANS ph KpHdeA KpHdeA 10 μmol /L ANS 100 μmol /L 150 mmol /L KH 2 PO 4 150 mmol /L NaCl 150 mmol /L NH 4 2 SO 4 ph 2. 0 KpHdeA LB 1 100 LB mmol /L IPTG 28 2 h ± SPSS14. 0 P < 0. 05 PCR KpHdeA 4. 0 7. 0 F-4500 Fig. 1 The relative expressions of the KpHdeA mrna 370 nm 420 ~ 590 under acid stress Klebsiella pneumoniae was grown in LB nm 25 medium ph7. 0 for 6 h and then transferred into ph 2. 0 or 1. 8 CD ph 4. 0-LB medium for 1 or 2 h. The expression levels of the 10 μmol /L KpHdeA ph KpHdeA mrna were quantified by RT-qPCR and normalized to the levels of 16S rrna. 2. 0 7. 0 0. 1 mol /L Tris-HCl n = 3. CD Jasco J815 spectrometer Jasco P < 0. 05 Data present mean ± S. D.

96 32 2. 2 KpHdeA KpHdeA 330 bp Fig. 2A PCR pet28a pet28a-kphdea Top10 Kana LB pet28-kphdea PCR KpHdeA Fig. 2B DNA GenBank CDL23413 Fig. 3 The expression and purification of recombinant KpHdeA protein The proteins were separated by 15% PAGE M Molecular weight marker 1 The proteins of BL21 / pet28a induced by IPTG 2 The proteins of BL21 /pet28a- KpHdeA induced by IPTG 3 The KpHdeA protein after purified by Ni 2 + affinity chromatography Fig. 2 Electrophoretic profile of KpHdeA amplification and PCR identification A Gene amplification of KpHdeA. 1 PCR product M DNA marker. B Identification of plasmid pet28a-kphdea by PCR. 1 PCR product M DNA marker 2. 3 KpHdeA pet28a-kphdea BL21 DE3 0. 5 mmol /L IPTG 28 KpHdeA Fig. 4 ADH aggregation is prevented by KpHdeA SDS-PAGE protein under acid stress The yeast alcohol 14 kd KpHdeA dehydrogenase ADH 10 μmol /L in the buffer solution Fig. 3 6 his containing 150 mmol /L KH 2 PO 4 150 mmol /L NaCl and 150 mmol /L NH 4 2 SO 4 ph 2. 0 was mixed with SDS-PAGE KpHdeA protein at the molar ratio of 1 0 1 1 1 2 and 1 14 kd 3 and incubated for 30 min. The aggregation of ADH was KpHdeA 98% Fig. 3 3 analyzed by the record of light scattering intensity. Data 2. 4 KpHdeA present mean ± S. D. n = 3. * P < 0. 05 ** P < 0. 01 ADH ph 2. 0 ADH 750 ph KpHdeA KpHdeA ph 2. 0 4. 0 500 KpHdeA 7. 0 ANS Fig. 5 ph KpHdeA 7. 0 KpHdeA ANS ADH KpHdeA KpHdeA ph Fig. 4 4. 0 ANS ph 2. 0 2. 5 KpHdeA ANS ph 4. 0 ph ANS KpHdeA ANS

1 HdeA 97 A 600 Fig. 7 Fig. 7 8 h 20 h 12 h 24 h KpHdeA Fig. 5 Fluorescence emission spectra of ANS 100 μmol /L in the presence of KpHdeA 10 μmol /L at different ph value ph 7. 0 4. 0 or 2. 0 after being excited at 395 nm 2. 6 KpHdeA CD KpHdeA ph Fig. 7 Over-expression of KpHdeA confers anti-acid ph 2. 0 7. 0 CD CD capability on E. coli The E. coli cells transformed by BL / Fig. 6 KpHdeA pet28a-kphdea plasmid and control cells transformed by BL / 208 nm 222 nm 2 pet28a empty vector were cultured in liquid LB at ph 3. 0. Cell growth was monitored by measuring the increase of A ph 2 KpHdeA 600 at the indicated hours. Data present mean ± S. D. n = 3 198 1 KpHdeA KpHdeA 3 Fig. 6 CD spectra of KpHdeA under different ph conditions The CD spectra from 190 nm to 250 nm of KpHdeA 10 μmol /L were recorded under ph 2. 0 and 7. 0 conditions respectively HdeA HdeA 2 7 HdeA HdeA HdeA HdeA 7-9 ph HdeA 12 HdeA HdeA 10 18 2. 7 KpHdeA BL /pet28a BL /pet28a- KpHdeA KpHdeA 19

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