Chlamydophila pneumonia and increased TLR4 gene expression in leukocytes are associated with acute myocardial infarction.

We investigated the relationship of the positivity for Chlamydophila pneumoniae (Cpn) and Mycoplasma pneumonia (Mpn), inflammatory and metabolic markers, and mRNA expression and polymorphisms of the TLR2, TLR4, IL-6 and TNFA genes with acute myocardial infarction (AMI). Two hundred and eighteen individuals (98 AMI and 120 non-AMI) were selected at two Clinical Centers. Blood samples were drawn to extract DNA and RNA and to measure laboratory variables including anti-Cpn IgM and IgG. Cpn and Mpn genomic DNA as well as TLR2, TLR4, IL-6 and TNFA mRNA expression were evaluated by quantitative real-time PCR (qPCR). Gene polymorphisms were detected by PCR-HRM. AMI patients had higher positivity for Cpn-DNA (17.3%) than non-AMI group (6.7%, p=0.018). In addition, Cpn-DNA positivity was an independent predictor of risk for AMI (OR: 2.56, CI: 1.08 - 6.04, p=0.031). Positivity for anti-Cpn IgG and Mpn-DNA was similar between AMI and non-AMI (> 0.05). TLR4 mRNA expression was higher in AMI than non-AMI individuals (p=0.005). CD14 -260C> T, TNFA -308A> G, TLR2 c.2258G> A, TLR4 c.896A> G and TLR4 c.1196> T variants were not associated with increased risk for AMI (p> 0.05). In the AMI group, individuals carrying CD14 -260CC genotype had higher hsCRP levels than CT/TT carriers (p=0.041). These results are suggestive that Cpn-DNA positivity and increased TLR4 mRNA expression in blood leukocytes may be associated with AMI and could be useful markers to evaluate the severity and progression of the atherosclerotic disease in AMI patients.

Proteomic identification of immunodominant chlamydial antigens in a mouse model.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen in the world. To identify new vaccine candidates a protein microarray was constructed by expressing the open reading frames (ORFs) from Chlamydia mouse pneumonitis (MoPn). C57BL/6, C3H/HeN and BALB/c mice were immunized either intranasally or intravaginally with live MoPn elementary bodies (EB). Two additional groups were immunized by the intramuscular plus subcutaneous routes with UV-treated EB, using CpG and Montanide as adjuvants to favor a Th1 response, or Alum, to elicit a Th2 response. Serum samples collected from the three strains of mice were tested in the microarray. The array included the expression of 909 proteins from the 921 ORFs of the MoPn genome and plasmid. A total of 530 ORFs were recognized by at least one serum sample. Of these, 36 reacted with sera from the three strains of mice immunized with live EB. These antigens included proteins that were previously described as immunogenic such as MOMP and HSP60. In addition, we uncovered new immunogens, including 11 hypothetical proteins. In summary, we have identified new immunodominant chlamydial proteins that can be tested for their ability to induce protection in animal models and subsequently in humans.

[Study on gene cloning of Chlamydial pneumonia CPn0308 and its endogenous localization].

OBJECTIVE: To clone CPn0308 gene from Clamyida pneumonia and express its fusion protein, to make antibodies to fusion protein GST-CPn0308, and to further localize endogenous protein preliminarily using antibodies raised with CPn0308 fusion protein. METHODS: The open reading frame (ORF) coding for CPn0308 in the Chlamydia pneumonia AR 39 genome was cloned into the pGEX6p2 vector after it was cloned using PCR and digested by the restriction enzymes BamHI and NotI. The recombinant plasmid pGEX6p2-CPn0308 was transformed into XL1-blue bacteria and the gene CPn0308 was expressed as fusion proteins with the glutathione-s-transferase (GST) tagged to the N-terminus. The GST-CPn0308 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CPn0308 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). RESULTS: The CPn0308 gene, which was 366bp in length,was successfully cloned and the GST fusion protein with molecular weight of 39kD was expressed. It was found that the hypothetical protein CPn0308 was located in the inclusion membrane of Chlamydia pneumonia-infected cells using IFA of mouse anti-fusion protein antibodies. CONCLUSIONS: Using antibodies raised with GST-CPn0308 fusion protein, the hypothetical protein CPn0308 was identified to be a Chlamydia pneumoniae inclusion membrane protein. It could be the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells.