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1 ISSN CN / Q http / /cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology ~ 98 DOI /j. cnki. cjbmb HdeA * 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 Hunan China 2 Shaoyang Municipal Center for Disease Control and Prevention Shaoyang Hunan China 3 Insititute of Pathogenic Biology University of South China Hengyang 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 No No. 14B164 * Tel ranhui81@ 163. com Received February Accepted May Supported by Key Laboratory of Hunan Province for Special Pathogens Prevention and Control Foundation under Grant No National Natural Science Foundation of China No and Outstanding Youth Project of Scientific Research Fund of Hunan Provincial Education Department No. 14B164 * Corresponding author Tel ranhui81@ 163. com

2 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 KpHdeA LB r /min 50 μl 5 ml LB ph h r /min ph min ph 2 4 LB 1 h 2 h 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 μl 50 ROX Reference Dye 0. 5 μl μl 10 μmol /L cdna Klebsiella pneumoniae 0. 5 μl RNase-free 25 μl s 95 5 s s KpHdeA 16S 15 RNA Ct KpHdeA KpHdeA HdeA GenBank KpHdeA KpHdeA F 5' -GGGCTAGCATGAAAGT TAAATCATTGG-3' NheⅠ NH12 30 s s 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 s pet28a-kphdea DNA pet28a KpHdeA

3 1 HdeA 95 NheⅠ BamHⅠ 190 ~ 250 nm CD pet28a KpHdeA T4 DNA nm 1. 0 nm h Top s 3 LB h 1. 9 PCR BL /pet28a BL /pet28a- Invitrogen 1. 5 KpHdeA pet28a-kphdea A BL21 Star DE3 BL / pet28a-kphdea LB r /min A LB ph LB mmol /L IPTG 37 A A mmol /L IPTG 28 6 h 3 PBS r /min min μm Ni mmol /L NaH 2 PO mol /L NaCl 40 mmol /L ph mmol /L NaH 2 PO mol /L NaCl 250 mmol /L 2 ph KpHdeA SDS-PAGE Bradford KpHdeA 1. 6 KpHdeA ADH ph h KpHdeA ADH Sigma KpHdeA 8 ph h ADH KpHdeA 10 Fig μmol /L 150 mmol /L KH 2 PO mmol /L NaCl 150 mmol /L NH 4 2 SO 4 ph min F nm KpHdeA ANS 1-Anilinonaphthalene-8-sulphonate ANS ph KpHdeA KpHdeA 10 μmol /L ANS 100 μmol /L 150 mmol /L KH 2 PO mmol /L NaCl 150 mmol /L NH 4 2 SO 4 ph 2. 0 KpHdeA LB LB mmol /L IPTG 28 2 h ± SPSS14. 0 P < PCR KpHdeA 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 mol /L Tris-HCl n = 3. CD Jasco J815 spectrometer Jasco P < Data present mean ± S. D.

4 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 DE 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 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 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 < ** P < ADH ph 2. 0 ADH 750 ph KpHdeA KpHdeA ph KpHdeA 7. 0 ANS Fig. 5 ph KpHdeA 7. 0 KpHdeA ANS ADH KpHdeA KpHdeA ph Fig ANS ph KpHdeA ANS ph 4. 0 ph ANS KpHdeA ANS

5 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 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 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 Cell growth was monitored by measuring the increase of A ph 2 KpHdeA 600 at the indicated hours. Data present mean ± S. D. n = 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 KpHdeA BL /pet28a BL /pet28a- KpHdeA KpHdeA 19

6 KpHdeA function HdeA 76% N strains of Listeria monocytogenes J KpHdeA KpHdeA pet28a-kphdea KpHdeA KpHdeA ADH ANS KpHdeA CD KpHdeA KpHdeA HdeA and growth ph J 2 12 KpHdeA HdeA KpHdeA KpHdeA KpHdeA KpHdeA KpHdeA References 1 Gorden J Small PL. Acid resistance in enteric bacteria J. Infect Immun Hong W Wu YE Fu X et al. Chaperone-dependent mechanisms for acid resistance in enteric bacteria J. Trends Microbiol HdeA HdeB J. Yu Y Q Yu Z C Li Y N. Mechanisms of molecular chaperones HdeA and HdeB J. Chin J Biochem Mol Biol Chattopadhyay MK Tabor H. Polyamines are critical for the induction of the glutamate decarboxylase-dependent acid resistance system in Escherichia coli J. J Biol Chem Karatzas KA Suur L Byrne CP. Characterization of the intracellular glutamate decarboxylase system analysis of its transcription and role in the acid resistance of various 6 Marshall BJ Barrett LJ Prakash C et al.. Appl Environ Microbiol Urea protects Helicobacter Campylobacter pylori from the bactericidal effect of acid J. Gastroenterology Gajiwala KS Burley SK. HDEA a periplasmic protein that supports acid resistance in pathogenic enteric bacteria J. J Mol Biol Valderas MW Alcantara RB Baumgartner JE et al. Role of HdeA in acid resistance and virulence in Brucella abortus 2308 J. Vet Microbiol Small P Blankenhorn D Welty D et al. Acid and base resistance in Escherichia coli and Shigella flexneri role of rpos. J Bacteriol Hong W Jiao W Hu J et al. Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach ph range by transforming into disordered conformation J. J Biol Chem Zhang M Lin S Song X et al. A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance J. Nat Chem Biol Wu YE Hong W Liu C et al. Conserved amphiphilic feature is essential for periplasmic chaperone HdeA to support acid resistance in enteric bacteria J. Biochem J Wu KM Li LH Yan JJ et al. Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044 a strain causing liver abscess and meningitis J. J Bacteriol 14 Garbati MA Al Godhair AI. The growing resistance of Klebsiella pneumoniae the need to expand our antibiogram case report and review of the literature J. Afr J Infect Dis Muzaheed Doi Y Adams-Haduch JM et al. Faecal carriage of CTX-M-15-producing Klebsiella pneumoniae in patients with acute gastroenteritis J. Indian J Med Res Maroncle N Rich C Forestier C. The role of Klebsiella pneumoniae urease in intestinal colonization and resistance to gastrointestinal stress J. Res Microbiol Coudeyras S Nakusi L Charbonnel N et al. A tripartite efflux pump involved in gastrointestinal colonization by Klebsiella pneumoniae confers a tolerance response to inorganic acid J. Infect Immun Malki A Le HT Milles S et al Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB J. J Biol Chem Bhat KG K Alex. Acid resistance in enteric bacteria J. Natl Med J India

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