Initial characterization of Chlamydophila (Chlamydia) pneumoniae cultured from the late-onset Alzheimer brain

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Abstract

Previous studies from this laboratory provided evidence that the intracellular bacterial pathogen Chlamydophila (Chlamydia) pneumoniae is present in the late-onset Alzheimer's disease (AD) brain. Here we report culture of the organism from two AD brain samples, each of which originated from a different geographic region of North America. Culturable organisms were detectable after one and two passages in HEp-2 cells for the two samples. Both isolates, designated Tor-1 and Phi-1, were demonstrated to be authentic C. pneumoniae using PCR assays targeting the C. pneumoniae-specific genes Cpn0695, Cpn1046, and tyrP. Assessment of inclusion morphology and quantitation of infectious yields in epithelial (HEp-2), astrocytic (U-87 MG), and microglial (CHME-5) cell lines demonstrated an active, rather than a persistent, growth phenotype for both isolates in all host cell types. Sequencing of the omp1 gene from each isolate, and directly from DNA prepared from several additional AD brain tissue samples PCR-positive for C. pneumoniae, revealed genetically diverse chlamydial populations. Both brain isolates carry several copies of the tyrP gene, a triple copy in Tor-1, and predominantly a triple copy in Phi-1 with a minor population component having a double copy. This observation indicated that the brain isolates are more closely related to respiratory than to vascular/atheroma strains of C. pneumoniae.

Introduction

Chlamydophila (Chlamydia) pneumoniae is a respiratory pathogen known to be a causative agent for community-acquired pneumonia (e.g. Grayston, 1992; Campbell and Kuo, 2002). Several studies have also implicated infection with this obligate intracellular eubacterium in more severe respiratory pathologies, including chronic obstructive pulmonary disease (Clementsen et al., 2002; Hahn, 1999). Importantly, infection with C. pneumoniae has been associated with several non-pulmonary diseases, including atherosclerosis, giant cell (temporal) arteritis, inflammatory arthritis, and others (Schumacher et al., 1999; Wagner et al., 2000; Belland et al., 2004; Watson and Alp, 2008; see also below). Epidemiologic study has demonstrated that the prevalence of infection with this organism is high in all adult populations examined, and that the prevalence increases with increasing age; multiple re-infections appear to be common (e.g. Leinonen, 1993).

As with all chlamydial species, C. pneumoniae undergoes a biphasic developmental cycle. In the first phase, the elementary body (EB), the infectious, metabolically inactive extracellular form of the organism, attaches to a target eukaryotic host cell; these are most often epithelial cells, but many other cell types can be infected (e.g. Hatch, 1999). The organism ends up in a cytoplasmic inclusion within which it reorganizes into the metabolically active growth form of the organism, the reticulate body (RB). RBs undergo several rounds of cell division, after which 70–80% reorganize back to the EB form. Newly formed EB exit the host cell via lysis or exocytosis or ‘extrusion’ to continue propagation of the infection (Hatch, 1999; Hybiske and Stephens, 2007). C. pneumoniae requires about 72 h to complete passage through the cycle. Many studies have shown that under certain conditions and/or within specific host cell types, C. pneumoniae can alter its biologic state to generate persistent, long-term infections. Chlamydiae undergoing such infections are morphologically aberrant and display an unusual transcriptional profile (e.g. Byrne et al., 2001; Gérard et al., 2001, Gérard et al., 2002, Gérard et al., 2004; Ouellette et al., 2006; Mäurer et al., 2007; see also below). The transcriptional responses of various host cell types actively or persistently infected with chlamydiae have also been investigated (e.g. Schrader et al., 2007; Rodriguez et al., 2007; Alvesalo et al., 2008). Importantly, reports indicate that the mechanisms of pathogenesis differ between actively and persistently infecting chlamydiae and that it is in the persistent state that these organisms elicit chronic disease (Hogan et al., 2004; Whittum-Hudson et al., 2004, Whittum-Hudson et al., 2007).

Alzheimer's disease (AD) is a neurodegenerative disorder associated with atrophy/death of neurons, leading ultimately to severe cognitive dysfunction. The disease occurs in two forms: an early-onset form that is genetically determined, and a more common late-onset (sporadic) form that is not (Keefover, 1996). The incidence of sporadic AD increases with increasing age and is now considered to be the most prevalent cause of senile dementia (Keefover, 1996). The means by which the neuropathology characteristic of late-onset AD is elicited is poorly understood, but much research indicates that neuritic senile plaques (NSP), one aspect of that pathology, are critical in neuronal degeneration (Citron, 2004). Indeed, the dominant hypothesis currently guiding AD research focuses on the role of NSP in disease genesis (Sommer, 2002). Neurofibrillary tangles (NFT) have also been demonstrated to be important in the neuropathogenesis of AD (e.g. Iqbal et al., 2003).

