DOI: 10.5061/dryad.m63xsj4c8
title: Chemokines kill bacteria by binding anionic phospholipids without triggering antimicrobial resistance

description: Classically, chemokines coordinate leukocyte trafficking during immune
 responses; however, many chemokines have also been reported to
 possess direct antibacterial activity in vitro. Yet, the bacterial killing
 mechanism of chemokines and the biochemical properties that define which
 members of the chemokine superfamily are antimicrobial remain
 poorly understood. Here 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 Escherichia coli and Staphylococcus aureus and that
 they exert rapid bacteriostatic and bactericidal effects against E. coli
 with a higher potency than the antimicrobial peptide beta-defensin 3.
 Furthermore, our data support the idea that bacterial membrane cardiolipin
 facilitates the antimicrobial action of chemokines. 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. Our results provide proof of principle
 for developing chemokines as novel antibiotics resistant to bacterial
 antimicrobial resistance mechanisms.
description: # Chemokines kill bacteria by binding anionic phospholipids without
 triggering antimicrobial resistance
 [https://doi.org/10.5061/dryad.m63xsj4c8](https://doi.org/10.5061/dryad.m63xsj4c8) ## Description of the data and file structure **DRYAD DATA DESCRIPTION** **Figure 2:** The quantification file contains the background-subtracted mean fluorescence intensity (a.u.) of CXCL11-AZ647 under each experimental condition, as indicated in the column titles (in the absence (just CXCL11) or presence of CXCL5,  CXCL1, CXCL20, or CCL11).Normalization and statistic analysis are also included in this file.  **Figure 3:** 3A CCL20 binding to bacteria liposome block by liposomes- confocal image.lsm 3B Quantification of A- Excel/prism file Quantifications 11-05-2024.pzfx The quantification file contains the background-subtracted mean fluorescence intensity (a.u.) of CCL20-AZ647 under each experimental condition, as indicated in the column titles (buffer control, or in the presence of PE, PG, or CL liposomes). Normalization and statistic analysis are also included in this file.  **Figure S2:** S2A CXCL11 binding to bacteria liposome block by liposomes- Confocal image.lsm S2B Quantification of S2A- Excel/prism file Quantifications 11-05-2024.pzfx  The quantification file contains the background-subtracted mean fluorescence intensity (a.u.) of CXCL11-AZ647 under each experimental condition, as indicated in the column titles (buffer control, or in the presence of PE, PG, or CL liposomes). Normalization and statistic analysis are also included in this file.  The binding of AZ647-labeled chemokines was tested by confocal microscopy. For this, bacteria were grown in TSB to mid-early log phase (OD600 = 0.4 - 0.6) and washed once in AAB-85. Then, bacteria (1 x 10+6 cfu) were incubated with 0.3 μM of fluorescent chemokine in 100 μl of AAB-85 at 37°C for 20 min. In Figure 2A: All bacterial samples were incubated with AZ647-labeled chemokines in microcentrifuge tubes. Then, samples were washed twice with 400 μl/sample of AAB-85 and bacteria were collected by centrifugation (9,000 x g, 3 min). For confocal microscopy analysis, washed bacteria were fixed with 2% PFA, immobilized on #1.5 coverslips of 0.17 ± 0.02 mm thickness previously coated with 0.1mg/ml poly-D-lysine following the manufacturer’s recommendations, and mounted using Prolong Diamond Antifade mountant with DAPI on superfrost plus microscope slides. Samples were imaged with a confocal laser scanning microscope Zeiss LSM 880 or LSM980. We used oil immersion alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective and Immersol 518F immersion media (ne=1.518 (30°C), Carl Zeiss). A z-stack of images was collected across the entire cell. Identical image acquisition settings, and optimal parameters for x, y, and z resolution were used in all samples from each independent experiment, and representative images for each condition in each experiment are shown with the same display range. In Figure 2B: Before the addition of the fluorescent chemokine, bacteria were preincubated with 0.3 μM of unlabeled chemokines in 100 μl of AAB-85 at 37°C for 5 min. Then, samples were washed twice with 400 μl/sample of AAB-85 and bacteria were collected by centrifugation (9,000 x g, 3 min). For confocal microscopy analysis, washed bacteria were fixed with 2% PFA, immobilized on #1.5 coverslips of 0.17 ± 0.02 mm thickness previously coated with 0.1mg/ml poly-D-lysine following the manufacturer’s recommendations, and mounted using Prolong Diamond Antifade mountant with DAPI on superfrost