Mitogen-activated protein kinase-dependent interleukin-1α intracrine signaling is modulated by YopP during Yersinia enterocolitica infection.

Yersinia enterocolitica is a food-borne pathogen that preferentially infects the Peyer's patches and mesenteric lymph nodes, causing an acute inflammatory reaction. Even though Y. enterocolitica induces a robust inflammatory response during infection, the bacterium has evolved a number of virulence factors to limit the extent of this response. We previously demonstrated that interleukin-1α (IL-1α) was critical for the induction of gut inflammation characteristic of Y. enterocolitica infection. More recently, the known actions of IL-1α are becoming more complex because IL-1α can function both as a proinflammatory cytokine and as a nuclear factor. In this study, we tested the ability of Y. enterocolitica to modulate intracellular IL-1α-dependent IL-8 production in epithelial cells. Nuclear translocation of pre-IL-1α protein and IL-1α-dependent secretion of IL-8 into the culture supernatant were increased during infection with a strain lacking the 70-kDa virulence plasmid compared to the case during infection with the wild type, suggesting that Yersinia outer proteins (Yops) might be involved in modulating intracellular IL-1α signaling. Infection of HeLa cells with a strain lacking the yopP gene resulted in increased nuclear translocation of pre-IL-1α and IL-1α-dependent secretion of IL-8 similar to what is observed with bacteria lacking the virulence plasmid. YopP is a protein acetylase that inhibits mitogen-activated protein kinase (MAP kinase)- and NF-κB-dependent signal transduction pathways. Nuclear translocation of pre-IL-1α and IL-1α-dependent secretion of IL-8 in response to Yersinia enterocolitica infection were dependent on extracellular signal-regulated kinase (ERK) and p38 MAP kinase signaling but independent of NF-κB. These data suggest that Y. enterocolitica inhibits intracellular pre-IL-1α signaling and subsequent proinflammatory responses through inhibition of MAP kinase pathways.

Generation and characterization of a large panel of murine monoclonal antibodies against vaccinia virus.

Vaccinia virus (VACV), the vaccine for smallpox, induces an antibody response that is largely responsible for conferring protection. Here, we studied the antibody response to VACV by generating and characterizing B cell hybridomas from a mouse immunized with VACV. Antibodies from 66 hybridomas were found to recognize 11 VACV antigens (D8, A14, WR148, D13, H3, A56, A33, C3, B5, A10 and F13), 10 of which were previously recognized as major antigens in smallpox vaccine by a microarray of VACV proteins produced with a prokaryotic expression system. VACV C3 protein, which was not detected as a target of antibody response by the proteome array, was recognized by two hybridomas, suggesting that selection of hybridomas based on immune recognition of infected cells has the advantage of detecting additional antibody response to native VACV antigens. In addition, these monoclonal antibodies are valuable reagents for studying poxvirus biology and protective mechanism of smallpox vaccine.

Transforming growth factor beta and CD25 are important for controlling systemic dissemination following Yersinia enterocolitica infection of the gut.

Infection of the gut by invasive bacterial pathogens leads to robust inflammatory responses that if left unchecked can lead to autoimmune disease and other sequelae. How the immune system controls inflammation and limits collateral damage to the host during acute bacterial infection is poorly understood. Here, we report that antibody-mediated neutralization of transforming growth factor beta (TGF-beta) prior to infection with the model enteric pathogen Yersinia enterocolitica reduces the mean time to death by 1 day (P=0.001), leads to rapid colonization of the liver and lung, and is associated with exacerbation of inflammatory histopathology. During Yersinia enterocolitica infection CD4+ cells are the source of de novo TGF-beta transcription in the Peyer's patches, mesenteric lymph nodes, and spleen. Correspondingly there is both antigen-specific and -independent expansion of CD4+ CD25+ Foxp3+ and TGF-beta+ T-regulatory cells (T-regs) after Yersinia infection that is reduced in ovalbumin T-cell receptor-restricted OT-II mice. Functional inactivation of CD25 by anti-CD25 treatment results in more rapid death, dissemination of the bacteria to the liver and lungs, and exacerbated inflammatory histopathology, similar to what is seen during TGF-beta neutralization. Altogether, these data suggest that TGF-beta produced by T-regs is important in restricting bacteria during the acute phase of invasive bacterial infection of the gut. These data expand the roles of T-regs to include tempering inflammation during acute infection in addition to the well-established roles of T-regs in chronic infection, control of immune homeostasis, and autoimmune disease.

