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Identification of heptapeptides targeting a lethal bacterial strain in septic mice through an integrative approach

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Animals

Ten- to twelve-week-old male-specific pathogen-free (SPF) C57BL/6 mice were obtained from Southern Medical University, Guangzhou, China. All mice were housed in a temperature-controlled room with 40–70% humidity and had free access to water and food. The animal procedures were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care and Use Committee of Southern Medical University.

Bacteria strain

Pseudomonas aeruginosa (ATCC27853) was purchased from American Type Culture Collection (ATCC) and cultured in Luria Broth (LB) medium with shaking at 37 °C. For construction of ampicillin-resistant MSI001 strain (MSI001-Ampr), the pET14b plasmid carrying Ampr marker gene was introduced into the wild type MSI001 strain. The MSI001-Ampr strain was cultured in LB medium supplemented with Ampicillin of 100 µg/ml at 37 °C.

CLP model

C57BL/6 mice were randomly divided into sham and CLP groups. The CLP model was prepared with mice as described previously.57,58 Briefly, mice were anesthetized with 1.5% pentobarbital (0.1 ml/20 g body weight), and a midline abdominal incision 1 cm in length was made. After careful isolation, the cecum was exposed, followed by cecal ligation and puncturing twice with a 22-gauge needle. The abdominal wall was closed after the cecum was returned to the abdominal cavity. Sham-operated mice underwent the same procedure but without ligation and puncture.

Identification of the lethal bacterial strain

Lethal bacteria were cultured in Luria Bertani (LB) broth at 37 °C. Genomic DNA was extracted by using a MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa, Japan) and commercially sequenced with a PacBio Sequel platform and Illumina NovaSeq PE150 at Beijing Novogene Bioinformatics Technology Co., Ltd. The genomic DNA sequence of the lethal strain MSI001 was blasted against the nucleotide (nt) sequence database using BLAST+ (v2.11.0) (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) with an E value cutoff of 10−5. The top 500 aligning sequences and the input DNA sequence were used to identify marker genes through the Genome Taxonomy Database Toolkit (GTDB-Tk, v1.5.0, https://github.com/Ecogenomics/GtdbTk/),59 and the result was input into IQ-TREE (v1.5.4, http://www.iqtree.org)60 for phylogenetic inference. The evolution distance tree was plotted through the Interactive Tree of Life (iTOL, v6, https://itol.embl.de).61

Survival study

Pathogenic bacterial clones were amplified by culturing in LB broth to an optical density at 600 nm wavelength (OD600) of approximately 1.0. After centrifugation at room temperature for 5 min, the bacterial precipitates were washed with Dulbecco’s phosphate-buffered saline (DPBS) and resuspended in normal saline (NS) for peritoneal injection into mice. To evaluate the pathogenicity of the bacterial clones isolated from the blood or peritoneal fluid of CLP mice, the mortality of mice was observed up to 48 h after low-dose (5 × 106 CFU/g body weight) or high-dose (1 × 107 CFU/g body weight) bacterial injection. To study the lethal bacterial strain MSI001, we randomly assigned a total of 30 C57BL/6 mice evenly to 3 groups, i.e., the low-dose group, high-dose group and control group, in which the mice were subjected to peritoneal injection with 5 × 106 or 1 × 107 CFU/g bodyweight bacteria or an equivalent volume of NS, respectively. The survival time of mice was observed for at least 48 h after bacterial injection.

To study the therapeutic effect of synthetic peptides, C57BL/6 mice were randomly divided into 4 groups. All mice were intraperitoneally injected with bacteria (6 × 106 CFU/g body weight) to reproduce a bacterial infection-induced sepsis model. After bacterial injection for 1 h, the mice were intravenously injected with the peptides LL37, VTK-LL37, or VTK at a dose of 0.2 nmol/g body weight. NS was used as control. The survival time of mice was observed for up to 168 h after bacterial injection.

Biopanning assay

After washing with DPBS 3 times, the cultured bacteria were resuspended in 250 μL of DPBS. Ten microliters of C7C phage library (New England Bio Labs) containing 2.0 × 1011 PFU was added to the purified bacteria and incubated for 10 min at room temperature. Unbound phages were removed by extensive washing with Tris buffered saline with Tween 20 (TBST) containing 0.1% Tween 20, and the phages bound to bacteria were eluted by glycine-HCl solution. Phage titration was performed by fold dilution, and phage DNA was extracted using a Quick Gene DNA Tissue Kit for M13 (BioTeke, China) following the procedure supplied by the manufacturer.

