Chapter 61 : The Pathogenesis of Nocardia

General characteristics of nocardiae. (A) Phase-contrast micrograph of Nocardia spp. grown on tryptone agar for 12 h. Note the typical branching, filamentous growth characterized as nocardioform morphology. Bar, 10 μm. (Reprinted from reference 12 with permission from the publisher.) (B) Typical colonial morphology of N. asteroides grown on glucose yeast extract agar at 37°C for 14 days. Bar, 1 cm. (C) Gram stain of N. asteroides in a smear from an abscess. Note the typical beaded appearance of the branching filaments. Bar, 15 μm.

Electron micrographs of phagosome-lysosome interactions in activated rabbit alveolar macrophages infected with N. asteroides GUH-2. The lysosomes were prelabeled with horseradish peroxidase for the purpose of ultrastructural and histochemical localization. (A) Section showing inhibition of phagosome-lysosome fusion and the intact appearance of the nocardiae. Many of the bacteria appear to be surrounded by a large granular zone (GZ) that prevents contact between the lysosome (L) and the phagosome (open arrow). The nocardiae were preincubated with 20% normal rabbit serum. Bar, 1.0 μm. (Reprinted from reference 19 with permission from the publisher.) (B) Phagosome-lysosome fusion and bacterial cellular damage are prominent (arrows) in the same preparation of macrophages shown in panel A, except that these phagocytes were incubated with specifically primed lymph node lymphocytes; the bacteria were preincubated with sera and pulmonary lavage fluid from immunized rabbits. Note that the extensive bacterial damage and enhanced phagosome-lysosome fusion presented in this figure did not occur if any one of the components (primed lymphocytes, antibody, or pulmonary lavage fluid from immunized rabbits) were deleted. Bar, 1.0 μm. (Reprinted from reference 24 with permission from the publisher.)

The comparative interactions of N. asteroides with different types of host cells grown in tissue culture. (A) Apical attachment of a log-phase nocardial filament to the surface of a HeLa cell. Note that this tip-associated adherence precedes penetration and invasion. (Reprinted from reference 8 with permission from the publisher.) (B) Apical penetration of the HeLa cell by three nocardial filaments (arrows). (Reprinted from reference 8 with permission from the publisher.) (C) Longitudinal adherence of a log-phase cell of N. asteroides to the surface of a HeLa cell. Note that only the bacterial filaments that attach by way of the filament tip appear to penetrate into the host cell. Bar, 1.0 μm. (Reprinted from reference 8 with permission from the publisher.) (D) Nocardial filament (N) tip invading through the surface (arrow) of an astrocytoma cell (CCF-STTG1) even after pretreatment of the tissue culture with cytochalasin. Bar, 1.0 μm. (Reprinted from reference 7 with permission from the publisher.) (E) Longitudinal association of stationary-phase bacilli to the microvilli of the HeLa cell. Bacteria attached in this manner do not invade the host cell. (Reprinted from reference 8 with permission from the publisher.) (F) Light micrograph of stationary-phase cells of N. asteroids GUH-2 adherent to the surface of type II astroglia (II). Bar, 10.0 μm. Note the arrangement of bacteria clustered around the nuclear region (N). Note, in contrast, the adjacent cuboidal type I astroglia ( 1 ) with a total absence of adherent nocardiae.

Scanning electron micrographs of differential attachment to and penetration of cells within the brain and lungs of mice by log-phase cells of N. asteroides GUH-2. (A) Apical penetration of capillary endothelial cells within an arteriole in the thalamus by nocardial filaments (arrows). Bar, 1.0 μm. (Reprinted from reference 11 with permission from the publisher.) (B) High-magnification view of two nocardial filaments penetrating into the endothelium of an arteriole in the region of the hypothalamus (arrows). Bar, 1.0 μm. (Reprinted from reference 11 with permission from the publisher.) (C) Nocardial interactions in the bronchiole of a C57BL/6 mouse 3 h after intranasal administration of log-phase cells of N. asteroides GUH-2. Note the association with nonciliated epithelial cells and apical penetration of Clara cells (arrowheads). Bar, 1.0 μm. (D) High-magnification view of nocardial penetration into bronchiolar epithelial cells as shown in panel C. Bar, 1.0 μm.

Comparative ultrastructure of N. asteroides GUH-2 growing within cells of the brain of a mouse and a monkey. Note that there is no inflammatory infiltration at the site of nocardial invasion in either animal. (A) Cell of N. asteroides GUH-2 growing within a neuron in the midbrain of a mouse 24 h after tail vein injection of a suspension of log-phase nocardiae. Note the numerous layers of membrane surrounding the ultrastructurally intact bacterium, with the innermost layer of membrane tightly adherent to the bacterial surface (arrow). N, nocardial filament. Bar, 0.5 μm. (Reprinted from reference 3 with permission from the publisher.) (B) A cross section of a nocardial cell growing within the brain of a monkey 48 h after i.v. injection (leg vein) of a suspension of log-phase cells of N. asteroides GUH-2 as in panel A. Note the numerous layers of membrane, with the innermost layer adherent to the bacterial surface (arrow). Bar, 0.5 μm.

(See the separate color insert for the color version of this illustration.) Confocal micrographs of nocardia-inducedapoptosis. (A) Apoptosis of dopaminergic neurons in the substantianigra in a head-shake mouse 14 days after infection.The red stain localizes dopaminergic neurons. Free 3′-OH endsof DNA from apoptotic nuclei were labeled with nucleotidesconjugated to fluorescein isothiocyanate (green). (B) Uninfectedcontrol. Dopaminergic neurons in the substantia nigrain a healthy uninfected mouse. The red stain localizes dopaminergicneurons. Note that there is no apoptosis. (Reproduced from reference 68 .)