A MyD88-dependent early IL-17 production protects mice against airway infection with the obligate intracellular pathogen Chlamydia muridarum.

We found that IL-17, a signature cytokine of Th17, was produced early in the innate immunity phase after an intranasal infection with the obligate intracellular pathogen Chlamydia muridarum. The airway IL-17, which peaked at 48 h after infection, was dependent on live chlamydial organism replication and MyD88-mediated signaling pathways. Treatment with antibiotics or knockout of the MyD88 gene, but not Toll/IL receptor domain-containing adapter-inducing IFN-beta, can block the early IL-17 production. Treatment of mice with an anti-IL-17-neutralizing mAb enhanced growth of chlamydial organisms in the lung, dissemination to other organs, and decreased mouse survival, whereas treatment with an isotype-matched control IgG had no effect. Although IL-17 did not directly affect chlamydial growth in cell culture, it enhanced the production of other inflammatory cytokines and chemokines by Chlamydia-infected cells and promoted neutrophil infiltration in mouse airways during chlamydial infection, which may contribute to the antichlamydial effect of IL-17. These observations suggest that an early IL-17 response as an innate immunity component plays an important role in initiating host defense against infection with intracellular bacterial pathogens in the airway.

Antibodies from women urogenitally infected with C. trachomatis predominantly recognized the plasmid protein pgp3 in a conformation-dependent manner.

BACKGROUND: C. trachomatis organisms carry a cryptic plasmid that encodes 8 open reading frames designated as pORF1 to 8. It is not clear whether all 8 pORFs are expressed during C. trachomatis infection in humans and information on the functionality of the plasmid proteins is also very limited. RESULTS: When antibodies from women urogenitally infected with C. trachomatis were reacted with the plasmid proteins, all 8 pORFs were positively recognized by one or more human antibody samples with the recognition of pORF5 protein (known as pgp3) by most antibodies and with the highest titers. The antibody recognition of the pORFs was blocked by C. trachomatis-infected HeLa but not normal HeLa cell lysates. The pgp3 fusion protein-purified human IgG detected the endogenous pgp3 in the cytosol of C. trachomatis-infected cells with an intracellular distribution pattern similar to that of CPAF, a chlamydial genome-encoded protease factor. However, the human antibodies no longer recognized pgp3 but maintained recognition of CPAF when both antigens were linearized or heat-denatured. The pgp3 conformation is likely maintained by the C-terminal 75% amino acid sequence since further deletion blocked the binding by the human antibodies and two conformation-dependent mouse monoclonal antibodies. CONCLUSION: The plasmid-encoded 8 proteins are both expressed and immunogenic with pgp3 as the most immunodominant antigen during chlamydial infection in humans. More importantly, the human anti-pgp3 antibodies are highly conformation-dependent. These observations have provided important information for further understanding the function of the plasmid-encoded proteins and exploring the utility of pgp3 in chlamydial diagnosis and vaccination.

The chlamydial plasmid-encoded protein pgp3 is secreted into the cytosol of Chlamydia-infected cells.

The chlamydial cryptic plasmid encodes eight putative open reading frames (ORFs), designated pORF1 to -8. Antibodies raised against these ORF proteins were used to localize the endogenous proteins during chlamydial infection. We found that the pORF5 protein (also known as pgp3) was detected mainly in the cytosol of Chlamydia-infected cells, while the remaining seven proteins were found inside the chlamydial inclusions only. The pgp3 distribution pattern in the host cell cytosol is similar to but not overlapping with that of chlamydial protease/proteasome-like activity factor (CPAF), a chlamydial genome-encoded protein known to be secreted from chlamydial inclusions into the host cell cytosol. The anti-pgp3 labeling was removed by preabsorption with pgp3 but not CPAF fusion proteins and vice versa, demonstrating that pgp3 is a unique secretion protein. This conclusion is further supported by the observation that pgp3 was highly enriched in cytosolic fractions and had a minimal presence in the inclusion-containing nuclear fractions prepared from Chlamydia-infected cells. The pgp3 protein was detected as early as 12 h after infection and was secreted by all chlamydial species that carry the cryptic plasmid, suggesting that there is a selection pressure for maintaining pgp3 secretion during chlamydial infection. Although expression of pgp3 in the host cell cytosol via a transgene did not alter the susceptibility of the transfected cells to the subsequent chlamydial infection, purified pgp3 protein stimulated macrophages to release inflammatory cytokines, suggesting that pgp3 may contribute to chlamydial pathogenesis.

