Bacterial phosphorylcholine decreases susceptibility to the antimicrobial peptide LL-37/hCAP18 expressed in the upper respiratory tract

Abstract

A number of pathogens of the upper respiratory tract express an unusual prokaryotic structure, phosphorylcholine (ChoP), on their cell surface. We tested the hypothesis that ChoP, also found on host membrane lipids in the form of phosphatidylcholine, acts so as to decrease killing by antimicrobial peptides that target differences between bacterial and host membranes. In Haemophilus influenzae, ChoP is a phase-variable structure on the oligosaccharide portion of the lipopolysaccharide (LPS). There was a bactericidal effect of the peptide LL-37/hCAP18 on a nontypeable H. influenzae strain, with an increasing selection for the ChoP(+) phase as the concentration of the peptide was raised from 0 to 10 microgram/ml. Moreover, constitutive ChoP-expressing mutants of unrelated strains showed up to 1,000-fold-greater survival compared to mutants without ChoP. The effect of ChoP on resistance to killing by LL-37/hCAP18 was dependent on the salt concentration and was observed only when bacteria were grown in the presence of environmental choline, a requirement for the expression of ChoP on the LPS. Further studies established that there is transcription of the LL-37/hCAP18 gene on the epithelial surface of the human nasopharynx in situ and inducible transcription in epithelial cells derived from the upper airway. The presence of highly variable amounts of LL-37/hCAP18 in normal nasal secretions (<1.2 to >80 microgram/ml) was demonstrated with an antibody against this peptide. It was concluded that ChoP alters the bacterial cell surface so as mimic host membrane lipids and decrease killing by LL-37/hCAP18, an antimicrobial peptide that may be expressed on the mucosal surface of the nasopharynx in bactericidal concentrations.

FIG. 1

Selection for LPS phase variants in the presence of LL-37/hCAP18. Nontypeable clinical isolate H233 shows a dose-dependent killing caused by LL-37/hCAP18. Numbers of bacteria surviving treatment for 60 min in the concentration of LL-37/hCAP18 indicated on the x axis are shown in panel A. The horizontal arrowhead along the y axis indicates the total number of bacteria present at the beginning of the incubation period (0 min). H233 is a mixed population of phase variants with (solid symbols) and without (open symbols) ChoP or Galα1-4Gal on their LPS (37). Survivors showed a dose-related increase in the proportion of ChoP⁺ compared to ChoP⁻ phase variants as determined by colony immunoblotting (B). There was, however, no selection for phase variants with or without the Galα1-4Gal structure (C). Results are the mean of a representative experiment in duplicate.

FIG. 2

Effect of ChoP on susceptibility to the bactericidal activity of LL-37/hCAP18. ChoP-on (solid symbols) and ChoP-off (open symbols) constitutive mutants were compared in unrelated strains with different LPS structures, unencapsulated type d strain Rd (A) and type b strain Eagan (B). Cells were treated for 60 min at 37°C with LL-37/hCAP18 at the concentrations indicated on the x axis, and the number of survivors was determined in viable counts. The horizontal arrowheads along the y axis indicate the number of bacteria of each type present at the beginning of the incubation period (0 min). Numbers represent the mean of at least four determinations + standard deviation.

FIG. 3

Effect of environmental choline on ChoP expression on the LPS and susceptibility to the bactericidal activity of LL-37/hCAP18. Constitutive ChoP-on mutant H491 was grown in a chemically defined medium for H. influenzae with (solid symbols) or without (open symbols) supplemental choline (5 μg/ml), and the sensitivity to killing by LL-37/hCAP18 (for 60 min at 37°C) was determined. The horizontal arrows along the y axis indicate the number of bacteria grown with or without choline present at the beginning of the incubation period (0 min). Western blot analysis (insert) of whole cell lysates using a MAb against ChoP shows the presence of ChoP on the LPS when grown with choline in the medium (lane 2) but not in the same medium without added choline (lane 1). Size markers are in kilodaltons.

FIG. 4

Effect of salt concentration on the bactericidal activity of LL-37/hCAP18. ChoP-on (H512; solid bars) and ChoP-off (H445; stippled bars) constitutive mutants of strain Eagan were treated for 60 min at 37°C with LL-37/hCAP18 (final concentration, 5 μg/ml), and the number of survivors was determined in viable counts. NaCl was added to medium E (16.7 mM Na⁺) in bactericidal assays at the final concentrations indicated at the bottom. Results are expressed in relation to the same conditions without peptide and are the mean of two determinations in duplicate.

FIG. 5

Binding of LL-37/hCAP18 to H. influenzae Rd with and without LPS-ChoP. Equivalent numbers of constitutive ChoP-off strain H446 (A) or ChoP-on strain H491 (B) were incubated with the concentration (in μg/ml) of LL-37/hCAP18 indicated above each lane. After removal of unbound peptide, the binding of LL-37/hCAP18 to the bacteria was determined in whole cell lysates. Following separation on SDS–18% polyacrylamide gels, the amount of bound peptide was determined by Western blotting using an antiserum raised against LL-37/hCAP18.

FIG. 6

In situ hybridization of LL-37/hCAP18 in a human nasal polyp. Tissue sections of the same polyp were hybridized with a [³⁵S]UTP-labeled RNA antisense (A) or sense (B) probe. The nasal epithelial surface is visualized by the blue fluorescence of cell nuclei with the Hoechst 33258 counterstain (B) and by the dense band of staining with hematoxylin and eosin (C). Magnification, ×10. (D) Western blot showing LL-37/hCAP18 in nonpurulent nasal secretions collected from the same individual over a 3-week period. Ten microliters of solubilized nasal secretions collected on the day indicated was separated on an SDS–18% polyacrylamide gel, transferred to a membrane, and immunoblotted with an antiserum raised to LL-37/hCAP18. The control consists of 0.5 μg of chemically synthesized peptide. + and − denote whether the untreated secretions were both grossly torpid and contained >5 mg of total protein per ml. Size markers are in kilodaltons.

FIG. 6

In situ hybridization of LL-37/hCAP18 in a human nasal polyp. Tissue sections of the same polyp were hybridized with a [³⁵S]UTP-labeled RNA antisense (A) or sense (B) probe. The nasal epithelial surface is visualized by the blue fluorescence of cell nuclei with the Hoechst 33258 counterstain (B) and by the dense band of staining with hematoxylin and eosin (C). Magnification, ×10. (D) Western blot showing LL-37/hCAP18 in nonpurulent nasal secretions collected from the same individual over a 3-week period. Ten microliters of solubilized nasal secretions collected on the day indicated was separated on an SDS–18% polyacrylamide gel, transferred to a membrane, and immunoblotted with an antiserum raised to LL-37/hCAP18. The control consists of 0.5 μg of chemically synthesized peptide. + and − denote whether the untreated secretions were both grossly torpid and contained >5 mg of total protein per ml. Size markers are in kilodaltons.

FIG. 7

Detection of LL-37/hCAP18 mRNA in H. influenzae-infected Detroit 562 pharyngeal carcinoma cells in culture. RNA was isolated from uninfected cells (lanes 1 and 3) or cells infected for 4 h with 10⁶ CFU of strain H491 per ml (lanes 2 and 4) and subjected to RT-PCR using primers specific to the gene for LL-37/hCAP18 (lanes 1 and 2). RT-PCR using primers to the glyceraldehyde 3-phosphate dehydrogenase gene served as controls for the quality of the template (lanes 3 and 4). The PCR products were visualized using ethidium bromide after separation on a agarose gel. Molecular weight markers consisted of a 100-bp ladder.