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Frontiers in Cellular and Infection Microbiology logoLink to Frontiers in Cellular and Infection Microbiology
. 2017 Feb 22;7:45. doi: 10.3389/fcimb.2017.00045

Colistin Resistance in Acinetobacter baumannii MDR-ZJ06 Revealed by a Multiomics Approach

Xiaoting Hua 1,2, Lilin Liu 1,2, Youhong Fang 3, Qiucheng Shi 1,2, Xi Li 4, Qiong Chen 5, Keren Shi 1,2, Yan Jiang 1,2, Hua Zhou 6, Yunsong Yu 1,2,7,*
PMCID: PMC5319971  PMID: 28275586

Abstract

Acinetobacter baumannii has emerged as an important opportunistic pathogen due to its ability to acquire resistance to most currently available antibiotics. Colistin is often considered as the last line of therapy for infections caused by multidrug-resistant A. baumannii (MDRAB). However, colistin-resistant A. baumannii strain has recently been reported. To explore how multiple drug-resistant A. baumannii responded to colistin resistance, we compared the genomic, transcriptional and proteomic profile of A. baumannii MDR-ZJ06 to the induced colistin-resistant strain ZJ06-200P5-1. Genomic analysis showed that lpxC was inactivated by ISAba1 insertion, leading to LPS loss. Transcriptional analysis demonstrated that the colistin-resistant strain regulated its metabolism. Proteomic analysis suggested increased expression of the RND efflux pump system and down-regulation of FabZ and β-lactamase. These alterations were believed to be response to LPS loss. In summary, the lpxC mutation not only established colistin resistance but also altered global gene expression.

Keywords: Acinetobacter baumannii, colistin, whole-genome sequencing, transcriptome, proteome

Introduction

Acinetobacter baumannii has emerged as an important opportunistic pathogen due to its ability to acquire resistance to most currently available antibiotics (Peleg et al., 2008; Howard et al., 2012; Antunes et al., 2014). Since current treatment options for multi-drug resistant (MDR) A. baumannii are extremely limited, colistin is often considered as the last line of the therapy for infections caused by MDR A. baumannii (Bae et al., 2016; Cheah et al., 2016b). However, colistin-resistant A. baumannii strain has recently been reported (Cai et al., 2012).

Colistin is a polycationic antimicrobial peptide that targets the polyanionic bacterial lipopolysaccharide (LPS) of Gram-negative bacteria. Two different colistin resistance mechanisms have previously been reported (Beceiro et al., 2014). The first mechanism inactivates the lipid A biosynthesis pathway, leading to the complete loss of surface LPS. Mutations in lpxC, lpxA, or lpxD are involved in the first mechanism. The pmrAB two-component system mediates the second resistance mechanism. Mutations in pmrA and pmrB induce the activity of pmrC, which adds phosphoethanolamine (PEtn) to the hepta-acylated form of lipid A (Beceiro et al., 2011). Further mutations in vacJ, pldA, ttg2C, pheS and a conserved hypothetical protein were reported to involve in reduced colistin susceptibility through novel resistance mechanisms (Thi Khanh Nhu et al., 2016). Four putative colistin resistant genes: A1S_1983, hepA, A1S_3026, and rsfS were also identified in our previous study (Mu et al., 2016).

The response to LPS alteration has been investigated via transcriptional analysis. In response to LPS alteration, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface (lpxA; Henry et al., 2012) or alters the expression of genes associated with outer membrane structure and biogenesis (pmrB; Cheah et al., 2016a). Moreover, the response to colistin is highly similar to the transcriptional alteration observed in an LPS-deficient strain (Henry et al., 2015). Colistin resistance was also explored using proteomic methods. There were 35 differentially expressed proteins. Most differentially expressed proteins were down-regulated in the colistin resistant strain, including outer membrane proteins, chaperones, protein biosynthesis factors, and metabolic enzymes (Fernandez-Reyes et al., 2009). However, the combination of genomic, transcriptomic, and proteomic methods to examine the colistin resistance mechanism in A. baumannii has rarely been reported. Furthermore, the strain used in this study was an MDR strain, but not laboratory strains (ATCC 19606, ATCC 17978) that do not represent clonal lineages in a clinical environment. Here, we used genome, transcriptome, and proteome to elucidate the colistin resistance mechanism in MDR A. baumannii. There was an ISAba1 insertion in lpxC (ABZJ_03720) in ZJ06-200P5-1 compared with the genome sequence of MDR-ZJ06, where lpxC encoded an UDP-3-O-acyl-N-acetylglucosamine deacetylase.

Materials and methods

Bacterial strains, media, and antibiotics

Restriction enzymes, T4 ligase, and Taq DNA polymerase were purchased from TaKaRa (Otsu, Shiga, Japan). The A. baumannii strain MDR-ZJ06 was isolated from the bloodstream of a patient in Hangzhou, China, in 2006. All A. baumannii cultures were grown at 37 °C in Mueller-Hinton (MH) agar and cation-adjusted MH broth (CAMHB) (Oxoid, Basingstoke, UK). Colistin was purchased from Sigma (Shanghai, China).

Generation of colistin-resistant mutant

A colistin-resistant mutant was generated in A. baumannii MDR-ZJ06 by a previously described method (Li et al., 2006). Briefly, first, MDR-ZJ06 was cultured in CAMHB containing colistin at 8 × minimum inhibitory concentration (MIC). After overnight incubation, the culture was diluted 1:1000 with CAMHB containing colistin at 64 × MIC and then incubated at 37 °C overnight. Finally, the culture was diluted 1:100 with CAMHB containing colistin at 200 × MIC. After overnight incubation, the culture was plated on plates containing 10 μg of colistin at an appropriate dilution, and then one of colistin resistant colonies was collected for further experiments and designated as ZJ06-200P5-1. MICs for colistin and tigecycline were determined by E-test (bioMérieux, France) on MH agar, and the antimicrobial activities of the other antimicrobial agents were detected by disk diffusion. The results were interpreted according to CLSI or EUCAST breakpoints.