Characterization of fifty putative inclusion membrane proteins encoded in the Chlamydia trachomatis genome.

Although the Chlamydia trachomatis genome is predicted to encode 50 inclusion membrane proteins, only 18 have been experimentally localized in the inclusion membrane of C. trachomatis-infected cells. Using fusion proteins and anti-fusion protein antibodies, we have systematically evaluated all 50 putative inclusion membrane proteins for their localization in the infected cells, distribution patterns, and effects on subsequent chlamydial infection when expressed ectopically, as well as their immunogenicity during chlamydial infection in humans. Twenty-two of the 50 proteins were localized in the inclusion membrane, and 7 were detected inside the inclusions, while the location of the remaining 21 was not defined. Four (CT225, CT228, CT358, and CT440) of the 22 inclusion membrane-localized proteins were visualized in the inclusion membrane of Chlamydia-infected cells for the first time in the current study. The seven intra-inclusion-localized proteins were confirmed to be chlamydial organism proteins in a Western blot assay. Further characterization of the 50 proteins revealed that neither colocalization with host cell endoplasmic reticulum nor inhibition of subsequent chlamydial infection by ectopically expressed proteins correlated with the inclusion membrane localization. Interestingly, antibodies from women with C. trachomatis urogenital infection preferentially recognized proteins localized in the inclusion membrane, and the immunodominant regions were further mapped to the region predicted to be on the cytoplasmic side of the inclusion membrane. These observations suggest that most of the inclusion membrane-localized proteins are both expressed and immunogenic during C. trachomatis infection in humans and that the cytoplasmic exposure may enhance the immunogenicity.

Intracellular interleukin-1alpha mediates interleukin-8 production induced by Chlamydia trachomatis infection via a mechanism independent of type I interleukin-1 receptor.

Chlamydia trachomatis infection induces a wide array of inflammatory cytokines and chemokines, which may contribute to chlamydia-induced pathologies. However, the precise mechanisms by which Chlamydia induces cytokines remain unclear. Here we demonstrate that the proinflammatory cytokine interleukin-1alpha (IL-1alpha) plays an essential role in chlamydial induction of the chemokine IL-8. Cells deficient in IL-1alpha expression or IL-1alpha-competent cells treated with IL-1alpha-specific small interfering RNA failed to produce IL-8 in response to chlamydial infection. However, neutralization of extracellular IL-1alpha or blockade of or deficiency in type I IL-1 receptor (IL-1RI) signaling did not affect chlamydial induction of IL-8 in cells capable of producing IL-1alpha. These results suggest that IL-1alpha can mediate the chlamydial induction of IL-8 via an intracellular mechanism independent of IL-1RI, especially during the early stage of the infection cycle. This conclusion is further supported by the observations that expression of a transgene-encoded full-length IL-1alpha fusion protein in the nuclei enhanced IL-8 production and that nuclear localization of chlamydia-induced precursor IL-1alpha correlated with chlamydial induction of IL-8. Thus, we have identified a novel mechanism for chlamydial induction of the chemokine IL-8.

Characterization of hypothetical proteins Cpn0146, 0147, 0284 & 0285 that are predicted to be in the Chlamydia pneumoniae inclusion membrane.