In previous work, we identified C. pneumoniae in a high proportion of brain tissue samples examined from patients with sporadic AD, and we showed that the organism was present in those samples almost exclusively in brain regions displaying the NSP and NFT characteristic of the disease; the organism was extremely rare in congruent brain samples from age- and sex-matched non-AD (control) individuals (Balin et al., 1998; Arking et al., 1999). We further showed that astrocytes and microglia were host cells for the organism in the AD brain, and in recent studies we and others have identified neurons as host cells for the organism as well (Balin et al., 1998; Gérard et al., 2006; Appelt et al., 2008).

In our earlier reports, we demonstrated culture of C. pneumoniae from infected AD brain tissues, but we did not provide a detailed analysis of that original isolate (Balin et al., 1998; Gérard et al., 2006). However, recent studies from a collaborating group showed that respiratory infection of mice with that brain isolate elicits some aspects of CNS neuropathology that resemble those characteristics of late-onset AD (Little et al., 2004). Isolates of C. pneumoniae and well-studied serovars of C. trachomatis have been shown to include a significant level of DNA sequence variation at several loci (e.g. Daugaard et al., 2001; Viratyosin et al., 2002; Gieffers et al., 2003; Dreses-Werringloer et al., 2003). In some cases, DNA sequence variants at specific loci have been shown to correlate with tissue tropism and/or pathogenicity for these organisms (Gieffers et al., 2003; Fehlner-Gardiner et al., 2002; Caldwell et al., 2003; Cochrane et al., 2005).

For reasons relating to the possible elicitation of pathogenesis, we are interested in determining whether C. pneumoniae in the late-onset AD brain displays a normal active or persistent growth phenotype in epithelial cells and in its host cells in the brain. Further, it is important to define whether DNA sequences in AD brain isolates show variation similar to that shown for other, well-studied strains/isolates of C. pneumoniae. We therefore cultured the organism from brain tissues of two AD patients, each from a different geographic region of North America. In this article, we provide an initial analysis of basic cell biologic aspects shown by the brain-derived isolates of C. pneumoniae during infection of three human cell lines, and we initiate the analysis of genetic variation in those isolates and among brain tissue samples from individuals with late-onset AD.

Section snippets

Cell culture, control C. pneumoniae

HEp-2 cells were maintained in Iscove's Modified Dulbecco's Medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS, 2 mM l-glutamine and 10 μg/ml gentamycin (Invitrogen). The human astrocytoma cell line U-87 MG (Lytle et al., 2005; gift of Dr. T.R. Reddy) and the human microglioma cell line CHME-5 (de Gannes et al., 2000; gift of Dr. D. Feinstein) were grown in Dulbecco's Modified Earle's Medium (Invitrogen) with supplements as above. C. pneumoniae strain AR-39 was grown in HEp-2 cells

Culture of C. pneumoniae from AD brain tissue samples

For the present study, tissue samples from the hippocampus (patient AD1, Table 1, Tor-1 isolate) and temporal cortex (patient AD4, Table 1, Phi-1 isolate) from individuals with documented AD were subjected to culture in HEp-2 cells. For the hippocampal specimen, inclusions were identified after a single growth cycle of 3 days. With that sample, an infection rate of 1–2% of host cells was achieved using 0.5 ml 1:10-diluted tissue homogenate as initial inoculum. For the temporal cortex sample, a

Discussion

In this report, we describe the successful culture of C. pneumoniae from postmortem brain tissues from two patients with sporadic AD, and we provide initial characterization of those two isolates. The observations presented demonstrate that this pathogen can be cultured from infected brain tissue of AD patients, that infection of cultured human astrocyte and microglial cells, as well as human epithelial cells, with each of the AD brain isolates displays an active, rather than a persistent,

Acknowledgments

This work was supported by NIH grants AI-44055 (A.P.H.), AR-47186 (H.C.G.), HL-71735 (R.J.B.), and AI-44493 and AR-48331 (both J.A.W.-H.) from the US National Institutes of Health. The Michigan Alzheimer's Disease Research Center is supported by NIH Grant AG-08671. We are grateful to the families who donated tissues to the sources listed here for research in Alzheimer's disease.

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    Current address: Litwin-Zucker Research Center for the Study of Alzheimer's Disease and Memory Disorder, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York, USA.

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