Whole genome DNA sequencing and analysis

ZJ06-200P5-1 cells were cultured from a single colony overnight at 37 °C in MH broth. The genomic DNA was extracted via a QIAamp DNA minikit (Qiagen, Valencia, CA) following the manufacturer's protocol. Agarose gel and a NanoDrop spectrophotometer were used to determine the quality and quantity of extracted genomic DNA. The 300 bp library for Illumina paired-end sequencing was constructed from 5 μg of genome DNA of ZJ06-200P5-1 by staff at Zhejiang Tianke (Hangzhou, China). Mapping and SNP detection were performed via Breseq (Deatherage and Barrick, 2014). The regions containing the detected SNPs were amplified by PCR. The PCR products were sent to Biosune (Biosune, Hangzhou, China) for Sanger sequencing.

Transcriptome analysis and real-time quantitative PCR verification

A. baumannii MDR-ZJ06 and ZJ06-200P5-1 were grown overnight at 37 °C in LB broth. Strains were subcultured 1/100 into fresh LB broth and grown at 37 °C for 2 h (OD600: 0.29 ± 0.02 for MDR-ZJ06, 0.26 ± 0.02 for ZJ06-200P5-1). The cells were collected at 4 °C, and the RNA was extracted using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA) after liquid nitrogen grinding. For RNA sequencing, wild type and mutants were sampled in triplicate. The subsequent RNA extraction, bacteria mRNA sequence library construction, transcriptome analysis and real-time quantitative PCR verification were performed by staff at Zhejiang Tianke (Hangzhou, China) as described previously in reference (Hua et al., 2014). Sequenced reads were mapped to the MDR-ZJ06 genome (CP001937-8) using Rockhopper (McClure et al., 2013). The output data was analyzed by edgeR (McCarthy et al., 2012). Data generated by RNA sequencing were deposited to the NCBI Sequence Read Archive with accession number SRR5234544 (the wild type) and SRR5234545 (the colistin resistant strain).

Proteomic analysis

A. baumannii MDR-ZJ06 and ZJ06-200P5-1 were grown overnight at 37 °C in LB broth. Strains were subcultured 1/100 into fresh LB broth and grown at 37 °C for 2 h (OD600: 0.29 ± 0.02 for MDR-ZJ06, 0.26 ± 0.02 for ZJ06-200P5-1). The cells were collected at 4 °C and sent to Shanghai Applied Protein Technology Co. Ltd. The cell pellets were washed twice with PBS, and 500 μl SDT lysis buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6) was added. After being sonicated for 2 mins on ice, the cells were centrifuged at 14,000 × g for 30 min at 4 °C. The protein concentration in the supernatant was determined by the BCA method.

In brief, 300 μg protein was added to 200 μl UA buffer (8 M urea, 150 mM Tris-HCl pH 8.0) and ultrafiltered (Sartorius, 10 kD) with UA buffer. To block reduced cysteine residues, 100 μl iodoacetamide (IAA) buffer (50 mM IAA in UA buffer) was added, centrifuged at 600 rpm for 1 min, and incubated for 30 min in the dark. The filter was washed twice with 100 μl UA buffer and twice with 100 μl Dissolution buffer (50 mM triethylammonium bicarbonate, pH 8.5). Finally, the proteins were digested with 2 μg trypsin (Promega) in 40 μl Dissolution buffer at 37 °C for 16–18 h. The peptides were collected as a filtrate, and its content was estimated at OD280.

For iTRAQ labeling, the peptides were labeled with the 4-plex iTRAQ reagent following the manufacturer's instructions (AB SCIEX). The peptides from MDR-ZJ06 were labeled with 114 and 116 isobaric reagents, and the peptides from ZJ06-200P5-1 were labeled with 115 and 117 isobaric reagents.

RP-HPCL online-coupled to MS/MS (LC-MS/MS) analysis of the iTRAQ-labeled peptides was performed on an EASY-nLC nanoflow LC system (Thermo Fisher Scientific) connected to an Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific). After the samples were reconstituted and acidified with buffer A (0.1% (v/v) formic acid in water), a set-up involving a pre-column and analytical column was used. The pre-column was a 2 cm EASY-column (100, 5 μm C18; Thermo Fisher Scientific), while the analytical column was a 10 cm EASY-column (75, 3 μm, C18; Thermo Fisher Scientific). The 120 min linear gradient from 0 to 100% buffer B (0.1% (v/v) formic acid and 80% acetonitrile) at a constant flow rate of 250 nl/min was as follows: 0–100 min, 0–35% buffer B; 100–108 min, 35–100% buffer B; 108–120 min, 100% buffer B. MS data were acquired using a data-dependent top 10 method, dynamically choosing the most abundant precursor ions from the survey scan (300–180 m/z) for HCD fragmentation. The Dynamic exclusion was set to a repeat count of 1 with a 30 s duration. Survey scans were acquired at a resolution of 30,000 at m/z 200, and the resolution for HCD spectra was set to 15,000 at m/z 200. The normalized collision energy was 35 eV, and the underfill ratio was defined as 0.1%.

The MS/MS spectra were searched using the MASCOT engine (Matrix Science, London, UK; version 2.2) against the A. baumannii MDR-ZJ06 FASTA database. False discovery rates (FDR) were calculated via running all spectra against the FASTA database using the MASCOT software. The following options were used to identify proteins: peptide mass tolerance = 20 ppm, fragment mass tolerance = 0.1 Da, Enzyme = Trypsin, Max missed cleavages = 2, Fixed modification: Carbamidomethyl (C), iTRAQ 4plex (N-term), iTRAQ 4plex (K), Variable modification: Oxidation (M). Quantification was performed based on the peak intensities of the reporter ions in the MS/MS spectra. The proteins were considered overexpressed when the iTRAQ ratio was above 1.5 and underexpressed when the iTRAQ ratio was lower than 0.67 (Wang et al., 2016). Functional classification of differentially expression genes were annotated using the KEGG databases. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaino et al., 2016) partner repository with the dataset identifier PXD005265 and 10.6019/PXD005265. Reviewer account details: Username: reviewer54242@ebi.ac.uk; Password: zR8mE9wu.

Growth rate determination

Four independent cultures per strain were grown overnight, diluted to 1:1000 in MH and aliquots placed into a flat-bottom 100-well plate in four replicates. The plate was incubated at 37 °C with agitation. The OD600 of each culture was determined every 5 min for 16 h using a Bioscreen C MBR machine (Oy Growth Curves Ab Ltd., Finland). The growth rate was estimated based on OD600 curves using an R script (Fang et al., 2016).

