TLR2 mediates autophagy through ERK signaling pathway in Chlamydia psittaci CPSIT_p7 protein-stimulated RAW264.7 cells.

Chlamydia psittaci is a zoonotic pathogen found in birds and humans. Macrophages, major components of the innate immune system, can resist chlamydial infections and trigger adaptive immune responses. However, the molecular mechanisms underlying the action of macrophages against C. psittaci infection are not well understood. This study investigated the roles and mechanisms of plasmid-encoded protein CPSIT_p7 of C. psittaci in regulating autophagy in RAW264.7 cells. The results demonstrated that stimulation of RAW264.7 with C. psittaci plasmid protein CPSIT_p7 induced the expressions of the autophagy signaling primary regulators LC3 and Beclin1, which could also significantly induce the phosphorylation levels of ERK, JNK, p38, and Akt. Next, siRNA knockdown of TLR2 resulted in significant downregulation of CPSIT_p7-triggered autophagy in RAW264.7 cells. Moreover, the extracellular regulated protein kinase (ERK) inhibitor PD98059 markedly reduced autophagy in CPSIT_p7-stimulated macrophages. In summary, these results indicated that TLR2 plays an essential role in the induction of autophagy through the ERK signaling pathway in CPSIT_p7-stimulated RAW264.7 cells.

Chlamydia psittaci hypothetical inclusion membrane protein CPSIT_0842 evokes a pro-inflammatory response in monocytes via TLR2/TLR4 signaling pathways.

Chlamydia psittaci (C. psittaci) is an obligate intracellular pathogen that resides within a membrane-bound compartment known as the inclusion. Upon entering the host cell, Chlamydiae secrete numerous proteins to modify the inclusion membrane. Inclusion membrane (Inc) proteins are important pathogenic factors in Chlamydia and play crucial roles in the growth and development of Chlamydia. In the present study, the C. psittaci protein, CPSIT_0842, was identified and shown to localize to the inclusion membrane. Temporal analysis revealed that CPSIT_0842 is an early expression protein of Chlamydia. Moreover, this protein was shown to induce the expression of pro-inflammatory cytokines IL-6 and IL-8 in human monocytes (THP-1 cells) via the TLR2/TLR4 signaling pathway. CPSIT_0842 increases the expression of TLR2, TLR4, and adaptor MyD88. Suppression of TLR2, TLR4, and MyD88 markedly attenuated CPSIT_0842-induced production of IL-6 and IL-8. MAP kinases and NF-κB, important downstream molecules of TLR receptors in inflammatory signaling pathways, were also confirmed to be activated by CPSIT_0842. CPSIT_0842-induced production of IL-6 was reliant on activation of the ERK, p38, and NF-κB signaling pathways while IL-8 expression was regulated by the ERK, JNK, and NF-κB signaling pathways. Specific inhibitors of these signaling pathways significantly decreased CPSIT_0842-mediated expression of IL-6 and IL-8. Together these findings demonstrate that CPSIT_0842 upregulates the expression of IL-6 and IL-8 via TLR-2/TLR4-mediated MAPK and NF-κB signaling pathways in THP-1 cells. Exploring these molecular mechanisms enhances our understanding of C. psittaci pathogenesis.

Chlamydia psittaci inclusion membrane protein CPSIT_0842 induces macrophage apoptosis through MAPK/ERK-mediated autophagy.

