ERK1/2 and the Bcl-2 Family Proteins Mcl-1, tBid, and Bim Are Involved in Inhibition of Apoptosis During Persistent Chlamydia psittaci Infection.

Chlamydia psittaci is an obligate intracellular pathogen that can cause zoonosis. Persistent C. psittaci infection can inhibit apoptosis in host cells, thus extending their survival and enabling them to complete their growth cycle. In this study, the antiapoptotic effects of persistent C. psittaci infection, induced by treatment with IFN-γ, were found to be associated with both the death receptor and the mitochondrial pathways of apoptosis. These effects were mediated by Bcl-2 family members, as evidenced by the decreased expression of proapoptotic proteins, such as tBid and Bim. Simultaneously, the antiapoptotic protein Mcl-1 was upregulated by persistent C. psittaci infection. Increased phosphorylation of ERK1/2 was observed; however, the expression of Bad, unlike that of other proapoptotic proteins, did not seem to be involved in this process. In summary, persistent chlamydial infection exerts antiapoptotic effects through both the death receptor and the mitochondrial pathways, in a process that is regulated by the ERK1/2 and apoptotic proteins of the Bcl-2 family.

Evaluation of Rv0220, Rv2958c, Rv2994 and Rv3347c of Mycobacterium tuberculosis for serodiagnosis of tuberculosis.

Tuberculosis (TB), the leading cause of death among infectious diseases worldwide, is caused by Mycobacterium tuberculosis (M. tuberculosis). Early accurate diagnosis means earlier prevention, treatment and control of TB. To confirm efficient diagnostic antigens for M. tuberculosis, the serodiagnosis value of four recombinant proteins including Rv0220, Rv2958c, Rv2994 and Rv3347c was evaluated in this study. The specificities and sensitivities of four recombinant proteins were determined based on enzyme-linked immunosorbent assay (ELISA) by screening sera from smear-positive pulmonary TB patients (n = 92), uninfected individuals (n = 60) and patients with Mycoplasma pneumoniae (n = 32) that potentially cross-react with M. tuberculosis. The ELISAs showed that Rv0220, Rv2958c, Rv2994 and Rv3347c exhibited high specificities and sensitivities in detecting immunoglobulin G (IgG) antibody, with 98.3/91.3%, 91.7/85.9%, 93.3/89.1% and 93.3/80.4% respectively. According to the receiver-operating characteristic (ROC) analysis, the area under the ROC of the target proteins was 0.988, 0.969, 0.929 and 0.945 respectively. Western blot was established to evaluate the immunoreactivities of target proteins to mice and human sera. Results demonstrated that Rv0220, Rv2958c, Rv2994 and Rv3347c could specifically recognize TB-positive sera and the sera of mice immunized with the corresponding protein. Thus, Rv0220, Rv2958c, Rv2994 and Rv3347c were valuable potential diagnostic antigens for M. tuberculosis.

Localization and characterization of two putative TMH family proteins in Chlamydia psittaci.

Chlamydia psittaci (C. psittaci), an obligate intracellular agent of psittacosis, causes an atypical pneumonia in humans. The transmembrane head proteins (TMH) of C. psittaci, putatively belong to the Inc family and presumably play similar roles. CPSIT_0844 and CPSIT_0846 were the putative TMH proteins of C. psittaci. To identify these two proteins, antisera were raised with fusion proteins which were prokaryotic expressed in Escherichia coli and purified. By immunofluorescence assay, CPSIT_0844 and CPSIT_0846 were localized in the inclusion membrane of C. psittaci-infected cells. By RT-PCR and western blot analysis to detect the temporal expression, CPSIT_0844 and CPSIT_0846 were detected as early as 12h post-infection (p.i.) and 6h p.i., separately; meanwhile, in secretions monitored with immunofluorescence assay, these proteins were observed in the inclusion membrane at 18h p.i. and remained in the inclusion membrane throughout the growth cycle. CPSIT_0844 and CPSIT_0846 could specifically be recognized by the antiserum of C. psittaci but failed to react with the antiserums of Chlamydia trachomatis and Chlamydia pneumoniae, which is consistent with the fact that they had no significant orthologs in C. trachomatis and C. pneumoniae. These results revealed that CPSIT_0844 and CPSIT_0846, the putative TMH family proteins, might be unique to C. psittaci and could be used to diagnose the infection caused by C. psittaci. Moreover, CPSIT_0844 and CPSIT_0846 could induce the expression of the inflammatory cytokines IL-1ß, IL-6 and TNF-α in THP-1 cells, which might contribute to chlamydia-induced inflammatory pathologies.