Results

Whole genome sequencing, minimum inhibitory concentration and growth rate

The colistin-resistant mutant ZJ06-200P5-1 generated from the culture in CAMHB containing colistin was sent for whole genome sequencing. There was an ISAba1 insertion in lpxC in ZJ06-200P5-1 compared with the genome sequence of MDR-ZJ06 (Figure 1). The MIC of MDR-ZJ06 and ZJ06-200P5-1 were detected and listed in Table 1. The MIC for colistin increased from 0.38 mg/L (MDR-ZJ06) to >256 mg/L (ZJ06-200P5-1). However, ZJ06-200P5-1 showed higher sensitivity to multiple antibiotics: β-lactams, carbapenem, tetracycline, and ciprofloxacin, but not aminoglycosides. Furthermore, ZJ06-200P5-1 showed a lower growth rate (0.81 ± 0.05) than wild type.

Figure 1.

Figure 1

Whole genome sequencing revealed the colistin-resistance mechanism in A. baumannii ZJ06-200P5-1. The gene lpxC was intact in MDR-ZJ06, while in ZJ06-200P5-1, lpxC was inactivated by the insertion sequence ISAba1.

Table 1.

Antibiotic susceptibility of A. baumannii MDR-ZJ06 and its colistin resistant mutant ZJ06-200P5-1.

Strains COa TGCa IPM MEM FEP CAZ CTX ATM PRL TZP SCF SAM CN AK TE MH CIP CT
MDR-ZJ06 0.38 mg/L 4 mg/L 8 8 6 6 6 6 6 6 16 10 6 6 6 10 6 14
ZJ06-200P5-1 >256 mg/L 0.5 mg/L 22 22 20 20 15 22 17 19 30 22 6 6 8 26 9 6

CO, colistin; TGC, tigecycline; IPM, imipenem; MEM, meropenem; FEP, cefepime; CAZ, ceftazidime; CTX, cefotaxime; ATM, aztreonam; PRL, Piperacillin; TZP, piperacillin/tazobactam; SCF, Cefoperazone/sulbactam; SAM, ampicillin/sulbactam; CN, gentamicin; AK, amikacin; TE, tetracycline; MH, minocycline; CIP, Ciprofloxacin; CT, colistin.

a

The MIC of colistin and tigecycline were determined by broth dilution method, while antimicrobial sensitivity of other antibiotics were detected by disk diffusion.

Transcriptome analysis

The transcriptome analysis of ZJ06-200P5-1 and MDR-ZJ06 was performed by Illumina RNA deep sequencing technology. Cells of the two strains were collected in the early exponential phase. A total of 137 genes showed significant differential expression [log2(FoldChange) > 1 or log2(FoldChange) < −1], among which 48 genes were upregulated and 89 were downregulated (Table 2). Sixteen selected genes, three up-regulated and thirteen down-regulated genes, were well-validated by RT-qPCR (Figure 2). After mapping the differentially expressed genes into the KEGG pathway, we observed that genes involved in Energy metabolism and Amino acid metabolism were down-regulated, while Carbohydrate metabolism was up-regulated.

Table 2.

Genes changed significantly in transcriptome.