Construction of His-tagged fusion protein expression plasmids

After enzyme digestion with BamHI, the pET14b/His-EGFP plasmid was linearized and used as the template for PCR amplification.62 The DNA sequences encoding heptapeptides were fused to the His-EGFP-encoding sequence to construct the expression plasmid pET14b/His-EGFP-C7C by PCR using the common forward primer 5’-TGCTAAGGATCCCAAAGCCCGAAAGGAAGCTGAGTTGG-3’ and a corresponding reverse primer (Supplementary Table 6). The PCR was conducted with initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 98 °C for 30 s, annealing at 62 °C for 30 s and extension at 68 °C for 1 min, with a final extension at 68 °C for 10 min. The amplified products were resolved by electrophoresis on a 1% agarose gel. PCR products were phosphorylated, ligated, and then transformed into E. coli BL21(DE3) for the production of His-EGFP-C7C proteins.

Affinity validation of binding peptides

The E. coli MSI001 bacterium was pelleted by centrifugation and washed with DPBS 3 times. Then, the His-EGFP and His-EGFP-C7C fusion proteins were added to 600 µL of a suspension containing 3 × 1010 CFU bacteria with a final concentration of 1 µmol/L. After incubation for 4 h, the sample was centrifuged at 4 °C and 10,000 rpm for 3 min, the supernatant was discarded, and the sediment was washed with DPBS 3 times and resuspended in 200 µL of DPBS. Finally, the fluorescence intensity of the protein binding to the bacteria was measured using a Spectramax M5 spectrometer (Molecular Devices, San Jose, CA, USA). Theoretically, a heptapeptide with a relatively high frequency should have a relatively large fluorescence intensity value. To test the correlation between the frequency of the heptapeptide and the fluorescence intensity, we performed bivariate correlation analysis with SPSS (v20.0).

In vitro antibacterial assay

The synthetic peptides LL37, VTK-LL37, LL37-VTK, KYY-LL37, LL37-KYY, ISS-LL37 and INS-LL37 were added to a bacterial suspension (1 × 103 CFU) at final concentrations of 0.25 μmol/L, 0.5 μmol/L, and 1 μmol/L. Sterile DPBS was used as control. The bacteria were incubated at 37 °C for 8 h, and the culture suspension was sequentially obtained for serial dilution with LB. The diluted suspension was plated on agar plates for culturing at 37 °C overnight, followed by calculating the bacterial colony-forming units (CFU).

Biofilm detection

When the OD600 of the bacterial suspension reached 1.0, the bacteria were transferred to a 96-well plate, and the VTK-LL37 or LL37 peptide was added at final concentrations of 0.5 μmol/L, 1 μmol/L, 2 μmol/L, or 4 μmol/L, followed by incubation at 37 °C. Replacement with fresh LB broth, as well as the peptides VTK-LL37 and LL37, was performed every 24 h. After culturing for 3 days, the suspended bacteria were discarded, and the plate was washed with DPBS 3 times. Bacterial biofilms were observed by microscopy and quantitated by spectrometry. In detail, after air-drying for 10 min, the biofilm attached to the bottom of a 96-well plate was stained with 1% crystal violet for 20 min. Then, the plate was washed with sterile water 3 times, followed by air drying. The precipitates in the plate were dissolved in 95% ethanol, followed by detection of the absorbance at 570 nm (A570) with a Spectramax M5 spectrometer.

In vivo antibacterial assay

Bacteria in the logarithmic growth stage were collected and washed with DPBS 3 times. Thirty-two C57BL/6 mice were evenly divided into 4 groups, i.e., the sepsis group, VTK-LL37 group, LL37 group and control group. Mice in the sepsis, LL37 and VTK-LL37 groups were intraperitoneally injected with E. coli MSI001 at a dose of 6 × 106 CFU/g bodyweight to reproduce the sepsis model. The control mice received an injection of an equivalent volume of NS. One hour after sepsis modeling, the mice of VTK-LL37 and LL37 groups were subjected to tail vein injection (TVI) of VTK-LL37 or LL37 at a dose of 0.2 nmol/g body weight, and the other group mice were injected with an equivalent volume of NS. Twelve hours later, blood was sequentially obtained from mice and diluted with LB broth. The diluted samples were plated on agar plates and cultured at 37 °C overnight to count the CFUs.

Histopathological examination

Twelve hours after modeling, the mice were anesthetized with 1.5% pentobarbital (0.1 ml/20 g), and the liver, kidney, heart and lung tissues were collected for routine pathological examination. The tissue samples were fixed with 4% formalin for 12 h and embedded in paraffin, followed by 3-μm-thick sectioning. Hematoxylin and eosin (H&E) staining was performed for observation under an Axio Imager Z2 microscope (Zeiss, Germany).63

Histopathological evaluation

Tissue sections of vital organs were stained with H&E and observed by microscopy. Multiple organ injury representations, including interalveolar septum thickened in the lung, tubular epithelial cell swelling in the kidney, inflammatory cell infiltration in the liver and heart. The detail criteria were based on a previously established method.64

Pull-down assay for heptapeptides

Membrane proteins were obtained from E. coli MSI001 by using a bacterial membrane protein extraction kit (BesBio, Shanghai, China). Biotinylated VTK or HEE heptapeptide was incubated with streptavidin-coated magnetic beads (Millipore, LSKMAGT) for 1 h. The unbound biotinylated heptapeptides were removed by washing with PBS 3 times. The heptapeptide-bound magnetic beads were incubated with the bacterial membrane proteins at 37 °C for 4 h to pull down the membrane proteins interacting with the heptapeptide.