BACKGROUND: Although more than 100 Chlamydia pneumoniae hypothetical proteins have been predicted to be inclusion membrane proteins, only a few have been experimentally demonstrated to be in the inclusion membrane. Using antibodies raised with fusion proteins, we characterized four such hypothetical proteins encoded by two gene clusters (Cpn0146-147 and Cpn0284-285) in the C. pneumoniae genome. RESULTS: Cpn0146 and 0147 were detected in the inclusion membrane while Cpn0284 and 0285 inside inclusion and mainly associated with reticulate bodies although all four proteins contain an N-terminal bi-lobed hydrophobic region, a signature motif assigned to inclusion membrane proteins. These four hypothetical proteins were only detected in cells infected with C. pneumoniae but not other chlamydial species, with Cpn0147 at 6 hours and Cpn0146, 0284 & 0285 at 24 hours after infection. Cpn0146 & 147 but not Cpn0284 and 285 co-localized with a host cell endoplasmic reticulum marker, a property known to be possessed by some chlamydial inclusion membrane proteins, when expressed in the host cell cytosol via transgenes. However, the endoplasmic reticulum localization of the C. pneumoniae inclusion membrane proteins did not result in inhibition of the subsequent C. pneumoniae infection. CONCLUSION: The hypothetical proteins Cpn0146 & 0147 were localized in the C. pneumoniae inclusion membrane while Cpn0284 & 0285 within the inclusion although all four were predicted to be Inc proteins, suggesting the need to experimentally characterize the predicted Inc proteins.

Characterization of the hypothetical protein Cpn1027, a newly identified inclusion membrane protein unique to Chlamydia pneumoniae.

The hypothetical protein Cpn1027 was detected in the inclusion membrane of Chlamydia pneumoniae-infected cells with antibodies raised with Cpn1027 fusion proteins in an indirect immunofluorescence assay. The inclusion membrane staining by the anti-Cpn1027 antibodies co-localized with the staining of an antibody recognizing a known inclusion membrane protein designated IncA and these membrane stainings were blocked by the corresponding but not irrelevant fusion proteins. Although Cpn1027 was not predicted to be an inclusion membrane protein, it contained a bi-lobed hydrophobic domain region at its N-terminus, a signature secondary structural motif possessed by most chlamydial inclusion membrane proteins. The Cpn1027 protein was detected as early as 12 h after C. pneumoniae infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of Cpn1027 via a transgene failed to affect the subsequent chlamydial infection. The anti-Cpn1027 polyclonal antisera failed to detect any significant signals in cells infected with chlamydial species other than C. pneumoniae, which is consistent with the sequence analysis result that no significant homologues of Cpn1027 were found in any other species. These experiments together have demonstrated that Cpn1027 is a newly identified inclusion membrane protein unique to C. pneumoniae.

Localization of the hypothetical protein Cpn0585 in the inclusion membrane of Chlamydia pneumoniae-infected cells.

Cpn0585, encoded by a hypothetical open reading frame in Chlamydia pneumoniae genome, was detected in the inclusion membrane during C. pneumoniae infection using both polyclonal and monoclonal antibodies raised with Cpn0585 fusion protein. The anti-Cpn0585 antibodies specifically recognized the endogenous Cpn0585 without cross-reacting with IncA (a known inclusion membrane protein of C. pneumoniae) or other control antigens. A homologue of Cpn0585 in the C. caviae species (encoded by the ORF CCA00156) was also localized in the inclusion membrane of the C. caviae-infected cells. The Cpn0585 protein became detectable 24h while CCA00156 as early as 8h after infection. Once expressed, both proteins remained in the inclusion membrane throughout the rest of infection course.

Localization of the hypothetical protein Cpn0797 in the cytoplasm of Chlamydia pneumoniae-infected host cells.

Using antibodies raised with chlamydial fusion proteins, we have localized a protein encoded by the hypothetical open reading frame Cpn0797 in the cytoplasm of Chlamydia pneumoniae-infected host cells. The anti-Cpn0797 antibodies specifically recognized Cpn0797 protein without cross-reacting with either CPAFcp or Cpn0796, the only two proteins known to be secreted into the host cell cytosol by C. pneumoniae organisms. Thus, Cpn0797 represents the third C. pneumoniae protein secreted into the host cell cytosol experimentally identified so far.

The secreted protease factor CPAF is responsible for degrading pro-apoptotic BH3-only proteins in Chlamydia trachomatis-infected cells.

