Ulrike Simnacher

Survival of Chlamydia pneumoniae-Infected Mono Mac 6 Cells Is Dependent on NF-κB Binding Activity

ABSTRACT The respiratory tract pathogen Chlamydia pneumoniae has been associated with atherosclerosis. Monocytes are supposed to serve as a vehicle for systemic dissemination of intracellular C. pneumoniae from the lung to the artery vessel wall. We were therefore interested in pathogen-induced cellular events associated with NF-κB, a crucial transcription factor for both inflammatory cytokines and antiapoptotic molecules. In this study we demonstrate by electrophoretic mobility shift assay that C. pneumoniaeinfection of the human monocytic cell line Mono Mac 6 induces activation of NF-κB over 48 h, with a maximum level at 1 h postinfection. As shown by supershift assay, the activated NF-κB complex consists of the subunits RelA (p65) and NF-κB1 (p50). Apoptotic host cells were not detected during the early stages of the infection when maximal activation of NF-κB was detected. Pretreatment of Mono Mac 6 with the antioxidant and NF-κB inhibitor PDTC (pyrrolidine dithiocarbamate) induced activation of caspase-3 and led to apoptotic cell death. The C. pneumoniae-induced activation of the NF-κB complex was reduced by PDTC, which in parallel resulted in an increased apoptosis, as quantified by annexin V labeling and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction. In the complete absence of activated NF-κB, when Mono Mac 6 cells were pretreated with the more potent NF-κB inhibitors MG-132 and parthenolide a C. pneumoniae-mediated rescue of cells from induced apoptosis could not be achieved. Our results indicate that activation of NF-κB inC. pneumoniae-infected Mono Mac 6 cells is associated with protection of Mono Mac 6 cells against apoptosis and might thereby contribute to systemic spread of the pathogen.

Identification and evaluation of a combination of chlamydial antigens to support the diagnosis of severe and invasive Chlamydia trachomatis infections

Chlamydia trachomatis is the most common sexually transmitted organism in industrialized countries. Nucleic acid amplification testing, using non-invasively collected specimens, is considered to be the method of choice for diagnosis of chlamydial infections of the urethra and the lower genital tract. Serological testing has the potential to circumvent the problem of specimen sampling in invasive C. trachomatis infections of the upper genital tract. However, only a few defined chlamydial antigens have been used in a standardized diagnostic assay format. In this study, we used serological two-dimensional proteomic analysis to broaden the spectrum of diagnostically relevant C. trachomatis proteins. The genes encoding an assortment of already known chlamydial antigens, as well as immunogenic proteins that have not been described before, were cloned, and the recombinant proteins were purified in order to compare their diagnostic usefulness in parallel with a newly developed line immunoassay. With 189 sera collected from patients with and without C. trachomatis infection, recombinant major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), outer membrane protein 2 (OMP2), translocated actin-recruiting protein, and polymorphic membrane protein D (PmpD) showed the highest level of diagnostic sensitivity and specificity. In patients suffering from ascending and invasive C. trachomatis infections, such as pelvic inflammatory disease and lymphogranuloma venereum, the sensitivity reached with these proteins ranged between 71% (PmpD) and 94% (OMP2), and the specificity ranged between 82% (PmpD) and 100% (MOMP and OMP2). Recombinant thio-specific antioxidant peroxidase, ribosomal protein S1 (RpsA) and hypothetical protein 17 showed lower sensitivity but comparably high specificity, ranging from 94% to 100%. The novel line immunoassay based on defined recombinant antigens has promise for improved serodiagnosis in severe and invasive C. trachomatis infections.

Profiling Antibody Responses to Infections by Chlamydia abortus Enables Identification of Potential Virulence Factors and Candidates for Serodiagnosis

Enzootic abortion of ewes (EAE) due to infection with the obligate intracellular pathogen Chlamydia (C.) abortus is an important zoonosis leading to considerable economic loss to agriculture worldwide. The pathogen can be transmitted to humans and may lead to serious infection in pregnant women. Knowledge about epidemiology, clinical course and transmission to humans is hampered by the lack of reliable diagnostic tools. Immunoreactive proteins, which are expressed in infected animals and humans, may serve as novel candidates for diagnostic marker proteins and represent putative virulence factors. In order to broaden the spectrum of immunogenic C. abortus proteins we applied 2D immunoblot analysis and screening of an expression library using human and animal sera. We have identified 48 immunoreactive proteins representing potential diagnostic markers and also putative virulence factors, such as CAB080 (homologue of the “macrophage infectivity potentiator”, MIP), CAB167 (homologue of the “translocated actin recruitment protein”, TARP), CAB712 (homologue of the “chlamydial protease-like activity factor”, CPAF), CAB776 (homologue of the “Polymorphic membrane protein D”, PmpD), and the “hypothetical proteins” CAB063, CAB408 and CAB821, which are predicted to be type III secreted. We selected two putative virulence factors for further characterization, i.e. CAB080 (cMIP) and CAB063, and studied their expression profiles at transcript and protein levels. Analysis of the subcellular localization of both proteins throughout the developmental cycle revealed CAB063 being the first C. abortus protein shown to be translocated to the host cell nucleus.