Chlamydia psittaci is a multi-host zoonotic pathogen, which mainly infects poultry and inflicts an appreciable economic burden on the livestock farming industry. C. psittaci inclusion membrane proteins are uniquely positioned at the host-pathogen interface and are important virulence proteins. We have previously confirmed that Incs regulate host cell survival to help Chlamydia sp. evade host-cell-mediated defense mechanisms. However, the role of the Inc, CPSIT_0842, in the regulation of cell death following the establishment of persistent C. psittaci infection remains unknown. This study explored the effect of CPSIT_0842 on the crosstalk between the autophagic and apoptotic pathways in macrophages. Results showed that CPSIT_0842 initiated autophagy and blocked autophagic flux in human macrophages, as indicated by autophagy-related protein LC3-II, Beclin-1, and p62 upregulation, autophagosome accumulation, and lysosomal protein LAMP1 diminution. We also showed that the disruption of autophagic flux had a regulatory effect on CPSIT_0842-induced apoptosis. Moreover, the suppression of autophagy initiation by 3-methyladenine attenuated CPSIT_0842-induced apoptosis. By contrast, the induction of autophagic flux by rapamycin did not significantly affect CPSIT_0842-induced apoptosis. Taken together, these findings demonstrate that CPSIT_0842 induced macrophage apoptosis by initiating incomplete autophagy through the MAPK/ERK/mTOR signaling pathway, which may be instrumental to the ability of C. psittaci to evade the host innate immune response and establish persistent infection. The improved understanding of the autophagic and cell death pathways triggered upon bacterial inclusion will likely help in the development of novel treatment strategies for chlamydia infection.

Characterization and comparison of differentially expressed genes involved in Chlamydia psittaci persistent infection in vitro and in vivo.

Chlamydia psittaci is an obligate intracellular zoonotic pathogen that can enter a persistence state in host cells. While the exact pathogenesis is not well understood, this persistence state may play an important role in chronic Chlamydia disease. Here, we assess the effects of chlamydial persistence state in vitro and in vivo by transmission electron microscopy (TEM) and cDNA microarray assays. First, IFN-γ-induced C. psittaci persistence in HeLa cells resulted in the upregulation of 68 genes. These genes are involved in protein translation, carbohydrate metabolism, nucleotide metabolism, lipid metabolism and general stress. However, 109 genes were downregulated following persistent C. psittaci infection, many of which are involved in the TCA cycle, expression regulation and transcription, protein secretion, proteolysis and transport, membrane protein, presumed virulence factor, cell division and late expression. To further study differential gene expression of C. psittaci persistence in vivo, we established an experimentally tractable mouse model of C. psittaci persistence. The C. psittaci-infected mice were gavaged with either water or amoxicillin (amox), and the results indicated that the 20 mg/kg amox-exposed C. psittaci were viable but not infectious. Differentially expressed genes (DEGs) screened by cDNA microarray were detected, and interestingly, the results showed upregulation of three genes (euo, ahpC, prmC) and downregulation of five genes (pbp3, sucB_1, oppA_4, pmpH, ligA) in 20 mg/kg amox-exposed C. psittaci, which suggests that antibiotic treatment in vivo can induce chlamydial persistence state and lead to differential gene expression. However, the discrepancy on inducers between the two models requires more research to supplement. The results may help researchers better understand survival advantages during persistent infection and mechanisms influencing C. psittaci pathogenesis or evasion of the adaptive immune response.

Polymorphic Membrane Protein 17G of Chlamydia psittaci Mediated the Binding and Invasion of Bacteria to Host Cells by Interacting and Activating EGFR of the Host.

Chlamydia psittaci (C. psittaci) is an obligate intracellular, gram-negative bacterium, and mainly causes systemic disease in psittacine birds, domestic poultry, and wild fowl. The pathogen is threating to human beings due to closely contacted to employees in poultry industry. The polymorphic membrane proteins (Pmps) enriched in C. psittaci includes six subtypes (A, B/C, D, E/F, G/I and H). Compared to that of the 1 pmpG gene in Chlamydia trachomatis (C. trachomatis), the diverse pmpG gene-coding proteins of C. psittaci remain elusive. In the present study, polymorphic membrane protein 17G (Pmp17G) of C. psittaci mediated adhesion to different host cells. More importantly, expression of Pmp17G in C. trachomatis upregulated infections to host cells. Afterwards, crosstalk between Pmp17G and EGFR was screened and identified by MALDI-MS and Co-IP. Subsequently, EGFR overexpression in CHO-K1 cells and EGFR knockout in HeLa 229 cells were assessed to determine whether Pmp17G directly correlated with EGFR during Chlamydial adhesion. Finally, the EGFR phosphorylation was recognized by Grb2, triggering chlamydial invasion. Based on above evidence, Pmp17G possesses adhesive property that serves as an adhesin and activate intracellular bacterial internalization by recognizing EGFR during C. psittaci infection.

