The Innate Immune Protein Calprotectin Interacts With and Encases Biofilm Communities of Pseudomonas aeruginosa and Staphylococcus aureus

Abstract

Calprotectin is a transition metal chelating protein of the innate immune response known to exert nutritional immunity upon microbial infection. It is abundantly released during inflammation and is therefore found at sites occupied by pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. The metal limitation induced by this protein has previously been shown to mediate P. aeruginosa and S. aureus co-culture. In addition to the transition metal sequestration role of calprotectin, it has also been shown to have metal-independent antimicrobial activity via direct cell contact. Therefore, we sought to assess the impact of this protein on the biofilm architecture of P. aeruginosa and S. aureus in monomicrobial and polymicrobial culture. The experiments described in this report reveal novel aspects of calprotectin's interaction with biofilm communities of P. aeruginosa and S. aureus discovered using scanning electron microscopy and confocal laser scanning microscopy. Our results indicate that calprotectin can interact with microbial cells by stimulating encapsulation in mesh-like structures. This physical interaction leads to compositional changes in the biofilm extracellular polymeric substance (EPS) in both P. aeruginosa and S. aureus.

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; biofilm architecture; calprotectin; extracellular polymeric substance (EPS); nutritional immunity.

Figure 1

Addition of CP to growth media leads to encapsulation of P. aeruginosa and S. aureus in a mesh-like structure. SEM images of P. aeruginosa and S. aureus biofilms grown as mono and co-cultures in +/- CP conditions- (A) P. aeruginosa monoculture (B) S. aureus monoculture (C) P. aeruginosa-S. aureus co-culture. Images show 2000X, 4500X, and 10,000X magnification and are representative of three independent experiments.

Figure 2

Calprotectin is a major component of the mesh-like structure that encapsulates P. aeruginosa and S. aureus biofilms. Intracellular DNA of microbial cells was detected by staining with the cell-permeant nucleic acid dye, Hoechst 33342; CP was detected by staining with Alexa fluor 488-tagged Goat anti-Rabbit antibody. (A) P. aeruginosa monoculture (B) S. aureus monoculture (C) P. aeruginosa-S. aureus co-culture. Images show 100X magnification and were processed using ImageJ.

Figure 3

Mouse CP signals overlap with bacterial signals in paraffin-embedded dorsal tissue sections. Non-injured skin control collected 12 days post-depilation display no background CP or bacterial signal, 12-day uninfected wound only data reveals minimal CP signal and no background bacterial signal. Duplicate samples of 12-day PAO1-infected wounds reveal both CP and bacterial signals. CP was detected by staining with Alexa fluor 488-tagged Goat anti-Rabbit antibody, PAO1 was detected by staining with Alexa fluor 594-tagged Goat Anti- Chicken antibody and nuclei were detected by staining with the cell-permeant dye, Hoechst 33342. Images are shown at 100X magnification and processed using ImageJ.

Figure 4

Addition of CP minimally impacts the relative abundance of eDNA in biofilm matrices of P. aeruginosa and S. aureus. This figure depicts confocal images that represent abundance of eDNA in P. aeruginosa and/or S. aureus biofilm communities in presence or absence of CP conditions. The fluorescence dyes Hoechst 33342, Alexa 488, and TOTO-3 iodide correspond to intracellular DNA of microbial cells, CP, and eDNA in the biofilm EPS matrix of- (A) P. aeruginosa monoculture (B) S. aureus monoculture (C) P. aeruginosa-S. aureus co-culture. (D) Quantification of eDNA per biomass in P. aeruginosa and S. aureus biofilms in presence or absence of CP. (E) Colocalization analysis of eDNA signals with cells and CP show minimal correlation of distribution of eDNA with cells and/or CP. Images show 100X magnification and were processed using ImageJ. Bars represent the mean of four biological replicates performed on two independent days. Error bars represent the standard error of mean of the biological replicates. Unpaired t-test (two-tailed) was used to measure statistical significance. Comparisons marked ns denote changes that were not found to be statistically significant.

Figure 5

Addition of CP correlates with subtle increase in the protein component of biofilm matrices. This figure depicts confocal images that represent abundance of proteins in P. aeruginosa and/or S. aureus biofilm communities in presence or absence of CP conditions. The fluorescence dyes Hoechst 33342, Alexa 488, and SYPRO Ruby correspond to intracellular DNA of microbial cells, CP, and matrix proteins in the biofilm EPS of- (A) P. aeruginosa monoculture (B) S. aureus monoculture (C) P. aeruginosa-S. aureus co-culture. (D) Quantification of proteins per biomass in P. aeruginosa and S. aureus biofilms in presence or absence of CP. (E) Colocalization analysis of protein signals with cells and CP show similar distribution of protein signals around cell signals and CP signals. Images show 100X magnification and were processed using ImageJ. Bars represent the mean of four biological replicates performed on two independent days. Error bars represent the standard error of mean of the biological replicates. Unpaired t-test (two-tailed) was used to measure statistical significance. Comparisons marked ns denote changes that were not found to be statistically significant.

Figure 6

Addition of CP leads to an increase in EPS carbohydrates in P. aeruginosa monoculture biofilms. This figure depicts confocal images that represent abundance of carbohydrates in P. aeruginosa and/or S. aureus biofilm communities in presence or absence of CP conditions. The fluorescence dyes Hoechst 33342, Alexa 488, and TRITC ConA correspond to intracellular DNA of microbial cells, CP, and carbohydrates in the biofilm EPS of- (A) P. aeruginosa monoculture (B) S. aureus monoculture (C) P. aeruginosa-S. aureus co-culture. (D) Quantification of carbohydrates per biomass in P. aeruginosa and S. aureus biofilms in presence or absence of CP. (E) Colocalization analysis of carbohydrate signals with cells and CP signals show higher correlation with CP compared to cells. Images show 100X magnification and were processed using ImageJ. Bars represent the mean of four biological replicates performed on two independent days. Error bars represent the standard error of mean of the biological replicates. Unpaired t-test (two-tailed) was used to measure statistical significance. **p≤ 0.01.