Chlamydia trachomatis has evolved a profound anti-apoptotic activity that may aid in chlamydial evasion of host defense. The C. trachomatis anti-apoptotic activity has been correlated with blockade of mitochondrial cytochrome c release, inhibition of Bax and Bak activation, and degradation of BH3-only proteins. This study presents evidence that a chlamydia-secreted protease factor designated CPAF is both necessary and sufficient for degrading the BH3-only proteins. When the C. trachomatis-infected cell cytosolic extracts were fractionated by column chromatography, both the CPAF protein and activity elution peaks overlapped with the BH3-only protein degradation activity peak. Depletion of CPAF with a CPAF-specific antibody removed the BH3-only protein degradation activity from the infected cell cytosolic extracts, whereas depletion with control antibodies failed to do so. Notably, recombinant CPAF expressed in bacteria was able to degrade the BH3-only proteins, whereas CPAF mutants similarly prepared from bacteria failed to do so. Finally, bacterium-expressed CPAF also degraded the human BH3-only protein Pumaalpha purified from bacteria. These results demonstrate that CPAF contributes to the chlamydial anti-apoptotic activity by degrading the pro-apoptotic BH3-only Bcl-2 subfamily members.

The hypothetical protein CT813 is localized in the Chlamydia trachomatis inclusion membrane and is immunogenic in women urogenitally infected with C. trachomatis.

Using antibodies raised with chlamydial fusion proteins, we have localized a protein encoded by hypothetical open reading frame CT813 in the inclusion membrane of Chlamydia trachomatis. The detection of the C. trachomatis inclusion membrane by an anti-CT813 antibody was blocked by the CT813 protein but not unrelated fusion proteins. The CT813 protein was detected as early as 12 h after chlamydial infection and was present in the inclusion membrane during the entire growth cycle. All tested serovars from C. trachomatis but not other chlamydial species expressed the CT813 protein. Exogenously expressed CT813 protein in HeLa cells displayed a cytoskeleton-like structure similar to but not overlapping with host cell intermediate filaments, suggesting that the CT813 protein is able to either polymerize or associate with host cell cytoskeletal structures. Finally, women with C. trachomatis urogenital infection developed high titers of antibodies to the CT813 protein, demonstrating that the CT813 protein is not only expressed but also immunogenic during chlamydial infection in humans. In all, the CT813 protein is an inclusion membrane protein unique to C. trachomatis species and has the potential to interact with host cells and induce host immune responses during natural infection. Thus, the CT813 protein may represent an important candidate for understanding C. trachomatis pathogenesis and developing intervention and prevention strategies for controlling C. trachomatis infection.

Inhibition of staurosporine-induced activation of the proapoptotic multidomain Bcl-2 proteins Bax and Bak by three invasive chlamydial species.

It has been reported that all major chlamydial species can inhibit host cell apoptosis and the Chlamydia trachomatis antiapoptotic activity is correlated with inhibition of activation of the proapoptotic multidomain Bcl-2 proteins Bax and Bak and degradation of BH3-only domain Bcl-2 proteins such as Puma. The current study is to test whether the more invasive species can also suppress host apoptosis through inhibition of Bax and Bak activation. We compared the effects of the three invasive chlamydial species C. muridarum, C. caviae, C. psittaci with that of C. trachomatis on host cell Bax and Bak activation. We found that these chlamydial species not only failed to activate Bax and Bak but also significantly inhibited Bax and Bak activation, mitochondrial cytochrome c release and caspase 3 activation induced by staurosporine. These results have demonstrated that inhibition of host cell apoptosis pathways mediated by Bax and Bak activation is a common property of the major chlamydial species.

Profiling of human antibody responses to Chlamydia trachomatis urogenital tract infection using microplates arrayed with 156 chlamydial fusion proteins.

