Listeria monocytogenes spreads within the brain by actin-based intra-axonal migration

Abstract

Listeria monocytogenes rhombencephalitis is a severe progressive disease despite a swift intrathecal immune response. Based on previous observations, we hypothesized that the disease progresses by intra-axonal spread within the central nervous system. To test this hypothesis, neuroanatomical mapping of lesions, immunofluorescence analysis, and electron microscopy were performed on brains of ruminants with naturally occurring rhombencephalitis. In addition, infection assays were performed in bovine brain cell cultures. Mapping of lesions revealed a consistent pattern with a preferential affection of certain nuclear areas and white matter tracts, indicating that Listeria monocytogenes spreads intra-axonally within the brain along interneuronal connections. These results were supported by immunofluorescence and ultrastructural data localizing Listeria monocytogenes inside axons and dendrites associated with networks of fibrillary structures consistent with actin tails. In vitro infection assays confirmed that bacteria were moving within axon-like processes by employing their actin tail machinery. Remarkably, in vivo, neutrophils invaded the axonal space and the axon itself, apparently by moving between split myelin lamellae of intact myelin sheaths. This intra-axonal invasion of neutrophils was associated with various stages of axonal degeneration and bacterial phagocytosis. Paradoxically, the ensuing adaxonal microabscesses appeared to provide new bacterial replication sites, thus supporting further bacterial spread. In conclusion, intra-axonal bacterial migration and possibly also the innate immune response play an important role in the intracerebral spread of the agent and hence the progression of listeric rhombencephalitis.

FIG 1

Specific topographical distribution pattern of microabscesses in listeric rhombencephalitis in 41 animals. Microabscesses are indicated as red dots in transverse sections at different levels of the brain, white matter is shown in dark gray, and gray matter is shown in light gray. Red circles indicate large areas of necrosis and suppuration. (a) Cerebral hemisphere with corpus striatum. Microabscesses are selectively located within white matter tracts of the capsula interna and corona radiata containing axonal fibers. (b) In the thalamus, microabscesses are located mainly within white matter tracts. (c) Midbrain. The commissura caudalis and the tegmental tract are predominantly affected. (d) Pons and middle cerebellar peduncle. (e) Rostral part of the medulla oblongata and caudal cerebellar peduncle. (f) Caudal part of the medulla oblongata rostral to the obex region. The fasciculus longitudinalis medialis, fasciculi tegmentalis, and caudal cerebellar peduncle are frequently affected. Note the severe lesions in the reticular formation with large areas of necrosis and suppuration.

FIG 2

Specific topography of microabscesses in listeric rhombencephalitis. Shown are H&E-stained brain sections. Bar, 200 μm. (a) Microabscesses selectively affect the white matter of the capsula interna (arrows). (b) Large microabscess (M) in the white matter of the fasciculi tegmenti (Forel) of the midbrain. The contralateral fasciculi tegmenti (Forel) are unaffected (*). A, aqueduct. (c) Chain of microabscesses in the caudal commissure (CC).

FIG 3

L. monocytogenes bacteria are predominantly associated with phagocytes within microabscesses. Shown are consecutive tissue sections of the midbrain area from a sheep containing a microabscess. (a) Immunofluorescence with antibodies against L. monocytogenes (red) and neurofilaments (green). Nuclei are stained in blue (DAPI). Bacteria are predominantly present in microabscesses, as indicated by agglomerations of nuclei. (b) Section view of the z-stack of high-magnification images from the same microabscess. L. monocytogenes bacteria are closely associated with segmented and unsegmented nuclei, indicating their intracellular localization within neutrophils and macrophages/microglia, respectively. (c) Immunofluorescence with antibodies against Iba-1 (red) and neurofilaments (green). Nuclei are stained in blue (DAPI). Cells with large oval nuclei are Iba-1 positive, indicating the presence of macrophages/microglia. The remaining cells have segmented nuclei, indicating the presence of neutrophils. (d) H&E staining of the corresponding area. Most cells within the microabscesses have condensed and segmented nuclei (neutrophils); the second population of cells have large round to oval nuclei (macrophages/microglia).

FIG 4

L. monocytogenes bacteria are associated with neurofilaments and within or aligned along axons. Shown are immunofluorescence images of L. monocytogenes (red) and neurofilaments (green) in brains of 2 sheep affected by listeriosis. Nuclei are stained in blue (TOTO-3). (a to c) Beyond microabscesses, bacteria are closely associated with cellular nuclei (arrowheads) or neurofilaments (arrows), with the latter indicating their intra-axonal location. Such bacteria are frequently aligned with the axonal axis (b and c). (d) Multiple bacteria are found in a fragmented axon (arrow) and associated with a cellular nucleus (arrowhead) within a dilated myelin sheath. (e) Section view of the z-stack of images from multiple L. monocytogenes bacteria within an axon (arrows). The z-planes of the section view clearly show the bacteria within the axon. Some of the bacteria partially colocalize with the neurofilaments. (f) Section view of the z-stack of images of a bacterium in close contact with the axonal neurofilaments.

