Mucociliary clearance defects in a murine in vitro model of pneumococcal airway infection.

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where--in pneumococcal infection--persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance

Pneumococci do not affect the ciliary beat frequency.

<p>In ciliated respiratory epithelial cells derived from mouse trachea that are induced to undergo ciliogenesis in air-liquid interface cultures, a typical ciliary beat frequency of 15±5 Hz (beats per second) was regularly observed. Slight random and temporary variations in beat frequencies occurred upon handling of the cultures and sporadically exceeded 25 Hz. In each individual experiment, the apical (ciliated) epithelial side of one culture was infected with viable bacteria and beat frequencies were measured in various epithelial areas at different time points. Each box indicates the mean beat frequency within a ∼30 min interval as indicated. Bars depict the standard derivation (SD) of results from individual experiments. Lines connect the results for each bacterial species or control, respectively. (<b>A</b>) Persistent beating (15±5 Hz) was observed upon <i>in vitro</i> infection with pneumococci (D39, wildtype strain; plyA(−), pneumolysin-deficient derivate) within 3 hours after addition of bacteria as in controls. D39∶199 measurements in 8 independent experiments; plyA(−) 66/3; no bacteria: 74/3 (<b>B</b>) Robust maintenance of the baseline ciliary beat frequency within 3 hours after infection with pathogenic and non-pathogenic <i>Escherichia coli</i> (HB101) as well as with the airway pathogens Group-B streptococci (GBS; NEM316), <i>Haemophilus influenza</i> and <i>Staphylococcus aureus</i>. GBS: 70 measurements in 2 independent experiments; <i>E.coli</i> path: 30/2; HB101∶28/1; <i>Haemoph</i>: 35/2; <i>S.aureus</i>: 41/2.</p

Pneumococci cause disintegration of the ciliated epithelium by F-actin reorganization and partial undocking of cilia.

<p>Respiratory epithelia were analyzed for morphological changes after infection by staining of cilia (green), F-actin (red) and nuclei (blue) with confocal Z-stack imaging (orthogonal views). Scale bar: 10 µm. (<b>A</b>) The respiratory epithelium is characterized by a strictly planar alignment of columnar-shaped cells, exclusive apical localization of cilia and basal positioning of the nuclei. Prominent F-actin is detected at the junctional and apical cell cortex (where the ciliary bases are anchored) and to a much lesser extent at the lateral cell borders. (<b>B</b>) Infection with pneumococci (D39, 2 h45 min) causes a disorganization of the polarized, planar epithelial structure, including the cell shapes and all analyzed sub-cellular compartments. The cytoplasmic space is frequently reduced and the basal positioning of nuclei is lost. Mislocalization of F-actin to the lateral cell borders occurs and a high proportion of cilia detach from the apical cell side, probably due to damage to the apical F-actin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059925#pone.0059925.s003" target="_blank">Figure S3</a>). Hence, persistent ciliary beating might only generate insufficient extracellular fluid flow.</p

<i>Ex vivo</i> pneumococcal airway infection causes distortion of the F-actin cytoskeleton and of the planar epithelial architecture.

<p>Freshly dissected mouse tracheae were untreated (left) or infected with pneumococci (3 h; right) and analyzed for morphological changes by confocal fluorescence microscopy. 3D illustrations were assembled from z-stack images. Scale bar: 10 µm. (<b>A</b>) The normal morphology of the F-actin cytoskeleton in ciliated tracheal epithelium is characterized by a robust F-actin cytoskeleton. In 3D side views, abundant F-actin staining is only observed at the apical cell sides (untreated control, left). In pneumococcal infected samples, massive mis-localization of F-actin throughout the entire cell body area occurs (right). (<b>B</b>) 3D profile views show that upon infection with pneumococci the stable F-actin cytoskeleton of the tracheal epithelium (left) is converted into an irregular structure (right). (<b>C</b>) The planar ciliated epithelial surface of the tracheal epithelium (left) is irregularly distorted in the infected sample (right). Single pneumococci sporadically adhered to the ciliated surface (3D profile view). (<b>D</b>) Overlay images, showing the collapse of the polarized, planar organization of the ciliated tracheal epithelium after pneumococcal infection (3D profile view).</p

Possible mechanism which could enable pneumococci to resist mucociliary clearance.

<p>(<b>A</b>) Columnar shaped epithelial cells generate powerful cilia strokes and efficient extracellular flow along the planar, mechanically stable surface. (<b>B</b>) Pneumococcal infection causes F-actin mislocalization, disintegration of the epithelial architecture, weakened mechanical stability, detachment of cilia and powerless cilia strokes. Persistent ciliary beating generates local turbulence. Impaired clearance is independent of reduction in ciliary beat frequency.</p

Pneumolysin-independent disruption of the F-actin cytoskeleton causes loss of planar epithelial surface architecture.

<p>Heavily ciliated respiratory epithelia were infected (3 h) <i>in vitro</i>. 3D profile views of the F-actin cytoskeleton alone (phalloidin staining, left panel) and with cilia (acetylated α-tubulin, right panel) were assembled from confocal Z-stack images. Bacteria, which were only found sporadically attached to the ciliated surface after sample processing and nuclei are not shown. Scale bar: 10 µm. (<b>A</b>) The normal planar epithelial morphology is built on a stable “honeycomb-like” F-actin cytoskeleton. It is composed of abundant F-actin at the junctional and apical cell cortex, and is maintained after control inoculation with non-invasive <i>E.coli</i>. (<b>B</b>) Upon pneumococcal infection (D39, parental wildtype strain), the F-actin cytoskeleton is converted into a non-stable “net-like” structure. Reduced apical F-actin indicates the loss of the mechanical support for efficient cilia strokes. The cell junctions remain closed, although severe distortions of the junctional F-actin occur. The non-planar alignment of aberrantly deformed, dome-shaped cells indicates that persistent ciliary beating generates fluid flow turbulence. (<b>C</b>) The comparable severity of epithelial damage after infection with pneumolysin-deficient, plyA(−), and -sufficient pneumococci, suggests a pneumolysin-independent mechanism.</p

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