Localization and characterization of a putative cysteine desulfurase in Chlamydia psittaci.

Chlamydia psittaci is an obligate intracellular pathogen with a biphasic developmental life cycle. It is auxotrophic for a variety of essential metabolites and obtains amino acids from eukaryotic host cells. Chlamydia can develop inside host cells within chlamydial inclusions. A pathway secreting proteins from inclusions into the host cellular cytoplasm is the type III secretion system (T3SS). The T3SS is universal among several Gram-negative bacteria. Here, we show that CPSIT_0959 of C. psittaci is expressed midcycle and secreted into the infected cellular cytoplasm via the T3SS. Recombinant CPSIT_0959 possesses cysteine desulfurase and PLP-binding activity, which removes sulfur from cysteine to produce alanine, and helps chlamydial replication. Our study shows that CPSIT_0959 improve the infectivity of offspring elementary bodies and seems to promote the replication by its product. This phenomenon has inhibited by the PLP-dependent enzymes inhibitor. Moreover, CPSIT_0959 increased expression of Bim and tBid, and decreased the mitochondrial membrane potential of host mitochondria to induce apoptosis in the latecycle for release of offspring. These results demonstrate that CPSIT_0959 has cysteine desulfurase and PLP-binding activity and is likely to contribute to apoptosis of the infected cells via a mitochondria-mediated pathway to improve the infectivity of progeny.

The JAK/STAT3 signaling pathway mediates inhibition of host cell apoptosis by Chlamydia psittaci infection.

The JAK-STAT3 signaling pathway is a key regulator of cell growth, motility, migration, invasion and apoptosis in mammalian cells. Infection with intracellular pathogens of the genus Chlamydia can inhibit host cell apoptosis, and here we asked whether the JAK-STAT3 pathway participates in chlamydial anti-apoptotic activity. We found that, compared with uninfected cells, levels of JAK1 and STAT3 mRNA as well as total and phosphorylated JAK1 and STAT3 protein, were significantly increased in C. psittaci-infected HeLa cells. Moreover, the apoptosis rate of infected cells was higher after treatment with the tyrosine kinase inhibitor AG-490 (2-cyano-3-(3, 4-dihydroxyphenyl)-N-(phenylmethyl)-2-propenamide). Immunoblotting of apoptosis-related proteins showed that C. psittaci infection reduces Bax, but increases Bcl-2, protein levels, resulting in reduced activation of caspase-3, caspase-7, caspase-9 and PARP; AG490 attenuates these effects. Together, our data suggest that the JAK/STAT3 signaling pathway facilitates the anti-apoptotic effect of C. psittaci infection by reducing the Bax/Bcl-2 apoptotic switch ratio, and by inhibiting the intracellular activation of key pro-apoptotic enzymes.

Recombinant protein CPSIT_0846 induces protective immunity against Chlamydia psittaci infection in BALB/c mice.

Chlamydia psittaci is an obligate intracellular bacteria that causes respiratory disease in poultry and humans. Currently, there are no licensed vaccines against chlamydial infection in humans. The transmembrane head protein CPSIT_0846 of C. psittaci is a putative member of the larger Inc protein family. In this study, we investigated immunogenicity and protective efficacy of the recombinant CPSIT_0846 protein in BALB/c mice. Mice immunized with CPSIT_0846 developed strong T-lymphocyte responses that were recalled by the immunogen CPSIT_0846 in an in vitro restimulation assay. These T cells displayed a strong Th1-biased cytokine profile with high levels of IFN-γ. At the same time, a strong humoral immune response was also detected in the immunized mice with high titers of Chlamydia psittaci-specific serum IgG antibodies. More importantly, the robust immune responses correlated well with significantly reduced chlamydial burden and inflammatory pathology in the mouse lungs upon an airway challenge infection. The above results together suggest that the CPSIT_0846 protein may be a potential vaccine candidate antigen for inducing protection against C. psittaci infection and disease in the airway.

Acute phase proteins as local biomarkers of respiratory infection in calves.

