Cytoskeletal requirements in Chlamydia trachomatis infection of host cells.

Infection of genital epithelial cells by the closely related sexually transmitted pathogens Chlamydia trachomatis serovars E and L2 results in different clinical disease manifestations. Following entry into target host cells, individual vesicles containing chlamydiae fuse with one another to form one large inclusion. At the cellular level, the only obvious difference between these serovars is the time until inclusion maturation, which is 48 h for the invasive serovar L2 and 72 h for serovar E. To begin to define the intracellular events of these pathogens, the effect of cytoskeletal disruption on early endosome fusion and inclusion development in epithelial (HEC-1B) and fibroblast (McCoy) cells was analyzed by fluorescence microscopy. Disruption of microfilaments with cytochalasin D markedly reduced serovar E, but not serovar L2, infection of both cell lines. Conversely, microfilament as well as microtubule disruption, with colchicine or nocodazole, had no effect on serovar E inclusion development but resulted in the formation of multiple serovar L2 inclusions per cell during early and mid-development. Later in serovar L2 inclusion development (> 36 h postinfection), vesicles containing chlamydiae fused to form one large inclusion in the absence of an intact cytoskeleton. These results imply that (i) C. trachomatis serovar E may utilize a different pathway for uptake and development from serovar L2; (ii) these differences are consistent in both epithelial cells and fibroblasts; and (iii) the cytoskeleton plays a unique role in the infection of host cells by these two genital pathogens.     

Role of gamma-delta T cells in murine Chlamydia trachomatis infection.

The role of gamma-delta T cells in host resistance to Chlamydia trachomatis was characterized by using a murine model of pneumonia caused by the mouse pneumonitis agent (MoPn), murine C. trachomatis. At days 3 and 7 after infection, gamma-delta T-cell-deficient knockout mice had significantly higher levels of MoPn in the lungs than did immunologically intact controls. At day 20, paradoxically, gamma-delta T-cell-deficient mice were more resistant to MoPn than were controls. This increased resistance was not due to an increased production of toxic cytokines or interleukin-10 in controls on that day. Gamma-delta T cells play a role in protection early in MoPn infection, but they may be deleterious later in infection, as has been observed in models of salmonella and trypanosome infection.     

Recruitment of myeloid and plasmacytoid dendritic cells in cervical mucosa during Chlamydia trachomatis infection

The mobilization of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) to the cervix during chlamydial infection is not fully understood, and the role of these cells in immunopathogenesis is largely unknown. As an effective vaccine to control chlamydial infection is currently unavailable, understanding the regulation of the local immune response becomes a necessity. Therefore, mDC and pDC populations were analysed in peripheral blood and cervical samples of controls and Chlamydia -positive women, with or without mucopurulent cervicitis (MPC). Cervical cytokines and C-reactive protein levels in serum were quantified by ELISA and the chlamydial infectious load by culture. Chlamydia trachomatis infection mobilized both mDCs and pDCs to the cervical mucosa. pDCs were recruited more often in women with MPC (p <0.05) and they correlated significantly with the chlamydial load, C-reactive protein levels and cervical interleukin-8 (IL-8) levels. Upregulation of surface expression of co-stimulatory molecules (CD80, CD83 and CD86) on cervical mDCs and pDCs was observed during chlamydial infection but was significant only for mDCs. Significantly higher levels of IL-1β, IL-6 and IL-8 were observed in Chlamydia- positive women with MPC; however, after therapy, IL-8 levels decreased significantly. Median numbers of mDCs after therapy were significantly higher in the cervix and blood of infected women as compared to the numbers of pDCs, which were found to be lower in the cervix after therapy. These results thus suggest that during chlamydial infection, both mDCs and pDCs are recruited to the cervix, but their number and possible immunological functions may differ with the pathological condition. pDCs were associated more often with MPC and inflammatory factors, suggesting that they may possibly be involved in the immunopathogenesis of infections due to Chlamydia .     

Frequency of Chlamydia trachomatis as the cause of pharyngitis.

We evaluated the incidence of Chlamydia trachomatis as the etiologic agent of uncomplicated pharyngitis by the cell culture procedure recommended by the Centers for Disease Control, Atlanta, Ga., and by the MicroTrak direct immunofluorescent stain (Syva Co., Palo Alto, Calif.) for elementary bodies on throat swabs collected from 126 symptomatic patients. Of the 126 cultures, 8% were positive for group A beta-hemolytic streptococci. Of 126 chlamydia cultures, none was positive. The MicroTrak test gave one borderline positive result. In contrast to a previously published report that C. trachomatis is the most frequent nonviral cause of adult pharyngitis (A. L. Komaroff, M. D. Aronson, T. M. Pass, C. T. Ervin, and W. T. Branch, Jr., Science 222:927-929, 1983), our data indicated an infection rate of less than 1%.     

