Midkine in host defence

Abstract

Midkine (MK) shares several features in common with antibacterial proteins of the innate immune system. These include growth factor properties, heparin-binding regions and effects on immune cells, such as recruitment and activation of neutrophils and macrophages. Indeed, recent research has demonstrated potent bactericidal and fungicidal activities of MK. This protein is constitutively expressed at relevant concentrations at barriers of the body, such as the skin and the large airways, where the body first encounters potential pathogens. The antibacterial properties of MK orthologues are preserved during evolution, as exemplified by miple2 of Drosophila. In addition to retinoic acid, promoters of MK gene expression include factors present at sites of infection, reactive oxygen species, activation of the transcription factor NF-κB and hypoxia. In the light of the development of resistance in pathogenic bacteria to conventional antibiotics, MK is an interesting molecule that could serve as a template in developing novel therapeutic strategies against bacterial and fungal infections, either alone or in combination with conventional antibiotics.

Linked articles: This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4.

Keywords: bactericidal; host defence; inflammation; innate immunity; midkine.

Figure 1

Distribution of bactericidal activity within the MK molecule. Using 20 amino acid long peptides and a radial diffusion assay (RDA) with Escherichia coli, the antibacterial activity was determined. In the assay, bacteria grow in solidified agar and peptides are added in wells that are punched out in the agar. After incubation, clearing zones around the wells can be measured, and the diameter corresponds to antibacterial activity. The primary sequence of MK is indicated and corresponding peptides are indicated (upper). The highest activities were recorded corresponding to peptide no. 5, associated with the NH₂ terminal domain and in the COOH terminal (e.g. peptide no. 12) as indicated (blue) in the model of MK (lower). (The figure is used with permission from Svensson et al., .)

Figure 2

Possible mechanism involved in the membrane-disrupting activity of MK. (A) Many antibacterial proteins adapt an α-helical, amphiphatic structure that may also be the case for the COOH terminal tail of MK when inserted in the membrane. To obtain this structure, cationic and hydrophobic amino acid residues are intermingled so that upon helix formation, one side becomes hydrophobic and one cationic. Electrostatic forces attract the cationic antibacterial protein to the anionic plasma membrane of bacteria. Thereafter, the amphiphatic character of the antibacterial protein executes a detergent-like or pore-forming activity, resulting in loss of bacterial integrity (adapted from Brogden, 2005). (B, C) In the lower part of the figure, negative staining and transmission electron microscopy have been used to investigate bacteria (Streptococcus pneumoniae) incubated in buffer, showing intact bacteria (left) and disrupted bacteria after exposure to an antibacterial protein (right).

Figure 3

Cholesterol-containing lipid bilayers of eukaryotic cells are protected against the membrane-disrupting activity of MK. The lytic activity of MK was compared in an assay using micelles containing cholesterol (corresponding to eukaryotic plasma membranes) and ergosterol (corresponding to fungal plasma membranes). The lytic activity, reflected as leakage of a fluorescent dye, is higher in the case of ergosterol-containing membranes. The values represent mean (±SD) of three separate experiments. (The figure is used with permission from Nordin et al., .)

Figure 4

MK is bactericidal against Streptococcus pneumoniae. (A) Using a viable count assay, MK shows strong bactericidal activity against Str. pneumoniae, a Gram-positive bacterium that is the most common cause of community-acquired pneumonia. In the assay, bacteria were incubated with MK at the indicated concentrations for 60 min, plated, and the number of colonies counted and compared with the number of colonies after incubation in buffer alone. MK is almost 10-fold more potent than the ‘classical’ antibacterial peptide LL-37. The values represent mean (±SD) of three separate experiments. (B) MK retains most of its bactericidal activity in the presence of salt at physiological concentrations, that is, sodium chloride at 140 mM, as reflected by the viable counts assay described above. The values represent mean (±SD) of three separate experiments. (C, D) Using scanning electron microscopy, intact bacteria (Str. pneumoniae) (C) can be compared with bacteria that have been incubated with MK (D), the latter showing disturbed integrity and leakage of intracellular contents. (The figure is used with permission from Nordin et al., .)