Synonym Product logFC logCPM P-value FDR
ABZJ_00055 hypothetical protein 8.308068 13.717 1.26E-78 4.54E-76
ABZJ_00068 hypothetical protein 6.4468 9.203574 2.14E-67 4.61E-65
ABZJ_00037 hypothetical protein 4.368832 9.669037 3.48E-68 9.36E-66
ABZJ_00056 hypothetical protein 4.349519 12.2059 6.03E-65 1.08E-62
ABZJ_00332 hypothetical protein 4.264896 9.455077 2.39E-53 2.86E-51
ABZJ_00036 hypothetical protein 3.449637 9.968726 9.61E-27 5.17E-25
ABZJ_01879 hypothetical protein 2.810666 6.769621 9.95E-35 7.65E-33
ABZJ_01880 putative transposase 2.758133 6.676606 5.52E-27 3.13E-25
ABZJ_01079 hypothetical protein 2.585295 6.001793 4.14E-10 6.55E-09
ABZJ_03753 hypothetical protein 2.318997 9.492231 2.51E-21 1.08E-19
ABZJ_00333 hypothetical protein 2.314205 5.437541 2.36E-11 4.53E-10
ABZJ_01881 transposase component 2.25458 8.338274 9.50E-21 3.93E-19
ABZJ_01133 heat shock protein 2.180889 13.35847 1.03E-25 5.06E-24
ABZJ_01180 putative phage-like protein 2.066152 3.22126 4.47E-06 3.56E-05
ABZJ_03752 PGAP1-like protein 2.014551 10.16569 2.49E-27 1.49E-25
ABZJ_00060 Thiol-disulfide isomerase and thioredoxin 1.894318 12.3252 7.68E-20 2.75E-18
ABZJ_00894 lactoylglutathione lyase-like protein 1.797874 6.779815 5.27E-15 1.62E-13
ABZJ_00054 N-alpha-acetylglutamate synthase (amino-acid acetyltransferase) 1.77044 10.25589 3.24E-20 1.27E-18
ABZJ_01151 hypothetical protein 1.634908 3.574211 4.88E-06 3.84E-05
ABZJ_03714 hypothetical protein 1.61859 8.500912 1.39E-08 1.85E-07
ABZJ_01900 acetoin:2,6-dichlorophenolindophenol oxidoreductase subunit alpha 1.527437 6.102611 2.98E-06 2.49E-05
ABZJ_01222 hypothetical protein 1.515854 2.111384 0.011897 0.034227
ABZJ_01191 hypothetical protein 1.46809 2.203352 0.011349 0.032877
ABZJ_01872 hypothetical protein 1.423713 7.613403 1.64E-08 2.10E-07
ABZJ_01187 hypothetical protein 1.423595 5.112417 2.82E-07 2.81E-06
ABZJ_01857 hypothetical protein 1.411761 2.566001 0.010144 0.029905
ABZJ_01829 Acyl-CoA dehydrogenase 1.402255 6.594396 4.45E-06 3.56E-05
ABZJ_01150 hypothetical protein 1.321675 3.205499 0.000936 0.003799
ABZJ_00028 lytic murein transglycosylase family protein 1.296752 10.96489 3.46E-14 9.79E-13
ABZJ_00976 hypothetical protein 1.295503 5.552053 1.46E-07 1.57E-06
ABZJ_01855 hypothetical protein 1.290522 2.587494 0.016132 0.044395
ABZJ_01186 hypothetical protein 1.249298 2.481015 0.013475 0.038054
ABZJ_00978 hypothetical protein 1.216859 3.038132 0.00684 0.021395
ABZJ_00977 hypothetical protein 1.209422 3.887522 0.000232 0.001118
ABZJ_00102 D-lactate dehydrogenase FAD-binding protein 1.170013 8.813908 1.91E-10 3.15E-09
ABZJ_01149 hypothetical protein 1.156232 3.314522 0.003302 0.011138
ABZJ_00053 alkanesulfonate transport protein 1.143156 6.421362 5.15E-06 3.99E-05
ABZJ_01275 hypothetical protein 1.122845 8.385252 1.31E-08 1.76E-07
ABZJ_03838 membrane-fusion protein 1.119324 7.708838 1.84E-08 2.33E-07
ABZJ_01901 acetoin:26-dichlorophenolindophenol oxidoreductase beta subunit 1.105826 6.349341 5.58E-05 0.000323
ABZJ_01899 lipoate synthase 1.08338 4.583472 0.003397 0.011422
ABZJ_00360 hypothetical protein 1.076106 8.065171 1.34E-07 1.46E-06
ABZJ_01210 hypothetical protein 1.065917 3.456549 0.011028 0.032156
ABZJ_01160 hypothetical protein 1.048988 3.144467 0.012194 0.034895
ABZJ_01148 hypothetical protein 1.048966 5.540519 1.77E-05 0.000122
ABZJ_00099 L-lactate permease 1.044891 10.0835 8.49E-08 9.61E-07
ABZJ_00901 major facilitator superfamily multidrug resistance protein 1.016944 9.235389 1.47E-08 1.91E-07
ABZJ_01775 6-pyruvoyl-tetrahydropterin synthase 1.014549 10.17374 3.05E-12 6.84E-11
ABZJ_03786 VirP protein −1.0004 6.133241 3.35E-06 2.73E-05
ABZJ_01269 TPR repeat-containing SEL1 subfamily protein −1.00222 4.702232 0.000305 0.001408
ABZJ_00120 hypothetical protein −1.00591 7.042084 6.25E-07 5.85E-06
ABZJ_00896 nucleoside-diphosphate sugar epimerase −1.0079 7.57903 9.80E-07 8.86E-06
ABZJ_01258 hypothetical protein −1.01127 4.48134 0.002855 0.009692
ABZJ_01260 metal ion ABC transporter substrate-binding protein/surface antigen −1.01249 9.488595 2.29E-08 2.86E-07
ABZJ_01120 urease accessory protein UreE −1.01439 6.914944 6.34E-07 5.88E-06
ABZJ_01873 hypothetical protein −1.01999 5.846082 1.89E-05 0.000128
ABZJ_03812 hypothetical protein −1.02082 4.567471 0.001409 0.005227
ABZJ_01101 hypothetical protein −1.03046 5.533349 0.001752 0.006282
ABZJ_01908 Zn-dependent hydrolase, including glyoxylase −1.03588 9.460654 2.53E-10 4.12E-09
ABZJ_03819 hypothetical protein −1.05745 9.905586 6.08E-11 1.11E-09
ABZJ_03796 putative acyltransferase −1.06273 6.680253 2.34E-07 2.42E-06
ABZJ_00947 hypothetical protein −1.0641 6.738813 1.36E-06 1.21E-05
ABZJ_01169 hypothetical protein −1.06442 8.404764 8.75E-07 7.98E-06
ABZJ_00345 hypothetical protein −1.06443 6.560939 2.47E-07 2.53E-06
ABZJ_03828 hypothetical protein −1.06567 4.05012 0.000406 0.001813
ABZJ_00922 hypothetical protein −1.07121 5.599955 7.64E-05 0.000424
ABZJ_01907 response regulator −1.07682 6.813752 2.94E-07 2.90E-06
ABZJ_03790 gamma-aminobutyrate permease −1.07931 8.18838 3.71E-05 0.000227
ABZJ_00882 hypothetical protein −1.07943 9.751157 2.22E-11 4.34E-10
ABZJ_01078 hypothetical protein −1.08109 10.14275 5.68E-14 1.49E-12
