Ultrastructural Analysis of Chlamydia Pneumoniae in the Alzheimer's Brain

Abstract

We recently reported identification of the bacterium Chlamydia pneumoniae in affected brain regions of patients with Alzheimer's disease (AD) (Balin et al., 1998). In this report, we extend those initial observations to demonstrate that, in addition to the frequently described, standard morphological forms of the organism, pleiomorphic forms are also present in the AD brain. All AD and control brain tissues examined were verified to be PCR-positive and negative, respectively, for the organism. DNA sequence determination of PCR products so derived from total DNA of infected AD brains, as well as from total DNA of cell lines infected with the organism following isolation from these same patient samples, confirmed the presence of organism in relevant samples. Various morphologic forms of C. pneumoniae were identified in PCR-positive tissues and these were characterized based on membrane structure, core density, size, and immunolabeling profiles. Structures identified include the typical pear-shaped elementary body, as well as larger, spherical and oblong reticulate bodies. Intact C. pneumoniae were found both intracellularly and extracellularly in the sampled autopsy brains. Intracellular organisms were located principally within microglia, astroglia, and presumptive pericytes. These results suggest that C. pneumoniae found in cells indigenous to the AD brain do not conform universally to the classical morphology observed in other infected cell types. This pleiomorphism may reflect an adaptive response and/or persistent state of infection for these organisms in Alzheimer's Disease.

FIGURE 1

Ultrastructural analyses of brain tissues from AD patients to characterize the morphologies of C. pneumoniae. AD patient samples were prepared for electron microscopy and IEM as described in Methods; the anti-chlamydial Ab used was a mAb against a C. pneumoniae outer membrane protein (see text). Panel A: Multiple EBs (arrows) and RBs (arrowheads) forms of C. pneumoniae are apparent in the hippocampus of an AD brain. Panel B: Extracellular area of the same region in Panel A showing a dividing reticulate body undergoing binary fission and exhibiting a distinct division plane (arrows). Panel C: Within the temporal cortex of an AD brain, alternate phenotypes of Chlamydia pneumoniae (large arrow) are present; these forms were apparent in numerous AD brains. IEM analysis with the anti-OMP mAB shows polar labeling on one end only (inset, small arrows), but not all organisms are immunoreactive. A neurofibrillary tangle bundle can be seen proximal to the organisms (arrowheads). Panel D: IEM of C. pneumoniae labeled with anti-OMP mAb (arrowheads) within the hippocampus from an AD brain demonstrates circumferential immunolabeling. Panel E: Equivalent area to that shown in D exhibiting an organism with central immunolabeling by anti-OMP mAb. Panel F: IEM (arrows) using the anti-OMP mAb targeting C. pneumoniae isolated from THP-1 cells following infection with AD brain homogenate. Bars: A, C = 1 μm; B, D, E = 0.5 μm; F = 0.25 μm

FIGURE 2

Analysis of C. pneumoniae-infected cells in AD blood vessels. Panel A: Blood vessel from the hippocampus from an AD brain showing an RB (arrowhead) within the cytoplasm of a presumptive monocyte/macrophage in the vessel lumen. Panel B: Blood vessel from the temporal cortex from an AD brain demonstrating two C. pneumoniae organisms (arrows) within the disrupted cytoplasm of a detached pericyte or perivascular macrophage on the abluminal surface of the vessel. Bars= 1 μm

FIGURE 3

Analysis of C. pneumoniae from AD brain homogenates. Panel A: THP-1 cells after infection with a homogenate from the temporal cortex from an AD brain 7 d post-incubation. Immunohistochemical analysis with an anti-OMP mAb shows organisms (arrows) within the cytoplasm of the cells. Bar=20 μm. Panel B: Ultrastructural analysis of infected THP-1 cells with C. pneumoniae from an AD brain homogenate 7 d post-incubation. Organisms demonstrating a more aberrant morphology can be seen in the cytoplasm of the host cell (arrow). The EBs and RBs show thickened cell walls and are variable in size. Since the bacteria are dependent on the host-cell's ATP, C. pneumoniae bodies are situated adjacent to mitochondria (arrowhead) which shows morphology indicative of mitochondria undergoing oxidative stress (Parker et al., 1990). Bar=1 μm. Panel C: Cloned 350 bp PCR product from the infected THP-1 cells shown in the previous two panels. (Left) Lane 1:100 bp standards. Lane 2: Uncut ligated M13 vector. Lane 3: Restriction digest of vector in lane 2; the lower band was sequenced and found to be Homologous to the C. pneumoniae omp A gene