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

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.

Fig. 1

Representative PCR screening assays targeting C. pneumoniae genes (Panel A), and genes from other chlamydial species (Panel B), using DNA prepared from the Tor-1 and Phi-1 isolates. EBs were prepared from each isolate as described in ‘Materials and methods’. DNA preparation from each isolate and PCR assay systems used in these analyses, also are described in ‘Materials and methods’ and Table 2. (A) Lanes in the gel in Panel A are: 1, 100 bp size standards; amplifications targeting 2, Cpn0695 (ompA) using C. pneumoniae strain AR-39 DNA as template (positive control); 3, Cpn0695 using water as template (negative control); 4, Cpn0695 using Tor-1 DNA, and 5, Phi-1 DNA as template; 6, Cpn1046 using C. pneumoniae strain AR-39 DNA as template (positive control); 7, Cpn1046 using water as template (negative control); 8, Cpn1046 using Tor-1 DNA, and 9, Phi-1 DNA as template. (B) Lanes in the gel in Panel B are: 1, 100 bp size standards; amplifications targeting 2, Cpn1046 using Tor-1 DNA as template; 3, Cpn1046 using Phi-1 DNA as template; 4, pCt02 (plasmid ORF4 C. trachomatis) using C. trachomatis serovar K, and 5, Tor-1, and 6, Phi-1 DNA as template; 7, incA (C. caviae) using C. caviae (GPIC), and 8, Tor-1, and 9, Phi-1 DNA as template; 10, Cps0300 (C. psittaci) using C. psittaci strain 6BC, and 11, Tor-1, and 12, Phi-1 DNA as template. Amplification product sizes are given in Table 2.

Fig. 1

Representative PCR screening assays targeting C. pneumoniae genes (Panel A), and genes from other chlamydial species (Panel B), using DNA prepared from the Tor-1 and Phi-1 isolates. EBs were prepared from each isolate as described in ‘Materials and methods’. DNA preparation from each isolate and PCR assay systems used in these analyses, also are described in ‘Materials and methods’ and Table 2. (A) Lanes in the gel in Panel A are: 1, 100 bp size standards; amplifications targeting 2, Cpn0695 (ompA) using C. pneumoniae strain AR-39 DNA as template (positive control); 3, Cpn0695 using water as template (negative control); 4, Cpn0695 using Tor-1 DNA, and 5, Phi-1 DNA as template; 6, Cpn1046 using C. pneumoniae strain AR-39 DNA as template (positive control); 7, Cpn1046 using water as template (negative control); 8, Cpn1046 using Tor-1 DNA, and 9, Phi-1 DNA as template. (B) Lanes in the gel in Panel B are: 1, 100 bp size standards; amplifications targeting 2, Cpn1046 using Tor-1 DNA as template; 3, Cpn1046 using Phi-1 DNA as template; 4, pCt02 (plasmid ORF4 C. trachomatis) using C. trachomatis serovar K, and 5, Tor-1, and 6, Phi-1 DNA as template; 7, incA (C. caviae) using C. caviae (GPIC), and 8, Tor-1, and 9, Phi-1 DNA as template; 10, Cps0300 (C. psittaci) using C. psittaci strain 6BC, and 11, Tor-1, and 12, Phi-1 DNA as template. Amplification product sizes are given in Table 2.

Fig. 2

Immunofluorescence analysis of inclusion size and shape in HEp-2 (Panels A–C), U-87 MG (Panels D–F), and CHME-5 (Panels G–I) cells infected with C. pneumoniae isolates Phi-1 (Group I[A–I], left) and Tor-1 (Group II[A–I], right) at 24 h p.i. (Panels A, D, G), 48 h p.i. (Panels B, E, H), and 72 h p.i. (Panels C, F, I). Cells were infected on glass cover slips at MOI 0.5 for HEp-2 cells and MOI 1 for U-87 MG and CHME-5 cells. Infected cells were stained with the Pathfinder™ mAb, which targets the chlamydial LPS. Infected cells were visualized using epifluorescence on a Nikon E600 microscope. The scale bar in Panel A in each group represents 10 µm, and all photomicrographs were taken at the same magnification in both large groups.

Fig. 2

Immunofluorescence analysis of inclusion size and shape in HEp-2 (Panels A–C), U-87 MG (Panels D–F), and CHME-5 (Panels G–I) cells infected with C. pneumoniae isolates Phi-1 (Group I[A–I], left) and Tor-1 (Group II[A–I], right) at 24 h p.i. (Panels A, D, G), 48 h p.i. (Panels B, E, H), and 72 h p.i. (Panels C, F, I). Cells were infected on glass cover slips at MOI 0.5 for HEp-2 cells and MOI 1 for U-87 MG and CHME-5 cells. Infected cells were stained with the Pathfinder™ mAb, which targets the chlamydial LPS. Infected cells were visualized using epifluorescence on a Nikon E600 microscope. The scale bar in Panel A in each group represents 10 µm, and all photomicrographs were taken at the same magnification in both large groups.

Fig. 3

Representative quantitative real-time PCR analyses of relative levels of chromosomal DNA from C. pneumoniae isolates Phi-1 and Tor-1 as a function of time post-infection in HEp-2 cells (Panel A), U-87 MG cells (Panel B), and CHME-5 cells (Panel C). Cells were infected, nucleic acids prepared and analyzed, as given in ‘Materials and methods’. Assays were run 3 times independently and in triplicate each time, from 2 independent experiments. Data were normalized to host 18S rDNA levels and indexed to the level at t=0. The bars indicate standard error.

Fig. 4

Recovery of infectious EB from HEp-2 cells (Panel A), U-87 MG cells (Panel B), and CHME-5 cells (Panel C) at 24, 48, and 72 h p.i. with the AR-39 strain of C. pneumoniae and the Tor-1 and Phi-1 isolates of that organism from AD brain tissue samples. Cultures were inoculated, and the infectious yields from each assessed as given in ‘Materials and methods’. Data are expressed as average IFU/ml of triplicate samples.

Fig. 5

Southern blot analysis of the tyrP locus in AR-39 and the brain isolates Tor-1 and Phi-1. DNA was prepared from the AD brain isolates, and blotting and analyses were done as given in ‘Materials and methods’. A 5623-bp fragment indicates a double copy, the 7300-bp fragment a triple copy of the gene. AR-39 and Phi-1 were mixtures of double and triple copy population; Tor-1 is a population carrying only the triple copy.