Chlamydia trachomatis infection induces cleavage of the mitotic cyclin B1

The obligate intracellular pathogen Chlamydia trachomatis interferes with a number of host cell processes, including cytoskeletal organization, vesicular trafficking, and apoptosis. In this study we report that C. trachomatis-infected cells proliferate more slowly than uninfected cells, suggesting that C. trachomatis may also manipulate the eukaryotic cell cycle. We further demonstrate that C. trachomatis infection destabilizes specific cell cycle proteins involved in the G2/M transition. C. trachomatis-infected cells, compared to uninfected cells, have lower levels of cyclin-dependent kinase 1. Additionally, C. trachomatis infection induces an N-terminal truncation of the mitotic cyclin B1. Manipulation of the host cell cycle may represent a strategy used by C. trachomatis to ensure a stable environment conducive to bacterial growth and replication.     

Cellular immune response during uncomplicated genital infection with Chlamydia trachomatis in humans.

Extraction of staphylococcal abscesses by the Folch procedure revealed that all of the staphylocidal activity was present in the lipid fraction. Further separation of the lipids indicated that the bactericidal activity resided in the free fatty acid pool. Lipids similarly extracted from mesenteric or epididymal fat tissue, either before of after activation, did not possess comparable activity. Myristic, palmitic, palmitoleic, linoleic, and oleic acids, as well as lysolecithin, also failed to exhibit the properties of the fatty acid fraction obtained from abscess homogenates. These findings suggest the staphylocidal fatty acid is not a common host lipid.     

Value of screening for oro-pharyngeal Chlamydia trachomatis infection.

AIMS--To determine whether oro-pharyngeal colonisation by Chlamydia trachomatis occurs in patients at risk of genital chlamydia infection; to determine whether screening pharyngeal specimens by polymerase chain reaction (PCR) increases detection of C trachomatis compared with isolation and the immune dot blot test; and to correlate the detection of C trachomatis and Neisseria gonorrhoeae in the pharynx with a history of oro-genital contact. METHODS--Thirteen homosexuals and 11 heterosexuals were included in the study. Urogenital and pharyngeal specimens were tested for C trachomatis and N gonorrhoeae using standard clinical diagnostic procedures. Two different PCR methodologies were also used to detect C trachomatis in the pharyngeal specimens. Results were correlated with the mode of sexual practice. RESULTS--Oro-genital sexual contact was practised by 64.9% (72/111) of heterosexuals in addition to penetrative penovaginal intercourse. Additionally, 62.1% (77/124) of all patients did not use any form of barrier protection. Of those who admitted to oro-genital sexual contact, 17.6% of patients with a genital chlamydial infection and 36.4% of those with genital gonorrhoea also had asymptomatic pharyngeal colonisation. C trachomatis was detected in three of 124 (2.4%) pharyngeal specimens by PCR which were reported as negative by chlamydial culture; one was positive by the immune dot blot test. CONCLUSION--The majority of patients practised unprotected oro-genital contact and significant pharyngeal colonisation by C trachomatis and N gonorrhoeae occurred if genital infection was present. Despite the use of PCR in a population at high risk of sexually transmitted disease, the prevalence of chlamydia in the pharynx was very low. This indicates that transmission of C trachomatis to the oro-pharynx does not pose a serious health risk and that screening of patients for oro-pharyngeal C trachomatis is not worthwhile.     

Variation in virulence among oculogenital serovars of Chlamydia trachomatis in experimental genital tract infection.

Seven different oculogenital serovars (D, E, F, G, H, I, and K) of Chlamydia trachomatis were inoculated intravaginally into CF-1 mice, and subsequent infection was monitored. The duration of infection was longest with serovars D and E. This may help to explain clinical surveys which demonstrate a high (50%) prevalence of these serovars. Furthermore, a comparison of the invasiveness of strains D and H demonstrated a much higher frequency of uterine horn infection with serovar D.     

Growth of Chlamydia trachomatis in enucleated cells.

Chlamydia trachomatis is an obligate intracellular parasite of eucaryotic cells. Little is known about the role of the host in supporting chlamydial replication beyond the facts that host cells provide ATP and that de novo host protein synthesis is not required for bacterial growth. To further explore potential contributions of host nuclear function to chlamydial development, we questioned whether murine C. trachomatis could grow in mouse L cells that had been enucleated with cytochalasin B. Following enucleation, cells were infected with chlamydiae and analyzed morphologically and biochemically. Late in infection, substantial numbers of chlamydiae of all developmental stages were seen within large cytoplasmic inclusions that were indistinguishable from those seen in infected intact cells. Normal numbers of infectious progeny particles were produced from enucleated cultures. We conclude that active host cell nuclear function is not required to support the growth of chlamydiae.     