The available chlamydial genome sequences have made it possible to comprehensively analyze host responses to all chlamydial proteins, which is essential for further understanding of chlamydial pathogenesis and development of effective chlamydial vaccines. Microplates arrayed with 156 Chlamydia trachomatis fusion proteins were used to evaluate antibody responses in women urogenitally infected with C. trachomatis. Based on both the antibody recognition frequency and titer, seven chlamydial antigens encoded by open reading frames (ORFs) CT089, CT147, CT226, CT681, CT694, CT795, and CT858, respectively, were identified as relatively immunodominant; six of these are encoded by hypothetical ORFs. Antibody binding to these chlamydial fusion proteins was blocked by C. trachomatis-infected but not by normal HeLa cell lysates or irrelevant bacterial lysates. These results have revealed novel immune-reactive chlamydial antigens, not only indicating that the hypothetical ORF-encoded proteins are expressed during chlamydial infection in humans but also providing the proof of principle that the fusion protein-based approach can be used to profile human immune responses to chlamydial infection at the whole-genome scale.

[Transfecting rat heart with human pacemaker gene in vivo to create a biological pacemaker].

OBJECTIVE: To test the ability and safety of injecting the hHCN gene into the ventricle of intact rats to create a novel biological pacemaker and to explore the duration of the hHCN over-expression in vivo. METHODS: Adenoviral constructs incorporating hHCN4 and green fluorescent protein (GFP) were subepicardially injected into the rats' left ventricular wall in situ (n = 10). Control group was injected with adenoviral constructs of GFP alone (n = 10). ECGs were recorded every day within the initial three days after operation. Up to seven days after injection, all rats were anesthetized and subjected to cervical vagal trunks stimulation to permit the emergence of escape rhythm. Then pace-mapping was performed by the hand-hold electrode. Finally, hearts were removed for histology and immunofluorescence study. RESULTS: Ventricular arrhythmic events were not found during the first three days. During vagal stimulation, the rate of spontaneous rhythm in the hHCN4-injected group was significantly more rapid than that in the control group (69 bpm +/- 6 bpm vs 41 bpm +/- 10 bpm, P < 0.001). Pace-mapping confirmed the escape rhythm in the hHCN4-injected group was originated near the injection site. Immunofluorescence study showed the overexpression of hHCN4 on the site of injection. However, the duration of hHCN overexpression was no more than one month after injection. CONCLUSION: The overexpression of hHCN4 provides ventricular escape rhythm with the physiologically acceptable rate. Long-term feasibility of this approach needs to be tested.

Degradation of the proapoptotic proteins Bik, Puma, and Bim with Bcl-2 domain 3 homology in Chlamydia trachomatis-infected cells.

We have previously correlated Chlamydia trachomatis antiapoptotic activity with the blockade of mitochondrial cytochrome c release and the inhibition of Bax and Bak activation. We now report that C. trachomatis infection leads to degradation of Bik, Puma, and Bim, three upstream proapoptotic BH3-only proteins of the Bcl-2 family that can transmit death signals to mitochondria by inhibiting the Bcl-2 antiapoptotic proteins and/or activating the Bcl-2 proapoptotic members, such as Bax and Bak. This observation has provided new information on the chlamydial antiapoptosis mechanisms.

Production of a proteolytically active protein, chlamydial protease/proteasome-like activity factor, by five different Chlamydia species.

We have previously identified a chlamydial protein, chlamydial protease/proteasome-like activity factor (CPAF), for degrading host transcription factors in cells infected with the human chlamydial species Chlamydia trachomatis or Chlamydia pneumoniae. We now report that functional CPAF was also produced during infection with the species Chlamydia muridarum, Chlamydia psittaci, and Chlamydia caviae, which primarily infect nonhuman hosts.

NF-kappa B activation is not required for Chlamydia trachomatis inhibition of host epithelial cell apoptosis.

Chlamydia trachomatis, an obligate intracellular bacterial species, is known to inhibit host cell apoptosis. However, the chlamydial antiapoptotic mechanism is still not clear. Because NF-kappaB activation is antiapoptotic, we tested the potential role of NF-kappaB activation in chlamydial antiapoptotic activity in the current study. First, no obvious NF-kappaB activation was detected in the chlamydia-infected cells when these cells were resistant to apoptosis induced via either the intrinsic or extrinsic apoptosis pathways. Second, inhibition of NF-kappaB activation with pharmacologic reagents failed to block the chlamydial antiapoptotic activity. Finally, NF-kappaB p65 gene deletion did not prevent chlamydia from inhibiting host cell apoptosis. These observations together have demonstrated that NF-kappaB activation is not required for the chlamydial antiapoptotic activity.