Clearance of Chlamydia trachomatis - induced polyserositis in SCID mice requires both CD4+ and CD8+ cells

Abstract To characterize the role of specific lymphocyte subsets in Chlamydia trachomatis infection, we established a murine model using the mouse pneumonitis agent (MoPn) of C. trachomatis and C.B-17 scid/scid (SCID) mice which lack functional B and T cells. After intraperitoneal inoculation with the bacteria, SCID mice developed polyserositis with pleuritis, pericarditis, and perihepatitis. Within 8 weeks post infection, SCID mice succumbed to the disease, whereas immunocompetent congenic C.B-17+/+ mice resolved the infection. Adoptive transfer of immune spleen cells into MoPn-infected SCID mice resulted in a complete elimination of the agent and prevention of polyserositis as measured by quantitative chlamydial culture, direct immunofluorescence and histopathological analysis. Selective reconstitution of MoPn-infected SCID mice with immune B lymphocytes, CD4+ T cells or CD8+ T cells alone did not influence the chlamydial load in the lung and liver of infected SCID animals, resulting in a polyserositis as observed in untreated MoPn-infected SCID mice. However, co-transfer of both CD4+ T cells and CD8+ T cells led to a significant reduction of chlamydiae in quantitative organ culture coupled with unremarkable histopathology. These data confirm that T cell-mediated immune responses are essential for immune protection in chlamydial infection, although total eradication of the agent could not be achieved. Further experiments are needed to stress the importance of a concerted action of B and T lymphocytes, as indicated by the complete protective efficacy of transferred splenocytes.

Decontamination of a Mycoplasma-infected Chlamydia pneumoniae strain by pulmonary passage in SCID mice

We describe a procedure to eliminate contaminating Mycoplasma from Chlamydia pneumoniae (C. pneumoniae) cultures by pulmonary passage in severe combined immunodeficiency mice (SCID). Four weeks after experimental infection only C. pneumoniae could be cultured from the lungs of the infected animals while Mycoplasma could not be detected any longer, as shown by PCR, culture and transmission electron microscopy (TEM).

Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay

ABSTRACT The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C. abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C. abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C. abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C. abortus infections.

Characterization of the potential virulence factors CAB063 and CAB821 of Chlamydia abortus

Introduction: The association between the major gastric pathogen Helicobacter pylori and humans dates back at least 100,000 years. Since that time H. pylori evolved in parallel with its human host, accompanying anatomically modern humans ‘out of Africa’ and differentiating into seven known biogeographic populations. Three of these are indigenous to Africa: hpNEAfrica, hpAfrica1, and hpAfrica2. The most divergent H. pylori population, hpAfrica2, is associated with the ancient San hunter-gatherers of southern Africa. This striking parallel between the oldest human and bacterial lineages led us to investigate the prevalence and genetic structure of H. pylori in another ancient hunter-gatherer population, the Cameroonian Baka Pygmies. Methods: Gastric biopsies were obtained for a total of 178 individuals, 77 of them belonging to the Baka Pygmy population and the remainder to neighboring agricultural Bantu communities. H. pylori was cultured from both antrum and corpus biopsies from each individual. The isolates were analyzed using MLST (Multilocus sequence typing). The population structure of distinct haplotypes was determined by Bayesian clustering using the program STRUCTURE. Phylogenetic analyses were perfomed using ClonalFrame. Results: The H. pylori prevalence among Baka pygmies (20%) was significantly lower compared to the non-Baka individuals (80%). MLST analysis of all isolates identified 113 unique haplotypes, ten of which were shared by more than one person. Subsequent STRUCTURE analyses assigned these isolates to either hpNEAfrica (67), hpAfrica1 (44), or hpEurope (2) populations. Among infected Baka and non-Baka individuals, the infection rates with hpNEAfrica and hpAfrica1 isolates were similar. In phylogenetic analyses using ClonalFrame we observed an intermediate position of the Cameroonian hpAfrica1 isolates between the already described hspSAfrica and hspWAfrica subpopulations corresponding to the geographic location of Cameroon between South and West Africa. Further STRUCTURE analyses of the hpNEAfrica population revealed a possible subdivision into an East African and a Central African subpopulation. Discussion: The results suggest that the Baka pygmies, which belong to one of the oldest branches of the human population tree and still live as hunter-gatherers, were initially H. pylori free and acquired H. pylori more recently from their neighboring Bantu communities.