DOI: 10.5061/dryad.dbrv15fc5
title: Chemokines kill bacteria without triggering antimicrobial resistance by binding anionic phospholipids

description: Classically, chemokines coordinate leukocyte trafficking; however, many
 chemokines also possess direct antibacterial activity. Yet, the bacterial
 killing mechanism of chemokines and the biochemical properties that define
 which members of the chemokine superfamily are antimicrobial remain poorly
 understood. We report that the antimicrobial activity of chemokines is
 defined by their ability to bind phosphatidylglycerol and cardiolipin, two
 anionic phospholipids commonly found in the bacterial plasma membrane. We
 show that only chemokines able to bind these two phospholipids kill
 bacteria and that they exert rapid bacteriostatic and bactericidal effects
 with a higher potency than the antimicrobial peptide beta-defensin 3. Both
 biochemical and genetic interference with the chemokine-cardiolipin
 interaction impaired microbial growth arrest, bacterial killing, and
 membrane disruption by chemokines. Moreover, unlike conventional
 antibiotics, E. coli failed to develop resistance when placed under
 increasing antimicrobial chemokine pressure in vitro. Thus, we have
 identified cardiolipin and phosphatidylglycerol as novel binding partners
 for chemokines responsible for chemokine antimicrobial action.
description: # Chemokines kill bacteria without triggering antimicrobial resistance by
 binding anionic phospholipids
 [https://doi.org/10.5061/dryad.dbrv15fc5](https://doi.org/10.5061/dryad.dbrv15fc5) ## Description of the data and file structure These are Excel, Prism files, and original FACS files **containing the raw numerical data** used to generate the final figures and FACS plots in the manuscript. The imaging data can also be found in the accompanying dataset 10.5061/dryad.m63xsj4c8.   ### Files and variables #### File: Figure\_1A.prism **Description:** Antimicrobial assays. Surviving number of *E. coli* colonies 2 h after treatment with increasing doses of protein/chemokine. Columns, treatment protein name. Rows, protein concentration in µM. #### File: Figure\_3D.pzfx **Description:** Antimicrobial assays. Surviving number *S. aureus* colonies 2 h after treatment with 5 µg/ml of CXCL9 or CXCL11 in the presence of liposomes containing 30% of cardiolipin (CL), phosphatidylglycerol (PG) or phosphatidylethanolamine (PE). Columns, type of liposome. Rows, liposome concentration in µM. #### File: Figure\_1B.pzfx **Description:** Antimicrobial assays. Surviving number of *E. coli* and *S. aureus* colonies 2 h after treatment with increasing doses of protein/chemokine. Columns, type (genera) of bacteria. Rows, chemokine concentration in µg/ml. #### File: Figure\_3C.pzfx **Description:** Antimicrobial assays. Surviving number of *E. coli* colonies 2 h after treatment with 5 µg/ml of CCL20 or CXCL11 in the presence of liposomes containing 30% of cardiolipin (CL), phosphatidylglycerol (PG) or phosphatidylethanolamine (PE). Columns, type of liposome. Rows, liposome concentration in µM. #### File: Figure\_4C.pzfx **Description:** Bacteria binding assays. Quantification of the median fluorescence intensity of the binding of fluorescently-labeled chemokines to W3110 and BKT12 strains of *E. coli*. Columns, *E. coli* strain name. Rows, chemokine name. #### File: Figure\_4E.pzfx **Description:** Antimicrobial assays. Surviving number of colonies for W3110 and BKT12 *E. coli* strains after combination and simultaneous incubation for 2 h with buffer alone or 1.2 µM of chemokine. Columns, *E. coli* strain name. Rows, treatment/chemokine name. #### File: Figure\_4F.prism **Description:** Antimicrobial assays. Surviving percentage of colonies for BW25113 and JW1241 *E. coli* strains 2 h after treatment with 1.2 µM of chemokine. Columns, *E. coli* strain name. Rows, treatment/chemokine name. #### File: Figure\_4D.pzfx **Description:** Antimicrobial assays. Surviving percentage of colonies for W3110 and BKT12 *E. coli* strains 2 h after treatment with 1.2 µM of chemokine. Columns, *E. coli* strain name. #### File: Figure\_4H.pzfx **Description:** Outer membrane permeabilization assay. Kinetics of the percentage of N-phenyl-1-phenylnapthylamine (NPN) fluorescence in W3110 and BKT12 *E. coli* strains treated (columns) with buffer alone, protamine, polymyxin B (PolB) or the chemokines CXCL9 and CXCL8, relative to the maximum fluorescence read measured after PolB treatment. Rows, time in minutes. #### File: Figure\_5B.pzfx **Description:** Bacterial plasma membrane integrity assay. Kinetics of the percentage of W3110 and BKT12 *E. coli* strains positive for SYTOX after treatment with 1.2 µM of human beta-defensin 3 (hBD3) or chemokines. Columns, *E. coli* strain name. Rows, time in minutes. #### File: Figure\_5A.pzfx **Description:** Bacterial growth assay. Kinetics of the growth of W3110 and BKT12 *E. coli* strains in the presence of buffer alone or 1.2 µM of human beta-defensin 3 (hBD3) or chemokines. Columns, *E. coli* strain name treated with buffer or chemokine. Rows, time in minutes. #### File: Figure\_5D\_quantification.pzfx **Description:** Bacterial plasma membrane integrity assay. Quantification of the percentage of SYTOX-positive W3110 and BKT12 *E. coli* strains 20 min after treatment with 4.8 µM of protamine or chemokines. Columns, *E. coli* strain name. Rows, treatment/chemokine name (Ptm, protamine). #### File: Figure\_5C.pzfx **Description:** Bacterial growth and killing assay. Quantification of the percentage of live (SYTOX-negative) and killed (SYTOX-positive) W3110 and BKT12 *E. coli* strains 1 h and 2 h after treatment with increasing doses of CXCL11 or CCL21. Columns, *E. coli* strain name live or killed. Rows, chemokine concentration in µM. #### File: Figure\_6A.prism **Description:** Calcein leakage assays. Kinetics of the percentage of calcein released from phophatidylethanolamine (PE) /phosphatidylglycerol (PG) /cardiolipin (CL) (70/25/5 mass ratio) liposomes in the presence of 1.2 µM protamine or chemokine relative to the maximum release detected after addition of Triton X-100. Columns, treatment/chemokine name. Rows, time in minutes.  #### File: Figure\_6C.xlsx **Description:**  Liposome SAXS assays. Peak positions (√) for the cubic phases (Pn3m, lm3m, and la3d) and mean negative gaussian curvature (k) detected by SAXS of phosphatidylglycerol/phosphatidylethamolamine (20:80 mass ratio) liposomes in the presence of increasing chemokine:liposome ratios. Blank cells indicate the absence of a cubic phase. Columns, type of cubic phase. Rows, chemokine name and chemokine:liposome ratio.  #### File: Figure\_6D.pzfx **Description:** Calcein leakage assays. Kinetics of the percentage of calcein released from phophatidylethanolamine (PE) /phosphatidylglycerol (PG) /cardiolipin (CL) (70/25/5 mass ratio), PE/PG (70/30 mass ratio) or PE/phosphatidylcholine (PC) (70/30 mass ratio) liposomes in the presence of 1.2 µM protamine or chemokine relative to the maximum release detected after addition of Triton X-100. Columns, type of liposome. Rows, time in minutes. #### File: Figure\_7C.pzfx **Description:** Minimum inhibitory concentration assay. Percentage of *E. coli* viability determined using the BacTiter-Glo kit after 18 h incubation with increasing doses of recombinant CCL20-His protein. Column, *E. coli* strain name. Rows, protein concentration in µM.  #### File: Figure\_6E.prism **Description:** Calcein leakage assays. Quantification of the percentage of calcein released from phophatidylethanolamine (PE) /phosphatidylglycerol (PG) /cardiolipin (CL) (70/25/5 mass ratio), PE/PG (70/30 mass ratio) or PE/phosphatidylcholine (PC) (70/30 mass ratio) liposomes 20 min after addition of increasing doses of protamine or the chemokines CCL21 and CXCL9 relative to the maximum release detected after addition of Triton X-100. Columns, type of liposome. Rows, protein concentration in µM. #### File: Figure\_7D.pzfx **Description:** Antimicrobial resistance assay. Fold change of the minimum inhibitory concentration (MIC) of ampicillin (Amp), tetracycline (Tet) and recombinant CCL20-His protein relative to their initial MIC after maintenance of E. coli cultures for 14 days in the presence of sublethal (0.5 x MIC) doses of the antimicrobial compounds. Columns, treatment/antimicrobial compound name. Rows, time in days. #### File: Figure\_7E.pzfx **Description:** Antimicrobial assays. Quantification (%) of the viability of the *E. coli* W3110 strains generated in Figure 7D, preconditioning for 14 days with ampicillin (Amp), tetracycline (Tet) and recombinant CCL20-His, 18 h after treatment with a dose corresponding to the initial minimum inhibitory concentration of each antimicrobial compound. Columns, *E. coli* strain name. Rows, treatment/antimicrobial compound name. #### File: Figure\_S4.prism **Description:** Bacterial growth assay. Kinetics of the growth of W3110 and BKT12 *E. coli* strains in the presence of buffer alone or 1.2 µM of protamine. Columns, *E. coli* strain name treated with protamine or buffer. Rows, time in minutes. #### File: Figure\_S6B.prism **Description:** Calcein leakage assays. Kinetics of the percentage of calcein released from phophatidylethanolamine (PE) /phosphatidylglycerol (PG) /cardiolipin (CL) (70/25/5 mass ratio), PE/PG (70/30 mass ratio) or PE/phosphatidylcholine (PC) (70/30 mass ratio) liposomes in the presence of 0.15 µM protamine or chemokine relative to the maximum release detected after addition of Triton X-100. Columns, type of liposome. Rows, time in minutes. #### File: Figure\_S3A.pzfx **Description:** Antimicrobial assays. Surviving percentage of *E. coli* colonies after 2 h treatment with 1.2 µM of CCL20, CXCL9 or human beta-defensin 3 (hBD3) in the presence of 0.85 or 85 mM NaCl. Columns, NaCl concentration in mM. Rows, treatment/chemokine name. #### File: Figure\_S7.pzfx **Description:** Antimicrobial assays. Surviving percentage of colonies for *E. coli* W3110 strain or the ampicillin-resistant strain (W3110-Amp) generated in Figure 7D after 2 h treatment with buffer alone, 1.2 µM of CXCL8 or CXCL9, or 5 µg/ml of ampicillin (Amp). Columns, *E. coli* strain name. Rows, treatment/chemokine name. #### File: Figure\_S5.prism **Description:** Bacterial plasma membrane integrity assay. Quantification of the percentage of SYTOX-positive W3110 and BKT12 *E. coli* strains 90 min after treatment with 4.8 µM of protamine (Ptm) or chemokines. Columns, *E. coli* strain name. Rows, treatment/chemokine name. #### File: Figure\_S3B.prism **Description:** Antimicrobial assays. Surviving percentage of *E. coli* colonies 2 h after treatment with increasing doses of protein/chemokine in the presence of 85 mM NaCl. Columns, treatment/chemokine name. hBD3, human beta-defensing 3. Rows, protein concentration in µM. #### File: Figure\_S6A.prism **Description:** Antimicrobial assays. Percentage of *E. coli* viability determined using the BacTiter-Glo kit after 2 h incubation with buffer alone or 1.2 µM of CCL5 or CCL19. Columns, treatment/chemokine name. #### File: Figure\_5D.wsp **Description:** Bacterial plasma membrane integrity assay. FlowJo file with the analysis performed to generate the contour plots of Figure 5D. Bacteria are stained with SYTO24 (FITC filter) and SYTOX-Orange (PE filter). #### File: Figure\_4B.wsp **Description:** Bacteria binding assay. FlowJo file with the analysis performed to generate the chemokine binding histograms of Figure 4B. Bacteria are stained with SYTO24 (FITC filter), SYTOX-Orange (PE filter) and AZ647-labeled chemokines (APC filter). Figure 4B shows chemokine binding to live bacteria (positive for SYTO24+, negative for SYTOX-Orange).  #### File: Figure\_4B\_FACS\_files.zip **Description:** Flow cytometry acquisition files used to generate Figure 4B. These are needed for Figure_4B.wsp.  #### File: Figure\_5D\_FACS\_files.zip **Description:**  Flow cytometry acquisition files used to generate Figure 5D. These are needed for Figure_5D.wsp.