Expression of two novel proteins in Chlamydia trachomatis during natural infection

Genes for a putative membrane associated protein (mvi -homologue) and a 48 kDa protein (ctr48) in Chlamydia trachomatis were characterized. The mvi -homologue has 12 transmembrane domains and shows considerable homology to the members of this gene family in various organisms. The ctr48 has a leader sequence and the C-proximal half is tryptophan-rich. The latter region shares 65% identity with the N-proxima third of C. pneumoniae 76 kDa protein over an overlap of 231 amino acid residues. The genes for the mvi -homologue and the ctr48 are present in the B, Ba, D, E, J and L2 serotypes of C. trachomatis. Immediately downstream from the ctr48 gene are multiple stop codons which are followed by a functional rho-independent terminator. The mvi -homologue and ctr48 genes are independently transcribed, albeit poorly in serotype B. However, protein products corresponding to these genes could not be detected by western blotting in HEp2 cells infected with C. trachomatis. Nevertheless, antibodies to peptides corresponding to these proteins were detected in sera with high micro-immunofluorescence titre against C. trachoImatic, collected from a Chlamydia -endemic population. These results suggest that the mvi -homologue and ctr48 are expressed by C. trachomatis during natural infection.     

沙眼衣原体感染诱导精子凋亡

目的:研究精液沙眼衣原体(CT)感染对男性生殖的影响.方法:选择精液CT阳性的男性不育者57例作为CT阳性组,另选择正常对照组15例.检测精液常规,免疫层析法检测CT感染情况,末端脱氧核苷酸转移酶(TdT)介导的TUNEL法检测凋亡精子.结果: CT阳性组精子凋亡率为33.03%±14.98%,正常对照组为9.68%±2.78%,CT阳性组精子凋亡率明显增高.结论:精液CT感染诱导精子凋亡率增加,精液沙眼衣原体感染是引起男性不育的原因之一.     

Antichlamydial specificity of conjunctival lymphocytes during experimental ocular infection.

Antigen-specific responses to chlamydiae have been demonstrated with lymphocytes isolated from the conjunctiva after primary ocular infection and after topical challenge of chlamydia-immune cynomolgus monkeys with noninfectious, Triton X-100-extracted antigen. Proliferative to viable elementary bodies homologous to the original infecting serovar were demonstrated. In addition, in vitro production of antichlamydial antibody by conjunctival B cells was demonstrated by enzyme-linked immunosorbent assay of culture supernatants collected after 7 to 21 days of culture. These findings demonstrate that antigen-specific lymphocytes appear in the conjunctiva as a result of ocular chlamydial infection and that a noninfectious chlamydial antigen stimulates their reappearance or expansion at the site of original infection.     

Reactivation of persistent Chlamydia trachomatis infection in cell culture.

Gamma interferon induces persistent chlamydial infections in cell culture. These infections are characterized by altered morphologic and biochemical features of the pathogen. These persistent forms are abnormally large and noninfectious and undergo unusual structural and functional changes, including production of a paucity of outer envelope constituents and normal levels of the chlamydial hsp60, an immunopathological antigen. The current investigation evaluates the events that occur during reactivation of infectious Chlamydia trachomatis from persistently infected cell cultures. Transfer of persistent chlamydial organisms to gamma interferon-free medium resulted in recovery of infectivity accompanied by an increase in levels of structural membrane proteins and reorganization of aberrant organisms to morphologically typical elementary bodies. In addition, reactivation of infectious organisms from persistent chlamydiae that were maintained in culture for several weeks was demonstrated. These studies show that persistent C. trachomatis maintains viability for extended periods, illustrate the reversibility of immunologically mediated persistent infections, and characterize reactivation at the ultrastructural and biochemical levels.     

Use of a Chlamydia trachomatis DNA probe for detection of ocular chlamydiae.