FIG 5

Neutrophils migrate between myelin lamella into the axonal space. Shown are transmission electron microscopy images of the inflammatory reaction in the axonal space. (a) Semithin section of a myelinated area in the brainstem in the vicinity of a microabscess. Numerous axons are swollen and surrounded by a thinned myelin sheath. Several myelin sheaths contain neutrophils within the axonal space (arrows). (b) Transmission electron microscopy of swollen axons such as those shown in panel a, demonstrating axonal degeneration with accumulation of neurofilaments (asterisk), mitochondria, and several large vacuoles. The surrounding myelin sheath is thinner but intact. (c) Morphologically normal axon surrounded by an intact myelin sheath of an appropriate thickness. A neutrophil has migrated inside a split between myelin lamellae (arrow). (d) Axon in the early stage of degeneration with accumulating organelles but an intact myelin sheath of a normal thickness. One neutrophil (asterisk) invaded the axoplasma. A second neutrophil seems to be located in the adaxonal space (arrow). (e) Swollen dystrophic axon surrounded by a thinned myelin sheath, which is split and contains a neutrophil between myelin lamellae (arrows). An adjacent axonal space contains three neutrophils (asterisk). (f) The axon is completely destroyed, but the axonal space is still surrounded by an intact thinned myelin sheath and contains numerous neutrophils. The latter contain intravacuolar and intracytoplasmic L. monocytogenes bacteria (arrows).

FIG 6

L. monocytogenes bacteria are located within axons and possess actin tails. (a) Semithin section in the periphery of a microabscess. The white matter is infiltrated by numerous inflammatory cells. Many axons are swollen and surrounded by a thinned myelin sheath. One severely swollen axon contains several bacteria (arrow). (b) Transmission electron microscopy image of a dystrophic axon containing one longitudinally sectioned and several cross-sectioned L. monocytogenes cells (arrows) below its axolemma. (c) Transmission electron microscopy image of a swollen axon with two intra-axonal L. monocytogenes cells, surrounded by a rim of electron-dense material consistent with an actin comet tail (arrows), amid bundles of neurofilaments. (d) Two cross-sectioned L. monocytogenes cells within an axon. One cell is clearly surrounded by a unipolar rim of electron-dense material consistent with an actin comet tail of condensed actin filaments (arrow). (e) High-magnification image of a cross-sectioned intra-axonal L. monocytogenes cell with the actin tail visible as a halo consisting of an electron-dense network of filamentous material. (f) High-magnification image of L. monocytogenes within an intra-axonal neutrophil. L. monocytogenes bacteria are within single-membrane-bound vacuoles. One L. monocytogenes cell is dividing (arrow).

FIG 7

L. monocytogenes bacteria are found in axon-like processes of fetal bovine brain cells (FBBC-1) with neuronal differentiation. Shown are IF images of L. monocytogenes-infected FBBC-1 cells at 24 h p.i., with L. monocytogenes (L146/2007) in green, neurofilaments in red, and actin in white. (a and b) L. monocytogenes bacteria without an actin tail colocalize with neurofilaments (yellow), demonstrating their localization within axon-like processes. (c) L. monocytogenes (green) without an actin tail is closely associated with the neurofilament of an axon-like process (red).

FIG 8

L. monocytogenes bacteria move within axon-like processes. Shown are live-cell images of FBBC-1 cells inoculated with GFP-expressing L. monocytogenes (L146/2007-GFP). One bacterium moves within the axon-like process (arrows). (a) Image from 19 h 33 min p.i. (b) Image from 19 h 35 min p.i. (c) Image from 19 h 37 min p.i. (d) Image from 19 h 39 min p.i. The complete film sequence is available as Video S1 in the supplemental material.

FIG 9

Schematic illustration of the intracerebral spread of L. monocytogenes exemplified by the sensory trigeminal system in a parasagittally sectioned sheep brain. The functional connection of structures affected by microabscesses indicates the transneuronal spread of intra-axonal L. monocytogenes (represented by black boxes). Arrows indicate the direction of intracerebral spread. V, nervus trigeminus. 1, ganglion trigeminale; 2, nucleus tractus spinalis nervi trigemini; 3, polysynaptic ascending tract through reticular formation; 4, nucleus sensibilis pontinus nervi trigemini; 5, fasciculi tegmenti; 6, thalamus; 7, capsula interna; 8, corpus striatum.