Transcriptional Analysis of 10 Selected Genes in a Model of Penicillin G Induced Persistence of Chlamydophila psittaci in HeLa Cells.

Chlamydophila psittaci is an important intracellular pathogen. Persistent infection is an important state of the host-parasite interaction in this chlamydial infection, which plays a significant role in spreading the organism within animal populations and in causing chronic chlamydiosis and serious sequelae. In this study, a C. psittaci persistent infection cell model was induced by penicillin G, and real-time quantitative PCR was used to study the transcriptional levels of 10 C. psittaci genes (dnaA, dnaK, ftsW, ftsY, grpE, rpsD, incC, omcB, CPSIT_0846, and CPSIT_0042) in acute and penicillin-G-induced persistent infection cultures. Compared with the acute cultures, the penicillin-G-treated cultures showed a reduced chlamydial inclusion size and a significantly decreased number of elementary body particles. Additionally, some enlarged aberrant reticulate body particles were present in the penicillin- G-treated cultures but not the acute ones. The expression levels of genes encoding products for cell division (FtsW, FtsY) and outer membrane protein E encoding gene (CPSIT_0042) were downregulated (p < 0.05) from 6 h post-infection onward in the persistent infection cultures. Also from 6 h post-infection, the expression levels of DnaA, DnaK, IncC, RpsD, GrpE, and CPSIT_0846 were upregulated (p < 0.05); however, the expression level of OmcB in the persistent infection was almost the same as that in the acute infection (p > 0.05). These results provide new insight regarding molecular activities that accompany persistence of C. psittaci, which may play important roles in the pathogenesis of C. psittaci infection.

Recombinant Cpn 0810 stimulates proinflammatory cytokine expression and apoptosis in human monocytes.

The aim of the present study was to express the recombinant Chlamydophila pneumoniae (C. pneumoniae) protein, Cpn 0810, in Escherichia coli (E. coli) BL21, and investigate the effects of Cpn 0810 on inflammatory and apoptotic processes in human monocytic (THP-1) cells. An ELISA was performed to detect the levels of the proinflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-6. In addition, Hoechst 33258 staining and annexin V binding analyses were performed to measure the rates of apoptosis. Purified glutathione S-transferase (GST)-Cpn 0810 recombinant proteins were obtained from the E. coli BL21 cells carrying the pGEX6p-2/Cpn 0810 plasmid, and were shown to stimulate the expression of TNF-α and IL-6 in the THP-1 cells in a dose- and time-dependent manner. TNF-α and IL-6 levels peaked at 24 h after GST-Cpn 0810 stimulation. Furthermore, GST-Cpn 0810 significantly promoted the apoptosis of THP-1 cells. In conclusion, recombinant GST-Cpn 0810 was shown to stimulate the expression of TNF-α and IL-6, inhibit proliferation and induce apoptosis in THP-1 cells. Therefore, Cpn 0810 may interact with host cells following C. pneumoniae infection, functioning as an important pathogenic factor.

Macrophage-activating lipopeptide-2 requires Mal and PI3K for efficient induction of heme oxygenase-1.

AIMS: This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes. METHODS: Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay. RESULTS: MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, LY294002. CONCLUSIONS: These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.

The immune effects of multiple antigen peptides containing the mimic epitopes of the adhesion protein of Mycoplasma genitalium.