ABZJ_01132 glutamate dehydrogenase/leucine dehydrogenase −1.08366 7.760303 2.14E-07 2.24E-06
ABZJ_03802 putative homogentisate 1,2-dioxygenase −1.08726 6.643847 0.000162 0.000822
ABZJ_00334 hypothetical protein −1.09533 6.571739 7.17E-08 8.25E-07
ABZJ_01250 outer membrane receptor protein −1.10965 7.442322 0.000193 0.000956
ABZJ_00367 hypothetical protein −1.11395 8.476819 9.04E-09 1.25E-07
ABZJ_00946 hypothetical protein −1.12668 5.862006 7.32E-06 5.59E-05
ABZJ_01265 hypothetical protein −1.12706 10.47521 4.03E-13 9.42E-12
ABZJ_01257 Zn-dependent protease with chaperone function −1.13229 6.680195 1.30E-05 9.11E-05
ABZJ_01110 putative hemolysin-related protein −1.13995 9.22038 1.74E-11 3.54E-10
ABZJ_03720 UDP-3-O-acyl-N-acetylglucosamine deacetylase −1.14429 8.585685 1.05E-05 7.52E-05
ABZJ_01960 isochorismate hydrolase −1.14761 5.633402 0.000121 0.000638
ABZJ_00942 hypothetical protein −1.15912 8.72549 8.38E-09 1.17E-07
ABZJ_03859 putative RND type efflux pump involved in aminoglycoside resistance (AdeT) −1.17363 8.75427 3.19E-05 0.000202
ABZJ_01874 hypothetical protein −1.17434 5.206346 2.41E-05 0.000159
ABZJ_01917 putative acyl carrier protein phosphodiesterase (ACP phosphodiesterase) −1.18991 7.045816 5.55E-08 6.50E-07
ABZJ_01861 membrane-fusion protein −1.20577 6.002924 1.77E-07 1.87E-06
ABZJ_03742 hypothetical protein −1.20817 3.772045 0.001579 0.005748
ABZJ_01262 hypothetical protein −1.21556 4.167491 8.53E-05 0.000466
ABZJ_01929 Aspartate ammonia-lyase (Aspartase) −1.21837 11.63816 9.24E-14 2.31E-12
ABZJ_00924 hypothetical protein −1.2423 8.464578 1.01E-10 1.79E-09
ABZJ_01155 hypothetical protein −1.2668 10.80427 1.20E-16 3.79E-15
ABZJ_00388 2-polyprenyl-6-methoxyphenol hydroxylase −1.26695 7.901741 1.19E-09 1.81E-08
ABZJ_01862 multidrug ABC transporter ATPase −1.27715 6.94382 4.78E-09 6.86E-08
ABZJ_00944 hypothetical protein −1.28276 5.658916 2.33E-08 2.88E-07
ABZJ_01156 hypothetical protein −1.28415 8.332979 5.92E-11 1.10E-09
ABZJ_01826 AraC-type DNA-binding domain-containing protein −1.29289 5.11387 5.57E-07 5.26E-06
ABZJ_03744 hypothetical protein −1.29678 8.720807 1.08E-08 1.47E-07
ABZJ_03737 hypothetical protein −1.30269 10.28829 3.31E-20 1.27E-18
ABZJ_00940 hypothetical protein −1.30722 6.280622 2.75E-07 2.79E-06
ABZJ_01218 hypothetical protein −1.30837 4.257169 9.06E-06 6.63E-05
ABZJ_00061 putative transcriptional regulator −1.31564 7.634498 1.67E-10 2.80E-09
ABZJ_01887 hypothetical protein −1.3281 6.449578 1.02E-07 1.14E-06
ABZJ_01025 homocysteine/selenocysteine methylase −1.33719 7.528478 3.07E-10 4.93E-09
ABZJ_00110 GNAT family acetyltransferase −1.33942 4.887691 1.06E-06 9.50E-06
ABZJ_01242 hypothetical protein −1.3506 7.369014 2.45E-09 3.61E-08
ABZJ_00895 hypothetical protein −1.35351 6.693904 7.37E-12 1.56E-10
ABZJ_03712 putative flavoprotein −1.38598 6.6067 2.04E-09 3.04E-08
ABZJ_00048 transcriptional regulator −1.40027 7.755295 9.36E-11 1.68E-09
ABZJ_03785 glutamate racemase −1.40496 7.417511 7.08E-12 1.52E-10
ABZJ_00938 hypothetical protein −1.40799 6.629998 1.09E-10 1.88E-09
ABZJ_01230 hypothetical protein −1.41279 10.19585 3.47E-19 1.20E-17
ABZJ_00124 glycine/D-amino acid oxidase (deaminating) −1.46015 13.3987 8.58E-14 2.20E-12
ABZJ_03791 histidine ammonia-lyase (Histidase) −1.49736 9.748038 2.37E-08 2.90E-07
ABZJ_03739 hypothetical protein −1.49749 13.98113 3.54E-13 8.47E-12
ABZJ_00881 glutamine amidotransferase −1.51327 8.144142 5.09E-14 1.37E-12
ABZJ_00988 hypothetical protein −1.54819 6.1324 7.44E-09 1.05E-07
ABZJ_01840 putative ferric siderophore receptor protein −1.55785 9.806018 9.74E-10 1.52E-08
ABZJ_00997 hypothetical protein −1.58106 5.257799 3.12E-08 3.77E-07
ABZJ_00339 HSP90 family molecular chaperone −1.6168 11.15864 7.57E-23 3.54E-21
ABZJ_00373 Type II secretory pathway, ATPase PulE/Tfp pilus assembly pathway, ATPase PilB −1.6419 6.706339 3.45E-14 9.79E-13
ABZJ_01845 phosphatase/phosphohexomutase −1.68301 7.222507 3.67E-12 8.06E-11
ABZJ_03793 urocanate hydratase −1.69267 10.89217 1.13E-07 1.25E-06
ABZJ_03754 Rhs element Vgr family protein −1.69503 8.757228 5.86E-18 1.97E-16
ABZJ_00945 hypothetical protein −1.72533 5.192791 2.02E-11 4.03E-10
ABZJ_01002 putative ABC oligo/dipeptide transport, ATP-binding protein −1.73182 6.449009 4.32E-14 1.19E-12
ABZJ_01259 hypothetical protein −1.75565 7.198513 1.30E-12 2.98E-11
ABZJ_00114 short chain dehydrogenase family protein −1.76754 7.176594 1.03E-13 2.52E-12
ABZJ_01177 hypothetical protein −1.8053 8.135954 6.06E-15 1.81E-13
ABZJ_03792 hypothetical protein −1.82418 6.284478 3.56E-06 2.88E-05
ABZJ_01219 hypothetical protein −1.86448 9.22858 7.68E-22 3.45E-20
ABZJ_01088 carbonic anhydrase −1.94984 9.430551 1.08E-27 6.83E-26
ABZJ_00346 hypothetical protein −2.03948 6.219886 1.15E-16 3.73E-15
ABZJ_01207 hypothetical protein −2.1746 7.126199 6.11E-20 2.27E-18
ABZJ_01886 hypothetical protein −2.33548 5.458495 1.05E-11 2.18E-10
ABZJ_03766 putative secretory lipase precursor −2.38284 9.073946 1.11E-31 7.47E-30
ABZJ_01206 hypothetical protein −3.28101 9.194837 2.48E-45 2.42E-43
ABZJ_03736 thiol:disulfide interchange protein −3.9361 9.872762 6.64E-41 5.50E-39

Figure 2.

Figure 2

Validation of the RNA sequencing results. The transcriptomic results obtained by RNA-seq were validated by quantitative RT-PCR analysis. The differential expression of 16 genes was detected in this study. Three biology replicates were used in this experiment. The results were presented as expression in ZJ06-200P5-1, relative to MDR-ZJ06. The reference gene rpoB was used for inter-sample normalization. Error bars denote standard deviation.

iTRAQ

A total of 1582 proteins were identified in the iTRAQ experiment. A protein ratio >1.5 or <0.67 (p <0.05) was considered to be differentially expressed. After filtration, 82 differentially expressed proteins were identified between ZJ06-200P5-1 and MDR-ZJ06. The detailed information is shown in Table 3.

Table 3.

Genes changed significantly in proteome.

Protein number NCBInr acession Gene tag Protein description Pep Count Unique PepCount Coverage (%) MW pI log2 of ratio (ZJ06-200P5-1 vs. MDR-ZJ06) p-value
233 384144952 ABZJ_03706 hypothetical protein 75 12 66.27 27649.89 4.59 1.65184 2.90E-20
1280 384143756 ABZJ_02510 hypothetical protein 1 1 10.18 17235.79 10.09 1.49121 8.79E-17
756 384144562 ABZJ_03316 hypothetical protein 27 4 34.13 13935.85 9.67 1.49075 8.99E-17
1032 384144568 ABZJ_03322 hypothetical protein 7 2 15.75 15550.26 10.03 1.39649 6.82E-15
565 384143898 ABZJ_02652 hypothetical protein 23 6 54.76 13282.22 8.99 1.15312 1.36E-10
594 384141430 ABZJ_00184 hypothetical protein 14 6 32.66 22273.87 4.56 1.131 3.05E-10
1241 384141854 ABZJ_00608 dehydrogenase 1 1 5.13 30137.1 8.79 1.11427 5.57E-10
1188 384141579 ABZJ_00333 hypothetical protein 5 1 10.66 11110.55 9.66 1.09309 1.18E-09
1076 384143755 ABZJ_02509 hypothetical protein 4 2 31.91 13701.31 10.29 1.09014 1.31E-09
147 384141823 ABZJ_00577 membrane-fusion protein 59 17 45.29 48231.1 9.44 0.9855 4.28E-08
1209 384142731 ABZJ_01485 dihydrodipicolinate synthase 2 1 2.89 33837.12 5.46 0.956837 1.05E-07
175 384143251 ABZJ_02005 membrane-fusion protein 50 15 47.22 43375.8 7.75 0.9115 4.13E-07
1281 384143760 ABZJ_02514 glycosyltransferase 1 1 3.37 48412.32 9.23 0.889123 7.92E-07
454 384141821 ABZJ_00575 putative outer membrane protein 18 8 21.57 54556.06 8.52 0.886277 8.60E-07
1009 384141578 ABZJ_00332 hypothetical protein 26 2 32.23 11005.53 9.93 0.859413 1.84E-06
1216 384143670 ABZJ_02424 hypothetical protein 2 1 25.58 4520.08 5.45 0.848157 2.51E-06
201 384143250 ABZJ_02004 cation/multidrug efflux pump 26 14 15.64 112744.8 7.6 0.801366 8.82E-06
885 384142076 ABZJ_00830 Outer membrane lipoprotein 12 3 18.75 21087.72 6.9 0.801241 8.85E-06
323 384144243 ABZJ_02997 putative porin protein associated with imipenem resistance 97 10 50.81 26505.22 4.8 0.770322 1.96E-05
1029 384141822 ABZJ_00576 peptide ABC transporter permease 7 2 3.77 71261.81 6.24 0.753391 2.98E-05
164 384144912 ABZJ_03666 NAD-dependent aldehyde dehydrogenase 41 16 43.15 51846.55 5.11 0.751721 3.11E-05
655 384144155 ABZJ_02909 hypothetical protein 27 5 33.48 26172.15 7.85 0.733875 4.80E-05
812 384142146 ABZJ_00900 multidrug resistance secretion protein 8 4 9.14 40956.99 6.56 0.691132 0.000131
852 384145008 ABZJ_03762 putative short-chain dehydrogenase 6 4 17.24 31854.29 9.26 0.688359 0.000139
539 384144680 ABZJ_03434 flavoprotein 10 7 15.52 55720.24 9.12 0.685088 0.00015
150 384144913 ABZJ_03667 4-aminobutyrate aminotransferase 55 17 50.23 45976.96 5.81 0.679784 0.000169
1306 384144561 ABZJ_03315 kinase sensor component of a two component signal transduction system 1 1 3.07 62690.76 6.3 0.672652 0.000198
600 384144948 ABZJ_03702 xenobiotic reductase 14 6 21.02 38725.16 5.08 0.608194 0.000783
315 384144930 ABZJ_03684 hypothetical protein 322 10 47.37 32732.07 4.71 0.602647 0.000876
603 384143541 ABZJ_02295 UDP-glucose 4-epimerase 13 6 28.06 38064.02 5.53 0.599175 0.000939
384 384142564 ABZJ_01318 Zn-dependent protease with chaperone function 35 9 48.66 27572.18 9.44 0.592971 0.001063
680 384143417 ABZJ_02171 hypothetical protein 14 5 40.65 17046.41 8.79 −0.59205 0.000855
996 384143586 ABZJ_02340 hypothetical protein 3 3 10.61 29941.63 6.85 −0.60757 0.000626
1007 384145105 ABZJ_03859 putative RND type efflux pump involved in aminoglycoside resistance (AdeT) 3 3 10.48 38641.56 9.71 −0.60779 0.000623
667 384144990 ABZJ_03744 hypothetical protein 18 5 21.99 27747.62 4.62 −0.60878 0.00061
820 384141318 ABZJ_00072 FKBP-type 22KD peptidyl-prolyl cis-trans isomerase 7 4 21.65 25217.38 9.06 −0.61264 0.000564
