Nonlethal infection of BALB/c mice with Nocardia asteroides GUH-2 (GUH-2) produces a variety of neurological signs, including an L-dopa-responsive movement disorder in 10 to 15% of the infected population. To study nocardial interactions with the brain, we characterized the attachment of GUH-2 within specific regions through the use of microdissection. Following an intravenous injection of a single-cell suspension of log-phase GUH-2, viable cells were recovered from all regions of the brain, and the distribution of the nocardiae was independent of the size of the inoculum. In addition, two mutants of GUH-2 were found to possess significantly altered binding characteristics with regard to both the percentage of the inoculum bound per brain and the relative distribution of adherence to regions of the brain, when compared with the parental strain. These results indicated that GUH-2 bound throughout the murine brain and suggested that GUH-2 utilized specific receptors to facilitate this attachment.
The growth of Nocardia asteroides GUH-2 and two mutants (NG-49 and I-38-syn) in regions of the brains of BALB/c mice was determined by microdissection and viable counting. GUH-2 grew throughout the murine brain but at different growth rates that depended on the specific location. The rate of increase in total CFU per brain during GUH-2 infection was unaffected by the inoculum size; however, in five of eight brain regions, an alteration in the inoculum size resulted in altered nocardial growth rates. Mutant NG-49 showed a significantly slower rate of increase in total CFU per brain than did the parental strain, GUH-2, and significantly decreased growth rates in seven brain regions. Mutant 1-38-syn showed a rate of increase in total CFU per brain similar to that of the parental strain; however, this mutant grew significantly faster in the cerebellum and pons-medulla. Growth appeared to be a necessary precursor to the cellular damage that resulted in the variety of neurological disorders observed in mice infected with N. asteroides GUH-2, because mutant NG-49 exhibited a decreased ability to grow in specific regions of the brain and did not induce signs of neurological damage. In
The attachment to and penetration of endothelial cells in the pons and midbrain (especially the substantia nigra) regions of the brains of BALB/c mice by log-phase Nocardia asteroides GUH-2 cells were determined by both scanning and transmission electron microscopic analysis. Within 15 min after exposure, the nocardiae attached to the surface of the endothelial cell membrane. This attachment occurred primarily at the growing tip of the nocardial filament, and the outermost layer of the nocardial cell wall had regions (electron-dense areas) that bound firmly to the cytoplasmic membrane of the host cell. There appeared to be specificity for this binding localized within the capillaries and arterioles because some regions had large numbers of bacteria bound, whereas adjacent areas had no bacterial cells. Nocardial filaments that attached by the apex induced a cuplike deformation of the endothelial cell membrane. This was followed by a rapid penetration of the endothelial cell so that within 25 min many of the bacteria were internalized within the host cell. These internalized bacteria remained within vesicles, and there was no ultrastructural evidence of damage to the nocardial cell during this process. Heat-killed GUH-2 cells still attached to endothelial surfaces (at a reduced frequency), but they did not penetrate into the endothelial cell. These data suggest that brain-invasive nocardiae possess both an adhesin for attachment to the membrane of endothelial cells and an invasion factor that promotes nocardial penetration of these cells.
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