The purpose of this study was to investigate the humoral and cellular immune responses stimulated by multiple antigen peptides (MAPs) containing the mimic epitopes of Mycoplasma genitalium adhesion protein (MgPa). Three MAPs containing the mimic epitopes of MgPa were synthesized on a branched polylysine matrix. After purification and characterization, these MAPs were used to immunize BALB/c mice. The immunoglobulin G (IgG) antibody and the subtype of IgG antibody in the serum of the immunized mice were detected by indirect ELISA (enzyme-linked immunosorbent assay). The proliferation of the spleen lymphocyte was detected using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. The gamma interferon (IFN-γ) and interleukin-4 (IL-4) levels in the cultured supernatant of spleen lymphocytes were measured by ELISA. The 3 different MAPs were prepared with high purity. Levels of IgG, IgG1, and IgG2a antibodies were elevated in the mice serum immunized by all 3 MAPs. The major antibody isotype was IgG2a. Importantly, mice immunized with a mixture of the 3 MAPs produced significantly more antibodies than those immunized with a single MAP (p < 0.05). Moreover, these MAPs could stimulate the proliferation of spleen lymphocytes of immunized mice and induce the production of IFN-γ and IL-4. The IFN-γ and IL-4 levels stimulated by the mixed MAPs were significantly higher than those stimulated by a single MAP (p < 0.01). The 3 different MAPs could induce strong cellular and humoral immune responses. The immunoreactivity of the mixed MAPs was stronger than that of the single MAP.

Mycoplasma fermentans MALP-2 induces heme oxygenase-1 expression via mitogen-activated protein kinases and Nrf2 pathways to modulate cyclooxygenase 2 expression in human monocytes.

Heme oxygenase-1 (HO-1) is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury and performs a vital function in the maintenance of cell hemostasis. Increasing numbers of reports have indicated that mycoplasma-derived membrane lipoproteins/lipopeptides, such as macrophage-activating lipopeptide-2 (MALP-2), function as agents that stimulate the immune system by producing various inflammatory mediators, such as cytokines and cyclooxygenase 2 (COX-2), which play roles in the pathogenesis of inflammatory responses during mycoplasma infection. Here, we report that MALP-2 induced HO-1 mRNA and protein expression and upregulated HO-1 enzyme activity in THP-1 cells. Specific inhibitors of mitogen-activated protein kinases (MAPKs), SB203580, PD98059, and SP600125, significantly abolished HO-1 expression. In addition, MALP-2 also induced NF-E2-related factor 2 (Nrf2) translocation, and the silencing of Nrf2 expression in THP-1 cells decreased the levels of MALP-2-mediated HO-1 expression. Furthermore, COX-2 protein expression levels were upregulated in THP-1 cells in response to MALP-2, and transfection with small interfering RNAs of HO-1 significantly increased COX-2 accumulation. These results demonstrate that MALP-2 induces HO-1 expression via MAPKs and Nrf2 pathways and, furthermore, that MALP-2-induced COX-2 expression was modulated by HO-1 in THP-1 cells.

Immunogenicity of Cpn0425 and its localization in cells infected with Chlamydophila pneumoniae.

The present study aimed to determine the intracellular localization of Cpn0425 in Chlamydophila pneumoniae-infected cells. The recombinant plasmid pGEX-6p/Cpn0425 was transformed into E.coli Bl21 cells to express the fusion protein. Following purification with glutathione S-transferase (GST) resin chromatography, the Cpn0425 fusion protein was used to induce immunity in mice to develop monoclonal and polyclonal antibodies, which were subsequently used to localize the endogenous Cpn0425 protein by indirect immunofluorescence assay (IFA). ELISA was used to determine the immunogenicity of the Cpn0425 plasmid protein by recognizing the pool sera of patients infected with Chlamydia trachomatis and the pool sera of mice immunized with the Cpn0425 fusion protein. The Cpn0425 gene was expressed as the GST-Cpn0425 fusion protein in E. coli and its antibody was prepared by immunizing mice with the fusion protein. An anti-GST-Cpn0425 antibody was used to localize the protein in cells infected with Chlamydophila pneumoniae AR-39 using an IFA. The anti-GST-CT058 antibody detected an inclusion signal in the IFA. Cpn0425 protein strongly reacted with antiserum. Although Cpn0425 protein is not a secreted protein, it has good immunogenicity. Therefore, this protein may be useful for developing vaccines against Chlamydophila pneumoniae infection.

Screening and identification of the mimic epitope of the adhesion protein of Mycoplasma genitalium.