767 384141553 ABZJ_00307 hypothetical protein 17 4 48.31 10746.92 5.3 −0.61297 0.00056
163 384144907 ABZJ_03661 hypothetical protein 47 16 39.91 49757.27 8.16 −0.61374 0.000551
865 384144338 ABZJ_03092 Zn-dependent hydrolase, including glyoxylase 5 4 15.00 35333.86 8.91 −0.62839 0.000407
780 384141775 ABZJ_00529 gluconate kinase 12 4 30.59 18924.48 4.88 −0.6352 0.000353
1259 384142716 ABZJ_01470 hypothetical protein 1 1 2.52 36304.38 9.04 −0.63588 0.000348
424 384142064 ABZJ_00818 3-oxoacyl-ACP reductase 42 8 45.90 26098.39 6.1 −0.64296 0.000299
825 384141812 ABZJ_00566 hypothetical protein 7 4 36.11 15329.44 9.46 −0.64431 0.00029
381 384141306 ABZJ_00060 Thiol-disulfide isomerase and thioredoxin 37 9 42.44 22825.09 9.58 −0.65529 0.000229
963 384142833 ABZJ_01587 dehydrogenase 4 3 9.93 31970.72 5.16 −0.6827 0.000125
645 384141583 ABZJ_00337 putative outer membrane protein W 52 5 28.64 22680.64 5.9 −0.69549 9.35E-05
329 384142063 ABZJ_00817 malonyl-CoA-[acyl-carrier-protein] transacylase 59 10 43.15 35339.2 5.22 −0.6997 8.49E-05
941 384142271 ABZJ_01025 homocysteine/selenocysteine methylase 5 3 12.33 32062.1 4.82 −0.71762 5.59E-05
716 384144502 ABZJ_03256 protein-disulfide isomerase 9 5 23.31 26361.06 9 −0.72106 5.15E-05
232 384144545 ABZJ_03299 acetylCoA carboxylase subunit beta 76 12 44.63 32971.73 5.85 −0.72297 4.93E-05
836 384144135 ABZJ_02889 hypothetical protein 7 4 38.57 15413.52 8.43 −0.72309 4.91E-05
207 384141892 ABZJ_00646 Acetyl-CoA carboxylase alpha subunit 87 13 75.09 29640.53 5.6 −0.72798 4.37E-05
1053 384144131 ABZJ_02885 LysR family transcriptional regulator 5 2 6.80 34516.26 6.26 −0.74843 2.67E-05
883 384142465 ABZJ_01219 hypothetical protein 14 3 26.54 17636.93 9.58 −0.75975 2.02E-05
791 384142700 ABZJ_01454 hypothetical protein 10 4 25.15 19116.5 5 −0.77251 1.47E-05
573 384144158 ABZJ_02912 putative fatty acid desaturase 20 6 17.03 42202.21 9.39 −0.77608 1.34E-05
663 384141673 ABZJ_00427 putative type III effector HopPmaJ 19 5 37.27 12074.21 5.41 −0.78501 1.07E-05
261 384141776 ABZJ_00530 NAD-dependent aldehyde dehydrogenase 28 12 22.69 60150.9 6.04 −0.80138 7.02E-06
166 384141820 ABZJ_00574 NADH-dependent enoyl-ACP reductase 142 15 64.24 31016.41 6 −0.81807 4.53E-06
280 384144728 ABZJ_03482 putative toluene tolerance protein (Ttg2D) 76 11 61.97 23513.33 9.83 −0.82764 3.51E-06
917 384142976 ABZJ_01730 hypothetical protein 7 3 14.80 21011.63 9.2 −0.8625 1.36E-06
192 384144009 ABZJ_02763 hypothetical protein 63 14 48.19 44493.93 8.79 −0.86539 1.25E-06
635 384144826 ABZJ_03580 putative penicillin binding protein (PonA) 8 6 8.23 94767.31 9.38 −0.88231 7.77E-07
292 384142962 ABZJ_01716 biotin synthetase 40 11 34.83 37136.95 5.45 −0.89634 5.20E-07
909 384142828 ABZJ_01582 putative 17 kDa surface antigen 8 3 44.76 12431.23 4.7 −0.93167 1.85E-07
483 384144247 ABZJ_03001 hypothetical protein 43 7 48.55 14704.84 9.54 −0.93498 1.67E-07
188 384142100 ABZJ_00854 beta-ketoacyl-ACP synthase 90 14 46.45 43130.17 5.2 −0.94675 1.17E-07
446 384144999 ABZJ_03753 hypothetical protein 22 8 39.09 28038.81 9.07 −0.95428 9.33E-08
401 384144159 ABZJ_02913 flavodoxin reductase (ferredoxin-NADPH reductase) family protein 1 23 9 31.46 39570.7 6.09 −0.95498 9.13E-08
489 384143515 ABZJ_02269 (3R)-hydroxymyristoyl-ACP dehydratase 39 7 50.93 17988.69 6.3 −0.97767 4.53E-08
459 384142835 ABZJ_01589 hypothetical protein 18 8 13.33 43721.35 4.96 −0.97866 4.39E-08
833 384143336 ABZJ_02090 hypothetical protein 7 4 37.91 17951.03 4.82 −1.00115 2.16E-08
586 384143810 ABZJ_02564 hypothetical protein 16 6 79.22 8718.62 5 −1.02063 1.15E-08
114 384143236 ABZJ_01990 beta-lactamase OXA-23 161 18 71.38 31385.05 8.37 −1.0965 8.98E-10
359 384143517 ABZJ_02271 putative outer membrane protein (OmpH) 25 10 57.49 18710.09 9.52 −1.22331 8.58E-12
606 384144983 ABZJ_03737 hypothetical protein 13 6 38.04 27580.52 4.68 −1.28419 7.75E-13
95 384144431 ABZJ_03185 putative DcaP-like protein 111 20 50.69 47278.17 6.37 −1.36068 3.23E-14
939 384141906 ABZJ_00660 putative lipoprotein precursor (VacJ) transmembrane 5 3 10.67 33499.98 4.85 −1.43792 1.09E-15
1289 384144099 ABZJ_02853 hypothetical protein 1 1 8.06 14811.21 4.39 −1.58122 1.26E-18
1186 384142065 ABZJ_00819 acyl carrier protein (ACP) 21 1 10.99 10132.23 4.11 −1.65109 3.70E-20
412 384142699 ABZJ_01453 hypothetical protein 14 9 46.52 25412.88 9.89 −1.66497 1.81E-20
113 384144004 ABZJ_02758 beta-lactamase 268 18 55.05 44683.92 9.28 −1.82062 3.88E-24