BACKGROUND: Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic inflammatory mediators, but are also expressed in different tissues as local reaction to inflammatory stimuli. The present study aimed to evaluate presence and changes in luminal lung concentrations of the APPs haptoglobin (Hp), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), and lactoferrin (Lf) in calves with an acute respiratory disease experimentally induced by Chlamydia (C.) psittaci. RESULTS: Intra-bronchial inoculation of the pathogen resulted in a consistent respiratory illness. In venous blood of the infected calves (n = 13), concentrations of plasma proteins and serum LBP were assessed (i) before exposure and (ii) 8 times within 14 days after inoculation (dpi). Increasing clinical illness correlated significantly with increasing LBP-and decreasing albumin concentrations in blood, both verifying a systemic acute phase response. Broncho-alveolar lavage fluid (BALF) was obtained from all 13 calves experimentally infected with C. psittaci at 4, 9 and 14 dpi, and from 6 uninfected healthy calves. Concentrations of bovine serum albumin (BSA), Hp, LBP, CRP and Lf in BALF were determined by ELISA. In infected animals, absolute concentrations of LBP and Hp in BALF correlated significantly with the respiratory score. The quotient [LBP]/[BSA] in BALF peaked significantly in acutely infected animals (4 dpi), showed a time-dependent decrease during the recovery phase (9-14 dpi), and was significantly higher compared to healthy controls. Concentrations of Hp and Lf in BALF as well as [Hp]/[BSA]--and [Lf]/[BSA]-quotients decreased during the study in infected animals, but were never higher than in healthy controls. CRP concentrations and [CRP]/[BSA]-quotient did not express significant differences between infected and healthy animals or during the course of infection. CONCLUSION: In conclusion, absolute concentrations of LBP in blood and BALF as well as the quotient [LBP]/[BSA] in BALF perfectly paralleled the clinical course of respiratory illness after infection. Beside LBP, the suitability of Hp and Lf as local biomarkers of respiratory infections in cattle and their role in the local response to pathogens is worth further investigation, while CRP does not seem to play a role in local defense mechanisms of the bovine lung.

The type III secretion system (T3SS) of Chlamydophila psittaci is involved in the host inflammatory response by activating the JNK/ERK signaling pathway.

Chlamydophila psittaci (C. psittaci) is a human zoonotic pathogen, which could result in severe respiratory disease. In the present study, we investigated the role and mechanism of the type III secretion system (T3SS) of C. psittaci in regulating the inflammatory response in host cells. C. psittaci-infected THP-1 cells were incubated with the specific T3SS inhibitor INP0007, inhibitors of ERK, p38, or JNK, and the levels of inflammatory cytokines were analyzed using Q-PCR and ELISA. The levels of ERK, p38, and JNK phosphorylation were analyzed by Western blot. Our results verified that INP0007 inhibited chlamydial growth in vitro, but the coaddition of exogenous iron completely reversed the growth deficit. INP0007 inhibited the growth of C. psittaci and decreased the levels of IL-8, IL-6, TNF-α, and IL-1ß. Exogenous iron restored the chlamydial growth but not the production of inflammatory cytokines. These results demonstrated that the expression of inflammatory cytokines during infection was associated with the T3SS which reduced by incubation with ERK and JNK inhibitors, but not with p38 inhibitor. We concluded that the T3SS elicited inflammatory responses by activating the JNK or ERK signaling pathways in the infection of C. psittaci.

Serological and molecular characterization of Chlamydophila psittaci strains using serovar-specific monoclonal antibodies and ompA sequence analysis.

In the present study, 21 avian Chlamydophila psittaci field isolates from 4 different European countries (Belgium, Germany, Italy and The Netherlands) were characterized using serovar-specific monoclonal antibodies as well as ompA sequence analysis, enabling the comparison between the two characterization methods for future epidemiological studies. The 21 European isolates included 6 isolates from the order Psittaciformes, 6 isolates from the Anseriformes, 5 isolates from the order Columbiformes and 4 Galliformes respectively. Only 19 on 21 isolates could be serotyped while all isolates were successfully genotyped. In addition, genotyping revealed the presence of mixed infections in 5 on 21 isolates while serotyping could only detect one of these 5 mixed infections. Interestingly, genotyping indicated the existence of a new genotype designated E/B. The E/B genotype is closely related to the genotypes A, B and E but the ompA gene of E/B strains shows a guanosine on position 1006 and 1021 in combination with a cytidine on position 1022 to be unique. Genotype E/B isolates reacted with both the serovar E- and B-specific monoclonal antibody leading to the conclusion of a mixed infection while only one specific ompA sequence was present in the sample. For epidemiological studies genotyping by use of ompA sequence analysis is to be preferred as it is more sensitive than serotyping and can distinguish genotype B, E and E/B strains.