Mycoplasma genitalium adhesion protein (MgPa) is the major adhesion protein of M. genitalium, and its C-terminal domain (amino acid 1075-1444) is the most immunogenic region. However, the exact epitopes of the adhesion protein of M. genitalium are still unclear. We used the purified polyclonal antibody against the recombinant adhesion protein to screen the mimic epitopes of MgPa using a random 12-peptide phage display library. Immunoscreening via the phage display peptide library revealed that 3 motifs (P-S-A-A/V-X-R-F/W-E/S-L-S-P, A-K-I/L-T/Q-X-T-L-X-L, and K-S-L-S-R-X-D-X-I) may represent 3 different mimotopes of MgPa. Results of bioinformatics analysis by MIMOX demonstrated that the key consensus amino acid residues in the aligned mimotopes may be S, A, and F for cluster 1; A, K, I, T, and L for cluster 2; and K, S, L, R, D, and I for cluster 3. Three representative phages could recognize sera from M. genitalium-positive patients to varying degrees, whereas they could not recognize the sera from Mycoplasma pneumoniae -positive patients or the sera from healthy people. These findings will help to clarify the mimic epitopes of MgPa to facilitate diagnosis of the antigen and to understand the antigenic structure of MgPa.

[Induction of proinflammatory cytokines and cell apoptosis by Chlamydia pneumoniae Cpn0810].

AIM: To expresse the Chlamydia pneumoniae Cpn0810 in E.coli BL21, and to study weather could it inducing proinflamatory cytokines including TNF-α and IL-6 in human monocytic (THP-1) and cell apoptosis. METHODS: Polymerase chain reaction(PCR) was used to amplify the Cpn0810 gene, PCR products were purified and cloned into the prokaryotic expression vector pGEX6p-2. The restriction plasmids pGEX6p-2/Cpn0810 confirmed by PCR and sequencing was transformed into E.coli BL21. The recombinant protein was purified with glutathione S-transferase (GST) resin chromatography of Novagen after renaturation. THP-1 cells were stimulated by different concentrations of Cpn0810 and for various durations to test the production and the expression of TNF-α and IL-6 by ELISA. Cell apoptosis was detected in C.pneumoniae Cpn0810 cells by Hoechst33258 fluorescence staining and Cell apoptosis was detected in THP-1 cells by Annexin-V-FITC-propidiu-m iodide (PI) staining. RESULTS: The restriction enzymes cleavage analysis and nucleotide sequencing showed the target gene was successfully inserted into pGEX6p-2 prokaryotic expression vector. Cpn0810 stimulated THP-1 cell to produce proinflamatory cytokines including TNF-α and IL-6 in a dose and time-dependent manner. After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy; apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810. CONCLUSION: Cpn0810 recombinant protein could stimulate THP-1 cell to produce and express proinflamatory cytokines including TNF-α and IL-6; After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810.

[Isolation of chlamydophila psittaci from avian samples and establishment of a respiratory infection murine model].

OBJECTIVE: To optimize the isolation and culture technique of Chlamydophila psittaci avian strains and to establish an animal model infected with C. psittaci. METHODS: C. psittaci ompA gene was amplified from DNA extracted from bird livers by polymerase chain reactions (PCR). For the PCR positive avian samples, the liver tissues were homogenized and used to incubated with HeLa or Vero cell monolayers for 72 h in different dilutions, and chlamydia inclusion bodies were detected by immunofluorescence or Giemsa staining. Different dose of the avian strains (2 x 10(4), 2 x 10(5), 2 x 10(6) IFUs) were used to attack C57BL/6 mice by intranasal injection,mice were sacrificed on day 5 or day 10 after infection, and the histopathology changes were analyzed by H&E and immunohistochemistry staining in different organs. RESULTS: Six of one hundred avian samples were positive by C. psittaci ompA gene amplification,and three were positive by cell culture. The C. psittaci avian strains were cultured in Vero or HeLa cells. Vero cells showed stronger tolerance of cytolysis after chlamydia infection and chlamydia inclusion bodies were larger and more dense. Successfully establish a murine model of intranasal infection with C. psittaci, and 2 x 10(5) IFU is the suitable amount of organisms to induce respiratory chlamydia infection. CONCLUSION: The isolation and culture condition was optimized for C. psittaci avian strains, and a murine model of respiratory tract infection by C. psittaci was successfully established, which can be applied to the clincal diagnosis of C. psittaci and epidemiological or pathogenetic study.