Abstract
The biofilm production of Pseudomonas aeruginosa (PA) is central to establishing chronic infection in the airways in cystic fibrosis. Epithelial cells secrete an array of innate immune factors, including antimicrobial proteins and lipids, such as human beta defensin 2 (HBD2) and cholesteryl lineolate (CL), respectively, to combat colonization by pathogens. We have recently shown that HBD2 inhibits biofilm production by PA, possibly linked to interference with the transport of biofilm precursors. Considering that both HBD2 and CL are increased in airway fluids during infection, we hypothesized that CL synergizes with HBD2 in biofilm inhibition. CL was formulated in phospholipid-based liposomes (CL-PL). As measured by atomic force microscopy of single bacteria, CL-PL alone and in combination with HBD2 significantly increased bacterial surface roughness. Additionally, extracellular structures emanated from untreated bacterial cells, but not from cells treated with CL-PL and HBD2 alone and in combination. Crystal violet staining of the biofilm revealed that CL-PL combined with HBD2 effected a significant decrease of biofilm mass and increased the number of larger biofilm particles consistent with altered cohesion of formed biofilms. These data suggest that CL and HBD2 affect PA biofilm formation at the single cell and community-wide level and that the community-wide effects of CL are enhanced by HBD2. This research may inform future novel treatments for recalcitrant infections in the airways of CF patients.
Highlights
Biofilm is a mixture of microbes, extracellular polysaccharides, DNA, and proteins and promotes bacterial residence establishment and resistance to host immune factors, as well as antimicrobial drugs [1,2]
We found that cholesteryl lineolate (CL) containing liposomes (CL-PL) alone and in combination with human beta defensin 2 (HBD2) significantly increased bacterial surface roughness
Combination treatment of Pseudomonas aeruginosa (PA) with CL was formulated in phospholipid-based liposomes (CL-PL) and HBD2 yielded significant increases in surface roughness, established by atomic force microscopy (AFM) studies and a loss of extracellular structures emanating from the bacterial cell. This was accompanied by significant effects on biofilm production and structure of the bacterial biofilm, consistent with a break-up of the microbial mat, as determined by CV stain. These data suggest that CL effects structural changes on the bacterial surface that interfere with biofilm formation, and these effects are further enhanced by HBD2
Summary
Biofilm is a mixture of microbes, extracellular polysaccharides, DNA, and proteins and promotes bacterial residence establishment and resistance to host immune factors, as well as antimicrobial drugs [1,2]. AMPs can be found in vertebrates, invertebrates, plants, and fungi Their chief antimicrobial action is pore formation on bacterial surfaces—a microbicidal mechanism, though their immunomodulatory activities are increasingly recognized [6,11]. AMPs have additional intracellular effects, such as interference with DNA/RNA and protein synthesis [7] They exhibit potent antimicrobial activity against gram-positive and gram-negative bacteria, and against fungi, protozoa, and viruses. We have recently reported that at low concentrations, HBD2 inhibits biofilm formation, without reducing bacterial metabolic activity This was accompanied by changes in the outer membrane protein profile and cell surface topology, suggesting that HBD2 induces structural changes that impair the proper function of the membrane-associated proteins involved in biofilm precursor transport into the extracellular environment [26]
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