Chlamydophila abortus (Chlamydia psittaci serotype 1) clearance is associated with the early recruitment of neutrophils and CD8(+)T cells in a mouse model.

The immune mechanisms in response to Chlamydophila abortus (Chlamydia psittaci serotype 1) infection were studied in C57BL/6 and CBA mice. The infection was monitored and the following aspects of the immune response were evaluated: the nature of the leucocyte infiltrate in the liver, the percentages of polymorphonuclear neutrophils (PMNs), macrophages and lymphocytes in the spleen, and the concentrations of cytokines in serum. In addition, the serum concentrations of IgG1 and IgG2a were determined. Both mouse strains showed a Th1-like immune response, with high concentrations of IFN-gamma and minimal levels of IL-4; however, C57 mice differed from CBA mice in showing milder clinical signs and earlier resolution of infection. The greater ability of C57 mice than CBA mice to eliminate chlamydophilae was related to the establishment of an earlier innate immunity, based on a more pronounced PMN response, and on a greater presence of CD8(+)T cells.

Recombinant ovine interferon gamma inhibits the multiplication of Chlamydia psittaci in ovine cells.

The local production of interferon-gamma (IFN-gamma) in sheep in response to Chlamydia psittaci was measured by cannulation of the efferent lymph duct draining the site of challenge inoculation. Peak production of IFN-gamma (256 U/ml) was detected 24 h after challenge. Based on these physiological data, functional studies were carried out in vitro to determine the effect of recombinant ovine (rOv) IFN-gamma on the multiplication of C. psittaci in ovine fibroblasts. IFN-gamma inhibited the multiplication of C. psittaci in ovine cells over a range of concentrations (250 U/ml to 2.5 U/ml) in a dose-dependent manner. The inhibition of chlamydial multiplication was most pronounced when cells were treated with rOvIFN-gamma for 24 h before infection. The addition of exogenous L-tryptophan (500 micrograms/ml) to cultures within 48th of infection abrogated the anti-chlamydial effect of rOvIFN-gamma thus suggesting that tryptophan deprivation is an anti-chlamydial mechanism induced by rOvIFN-gamma in these ovine cells.

Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body.

Ocular infection of guinea pigs with the guinea pig inclusion conjunctivitis (GPIC) strain of Chlamydia psittaci produces a clinical condition representative of acute chlamydial conjunctivitis in humans. Guinea pigs which had recovered from two challenges with GPIC were used as a source of sera for the identification of antigens present in GPIC-infected tissue culture cells but absent in the infectious elementary body (EB). Immunoblots of lysates of infected HeLa cells probed with the convalescent-phase sera identified protein antigens of 22, 34, and 52 kDa (p22, p34, and p52, respectively) that were not detected in lysates of purified EB or in uninfected HeLa cells. Protein p22 was also not detected in lysates of purified reticulate bodies. Immunoblotting of lysates of HeLa cells infected with other chlamydiae demonstrated that the antigenicity of p22 and p34 was subspecies specific. Immunoblotting was also used to detect p22 and p34 in lysates of the conjunctivae of infected guinea pigs. Adsorption of convalescent-phase sera with GPIC EB produced a reagent with dominant reactivity toward p22, p34, and a 28-kDa EB protein. Immunofluorescent staining of GPIC-infected HeLa cells demonstrated that these adsorbed sera labeled the inclusion and inclusion membrane, with no apparent reactivity toward EB or reticulate bodies. Collectively, these data identify non-EB chlamydial components which may be released into the inclusion during intracellular growth.