The expression of AdeABC was up-regulated in the LPS-loss ZJ06-200P5-1 strain. The AdeABC efflux pump confers resistance to various antibiotics classes. The expression of AdeABC genes was increased approximately two-fold in ZJ06-200P5-1 (Figure 3A). However, ZJ06-200P5-1 showed higher susceptibility to multiple antibiotics than MDR-ZJ06 (Table 1).

Figure 3.

Figure 3

ITRAQ analysis showed that AdeABC were up-regulated, and the fatty acid biosynthesis pathway was down-regulated in ZJ06-200P5-1. (A) AdeABC efflux pump, (B) fatty acid biosynthesis pathway. Green shows genes with significantly reduced expression levels, and red shows genes with significantly increased expression levels.

The fatty acid biosynthesis pathway was down-regulated in the ZJ06-200P5-1 strain (Figure 3B). The expression of FabZ was decreased by approximately two-fold in ZJ06-200P5-1. The β-lactamases blaOXA−23 and blaADC−25 were down-regulated in ZJ06-200P5-1 strain. The expression levels of blaOXA−23 and blaADC−25 were decreased two- to four-fold in ZJ06-200P5-1.

Common genes altered expression in both transcriptome and proteome

A total of 15 differentially expressed genes (or proteins) were identified in both transcriptome and proteome (Table 4). Among them, three genes were both up-regulated, and nine genes were both down-regulated. Although there was correlation between transcriptome and proteome data, the absolute expression difference values in transcriptome data was higher than those in proteome data. In addition, the result of three gene/proteins were contradictory (highlighted in red letters in Table 4). The contradictory result might be caused by post-transcriptional regulation.

Table 4.

Common genes altered expression both in transcriptome and proteome.

Synonym Product Fold change (log2, Transcriptome) Fold change (log2, Proteome)
ABZJ_00332 hypothetical protein 4.26489563 0.859413
ABZJ_03753 hypothetical protein 2.318997325a −0.95428
ABZJ_00333 hypothetical protein 2.314204886 1.09309
ABZJ_01133 heat shock protein 2.180888936 0.532117
ABZJ_00060 Thiol-disulfide isomerase and thioredoxin 1.894317881a −0.65529
ABZJ_00028 lytic murein transglycosylase family protein 1.296751692a −0.57293
ABZJ_01078 hypothetical protein −1.081092562 −0.44448
ABZJ_03720 UDP-3-O-acyl-N-acetylglucosamine deacetylase −1.144287283 −0.48378
ABZJ_03859 putative RND type efflux pump involved in aminoglycoside resistance (AdeT) −1.173634714 −0.60779
ABZJ_03744 hypothetical protein −1.296782077 −0.60878
ABZJ_03737 hypothetical protein −1.302692756 −1.28419
ABZJ_01025 homocysteine/selenocysteine methylase −1.337189269 −0.71762
ABZJ_01219 hypothetical protein −1.864476303 −0.75975
ABZJ_01088 carbonic anhydrase −1.949843631 −0.56001
ABZJ_01206 hypothetical protein −3.281014801 −0.4346
a

The result of three gene/proteins were contradictory.

Discussion

Due to the limitation of antimicrobial agents in clinical use, it is urgent to extend our understanding of the emergence of colistin resistance in A. baumannii. A. baumannii MDR-ZJ06, a multidrug-resistant clinical strain isolated from bloodstream, has been sequenced and was considered an ideal strain for examining the colistin-resistant mechanism in A. baumannii (Zhou et al., 2011). In this study, colistin-resistant strain was rapidly obtained, and its resistance mechanism was LPS loss caused by ISAba1 insertion in lpxC. This result confirmed a previous finding (Moffatt et al., 2010). The rapid isolation of colistin-resistant mutant from multiple drug-resistant A. baumannii indicated a high risk of A. baumannii evolving resistance to colistin in clinical use.

We successfully detected the whole transcriptional profile of A. baumannii strain MDR-ZJ06 and its colistin-resistant mutant ZJ06-200P5-1 via Illumina RNA-sequencing. In another transcriptome study (Henry et al., 2012), A. baumannii ATCC 19606 and its lpxA mutant were used. Although both the lpxC and lpxA mutation lead to LPS loss, the different transcriptional response may be due to differences in the strain genetic background and the resistant mutation. In transcriptional analysis, we observed that genes involved in Energy metabolism and Amino acid metabolism were down-regulated, while Carbohydrate metabolism was up-regulated.

The expression of AdeABC was up-regulated in the LPS-loss ZJ06-200P5-1 strain. Similar results were also observed in all polymyxin-treated samples (Cheah et al., 2016a). In addition, the expression levels of adeIJK and macAB-tolC were up-regulated in the LPS loss mutant (Henry et al., 2012). Increased expression of the RND efflux pump system (AdeABC) was a common finding across all experiments in colistin exposure. The up-regulation of AdeABC indicated the diminished integrity and barrier function of the outer membrane in colistin-resistant A. baumannii (Henry et al., 2015; Cheah et al., 2016a). However, ZJ06-200P5-1 showed higher susceptibility to multiple antibiotics than MDR-ZJ06. The higher susceptibility might result from the higher outer membrane permeability of ZJ06-200P5-1 due to LPS-loss. The increased expression of the efflux pump was thought to be a response to toxic substances that accumulated in the cells due to the increased membrane permeability (Henry et al., 2012).

The fatty acid biosynthesis pathway was down-regulated in the ZJ06-200P5-1 strain. In E. coli, it is important to balance LPS and fatty acid biosynthesis to maintain cell integrity. FabZ, which dehydrates R-3-hydroxymyristoyl-acyl carrier protein in fatty acid biosynthesis, plays an important role in rebalancing lipid A and fatty acid homeostasis (Bojkovic et al., 2016). The decrease in FabZ was considered to be a response to LPS-loss in ZJ06-200P5-1. The β-lactamases blaOXA−23 and blaADC−25 were down-regulated in the ZJ06-200P5-1 strain. Decreased expression levels of blaOXA−23 and blaADC−25 were also observed in A. baumannii MDR-ZJ06 under a subinhibitory concentration of tigecycline (Hua et al., 2014). Meanwhile, the strain under tigecycline stress showed a lower MIC of ceftazidime (Hua et al., 2014). The decrease in blaOXA−23 and blaADC−25 might contribute to the increased sensitivity to β-lactam antimicrobial agents.

A multi-omics approach was adopted to obtain a more global view of colistin-resistant A. baumannii. Genomic analysis showed that lpxC was inactivated by ISAba1 insertion, leading to LPS loss. Transcriptional analysis demonstrated that the colistin-resistant strain regulated its metabolism. Metabolic change and LPS loss were concomitant. Proteomic analysis suggested increased expression of the RND efflux pump system and the down-regulation of FabZ and β-lactamase. These alterations are believed to be responses to LPS loss. Together, the lpxC mutation not only confirmed colistin resistance but also altered global gene expression.

Nucleotide sequence accession numbers

The whole-genome shotgun sequencing results for A. baumannii ZJ06-200P5-1 have been deposited at DDBJ/EMBL/GenBank under the accession number MIFW00000000.

Author contributions

XH and YY conceived and designed the study. XH, LL, YF, QS, XL, QC, KS, YJ, and HZ performed the experiments. XH and YY performed data analysis and drafted the manuscript. All authors reviewed and approved the final manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81230039, 31670135, 81378158), the 973 Preliminary Research Program (2014CB560707), the Natural Science Foundation of Zhejiang province, China (LY15H190004, Y16H190013) and the Zhejiang Province Medical Platform Backbone Talent Plan (2016DTA003).

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