Sample preparation for LC-MS/MS

Protein samples were prepared in accordance with a standard protocol for filter-aided sample preparation (FASP). Briefly, the protein sample (100 µg) in a Vivacon 500 filtrate tube (Sartorius Stedim Biotech GmbH, Goettingen, Germany) was mixed with 300 µL of 8 mol/L urea in 0.1 mol/L NH4HCO3 (pH 8.5), followed by centrifugation at 14,000 × g at room temperature for 15 min. The sample was washed with 300 µL of 8 mol/L urea in 0.1 mol/L NH4HCO3 (pH 8.5), and irrelevant components were removed by centrifugation. After repeating this procedure three times, the sample was washed with 300 µL of 0.1 mol/L NH4HCO3 (pH 8.5).

The sample was incubated with 5 μL of 0.5 mol/L dithiothreitol (DTT) in 250 μL of 100 mmol/L NH4HCO3 solution at 56 °C for 30 min, followed by incubation with 10 μL of 0.5 mol/L iodoacetamide (IAA) in the dark for 30 min. After three washes with 300 μL of 100 mmol/L NH4HCO3, the protein sample was digested in a filter with 2 μg of trypsin (Cat# v5280, Promega, Madison, WI, USA) in 300 μL of 0.1 mol/L NH4HCO3. After incubation at 37 °C for 16–18 h, the peptides were collected through centrifugation at 14,000 × g for 15 min. Finally, the concentrated peptides were desalted through a C18 column (Cat# S181001; Agela Technologies, Torrance, CA, USA).

LC-MS/MS analyses

The peptide sample (1 µg) was analyzed by using an EASY-nLC1200 instrument connected to an Orbitrap fusion mass spectrometer (Thermo Scientific, Waltham, MA, USA). The peptides were separated by a linear gradient of 5–30% acetonitrile (ACN) with 0.1% formic acid (FA) at 300 nL/min for 48 min, which was then linearly increased to 100% ACN over 7 min and maintained at 100% for 5 min. The column was re-equilibrated with 6 µL of 0.1% FA. The data-dependent acquisition (DDA) scheme was performed with a full MS survey scan in the range of m/z 350 to 1500 at a resolution of 120,000 full width at half maximum (FWHM) and the automatic gain control (AGC) set to 2.0E5, followed by a top speed data acquisition model at a resolution of 30,000 FWHM with the AGC set to 5.0E4.

MS raw data were processed by MaxQuant software (v1.6.10.43) (https://www.maxquant.org/maxquant/, Max Plank Institute of Biochemistry, Planegg, Germany), and fragments were searched against the UniProt database of Mus musculus. The false discovery rate (FDR) was set to 0.01, and a minimum length of 7 AAs for peptides was specified. The search results were processed with Perseus software (v1.6.10.50) (https://www.maxquant.org/perseus/).

Bioinformatic analysis for heptapeptide binding proteins

CPCoA based on the Bray-Curtis distance matrix was conducted by online ImageGP software (http://www.ehbio.com/ImageGP/). Visual hierarchical cluster analysis was performed after generating volcano plots and heatmaps in ImageGP software (http://www.ehbio.com/ImageGP/index.php/Home/Index/index.html). Protein sequence alignment was performed on UniProt (https://www.uniprot.org/).

SPRi analysis

To measure the binding affinity of peptides with targeted proteins, a PlexArray HT A100 (Plexera, USA) Surface Plasmon Resonance imaging (SPRi) system was used to monitor the whole procedure in real time. Briefly, a chip with a well-prepared peptide microarray was assembled with a plastic flow cell for sample loading. The operation details for the PlexArray HT were as described previously.65 The purified protein samples were prepared at determined concentrations in PBS running buffer, while glycine-HCl buffer (10 mmol/L, pH 2.0) was used for regeneration. Binding data were collected and analyzed by SPR Data Analysis Module software (Plexera, USA). The KD values were obtained using a 1:1 Langmuir binding model via the BIAevaluation Software ver4.1 (Biacore AB, Germany).

Statistical analysis

Results are expressed as the mean ± SEM except other description. For statistical analysis, significant differences between groups which pass both normality (Shapiro-Wilk test) and equal variance test (Levene’s test) were evaluated using the one-way ANOVA follow by Bonferroni post hoc test for multiple comparisons, or the unpaired two-tailed Student’s t-test for comparison between two groups. Welch correction was used if the data failed the test of equal variance and followed by Dunnett T3 post hoc test for multiple comparisons. Survival differences were analyzed by Kaplan-Meier survival curves. Bivariate regression analysis was performed to determine the correlation between two variables. All statistics were analyzed using Statistical Package for the Social Sciences (SPSS) software (v22.0), and P < 0.05 was considered statistically significant.


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