plus microscope slides. Samples were imaged with a confocal laser scanning microscope Zeiss LSM 880 or LSM980. We used oil immersion alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective and Immersol 518F immersion media (ne=1.518 (30°C), Carl Zeiss). A z-stack of images was collected across the entire cell. Identical image acquisition settings, and optimal parameters for x, y, and z resolution were used in all samples from each independent experiment, and representative images for each condition in each experiment are shown with the same display range. Figure 2C shows quantification of Figure 2B data. We measured the AZ647 fluorescence intensity of each bacterium by generating a region of interest (ROI) around the cell using the oval tool. An equivalent ROI was generated at a region outside the bacterium, considered as background and subtracted from the cell fluorescence intensity. The data were further analyzed and normalized against the control mean using GraphPad Prism 9. Figure 2D shows localization of bacteria-bound CXCL9 was analyzed by Airyscan confocal microscopy in W3110 E. coli bacteria co-stained with Nonyl Acridine Orange (NAO, Thermo Fisher Scientific). For this, bacterial cultures grown overnight were diluted 1:30 in TSB in the presence of 2 μM NAO and cultured in a lab shaker at 37°C and 220 rpm until OD600 ≥ 0.55. Then, bacteria were washed once with AAB-85 and 40 x 10+6 cfu were incubated with 0.3 μM CXCL9-AZ647 in 100 μl of AAB-85 at 37°C for 15 min. Bacteria were washed, fixed, immobilized onto poly-D-lysine coated coverslips and mounted on microscope slides, as explained above. A z-stack of images was collected across the entire cell on a LSM980 confocal microscope equipped with Airyscan 2 detector (Carl Zeiss) using the super resolution settings in frame mode and optimal parameters for x, y, and z resolution. NAO-524 was imaged with a 488 nm, an MBS 488/561 and SBS SP 550 while a 561 nm argon laser, an MBS 488/561 and a SBS LP 525 was used to image NAO-630. CXCL9-AZ647 was imaged with a 639 nm laser, an MBS 488/561/639 and a SBS LP 640. Airyscan postprocessing was performed using the standard parameters. A line was created along a bacterium and the fluoresce profiles for each channel was generated using ImageJ and further normalized for the minimum and maximum florescence intensity of each independent channel. In Figure 3A and supplementary Figure 2A, AZ647-labeled chemokines CCL20 (Figure 3A-B) or CXCL11 (Fig. Supp2A-B) were preincubated with 100 μM of PC liposomes containing 30% of PE, PG or CL in 50 μl of AAB-85 at room temperature for 5 min. Then, 1 x 10+6 cfu of bacteria in 50 μl of AAB-85 were added to the liposome-chemokine mix and incubated at 37°C for 20 min. Then, samples were washed twice with 400 μl/sample of AAB-85 and bacteria were collected by centrifugation (9,000 x g, 3 min). For confocal microscopy analysis, washed bacteria were fixed with 2% PFA, immobilized on #1.5 coverslips of 0.17 ± 0.02 mm thickness previously coated with 0.1mg/ml poly-D-lysine following the manufacturer’s recommendations, and mounted using Prolong Diamond Antifade mounting media with DAPI on superfrost plus microscope slides. Samples were imaged with a confocal laser scanning microscope Zeiss LSM 880 or LSM980. We used oil immersion alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective and Immersol 518F immersion media (ne=1.518 (30°C), Carl Zeiss). A z-stack of images was collected across the entire cell. Identical image acquisition settings, and optimal parameters for x, y, and z resolution were used in all samples from each independent experiment, and representative images for each condition in each experiment are shown with the same display range. Microscopy data processing, analysis, and quantification were done in ImageJ. Figure 3B or S2B shows quantification of the CCL20 or CXCL11 bacterial binding data showed in Figure 3A or Figure S2A, respectively. We measured the AZ647 fluorescence intensity of each bacterium by generating a region of interest (ROI) around the cell using the oval tool. An equivalent ROI was generated at a region outside the bacterium, considered as background and subtracted from the cell fluorescence intensity. The data were further analyzed and normalized against the control mean using GraphPad Prism 9. **Quantifications 11-05-2024.pzfx: ** Self-explanatory file containing titration results, absorbance readings, statistical tests like t-tests, ANOVA, and nonlinear regression. ## Code/software Image J or Fiji with the Bioformat plugging is needed to open and visualize the confocal images (.lsm or .tif files) Prism is needed to open the .pzfx file