We examined the efficacy of a Chlamydia trachomatis DNA probe in detecting ocular chlamydiae by comparing it with tissue culture isolation, direct fluorescent-antibody cytology, and clinical eye exams. In a trachoma-endemic area of Nepal, 430 Nepalese villagers were examined according to the World Health Organization trachoma grading scale. Upper tarsal conjunctival specimens from each subject were obtained for DNA probing, tissue culture, and fluorescent-antibody screening. Moderate to severe intensity of inflammation was found in 85 (21%) of 430 people studied. An additional 25 (7.2%) of 345 people with low or no intensity of inflammation also had microbiologically proven infection, which may reflect asymptomatic carriage. Compared with culture, the DNA probe had a sensitivity of 86.9% and a specificity of 91%. For direct fluorescent antibody versus culture, the values were 47.8 and 96.9%, respectively. Results from this study indicate that the DNA probe for C. trachomatis might be considered a valuable epidemiologic tool in screening trachoma-endemic populations for ocular chlamydiae.     

Activation of lipid metabolism contributes to interleukin-8 production during Chlamydia trachomatis infection of cervical epithelial cells

Chlamydia trachomatis infection is the most common cause of bacterial sexually transmitted diseases. Infection of the urogenital tract by C. trachomatis causes chronic inflammation and related clinical complications. Unlike other invasive bacteria that induce a rapid cytokine/chemokine production, chlamydial infection induces delayed inflammatory response and proinflammatory chemokine production that is dependent on bacterial growth. We present data here to show that the lipid metabolism required for chlamydial growth contributes to Chlamydia-induced proinflammatory chemokine production. By gene microarray profiling, validated with biochemical studies, we found that C. trachomatis LGV2 selectively upregulated PTGS2 (COX2) and PTGER4 (EP4) in cervical epithelial HeLa 229 cells. COX2 is an enzyme that catalyzes the rate-limiting step of arachidonic acid conversion to prostaglandins, including prostaglandin E2 (PGE2) and other eicosanoids, whereas EP4 is a subtype of cell surface receptors for PGE2. We show that Chlamydia infection induced COX2 protein expression in both epithelial cells and peripheral blood mononuclear cells and promoted PGE2 release. Exogenous PGE2 was able to induce interleukin-8 release in HeLa 229 epithelial cells. Finally, we demonstrated that interleukin-8 induction by Chlamydia infection or PGE2 treatment was dependent on extracellular signal-regulated kinase/mitogen-activated protein activity. Together, these data demonstrate that the host lipid remodeling process required for chlamydial growth contributes to proinflammatory chemokine production. This study also highlights the importance of maintaining a balanced habitat for parasitic pathogens as obligate intracellular organisms.     

Cultivation of Chlamydia trachomatis in cycloheximide-treated mccoy cells.

An isolation technique for Chlamydia trachomatis using McCoy cells is described. In contrast to earlier techniques employing such cells, no pretreatment of the cells was used. The glutarimide antibiotic cycloheximide was added to the culture medium used for incubating the cells after infection. Cycloheximide was used at concentrations that depressed, but did not completely inhibit, the metabolism of the eucaryotic host cells. In studies on different immunotypes of C. trachomatis cultured in the yolk sac of embryonated hen eggs, the cycloheximide technique was compared with a method using pretreatment of cells with 5-iodo-2-deoxyuridine. The cycloheximide method gave greater numbers of inclusion-forming units per cover slip for all the immunotypes of trachoma-inclusion conjunctivitis agents tested, i.e., A through I. In a study of 194 cervical and urethral specimens from women, cycloheximide treatment of McCoy cells was found to be more efficient than 5-iodo-2-deoxyuridine treatment for the isolation of C. trachomatis.     

Gamma interferon-mediated cytotoxicity related to murine Chlamydia trachomatis infection.

After infection with the mouse pneumonitis agent (MoPn; murine Chlamydia trachomatis), heterozygous (nu/+) but not nude athymic (nu/nu) mice produced enhanced amounts of gamma interferon (IFN-gamma) in vitro in response to MoPn antigen that exhibited cytotoxic activity when added to host cells already infected with chlamydiae. Antibody-complement lysis showed the cytotoxic activity to be dependent, at least in part, on L3T4+ T cells for production. The cytotoxic responses were directed primarily against Chlamydia-infected target cells, but a second type of toxicity was demonstrable against uninfected target cells after treatment of the generating cell population with anti-Lyt-2 antibody plus complement at certain time points after infection. This additional nonspecific cytotoxic activity was presumably due to a second factor (factor X) acting in concert with IFN-gamma. Lyt-2+ cells, however, also were shown to play a role in IFN-gamma production and cytotoxicity directed against infected targets at later time points after infection. Neutralization of IFN-gamma in the samples containing cytotoxic activity abrogated the cytotoxicity against both infected and uninfected targets, but cloned murine IFN-gamma exhibited toxicity in a dose-dependent manner only against infected target cells. The data provides evidence that cytotoxicity against infected targets is due to antigen-specific induction of IFN-gamma, but other cytokine activity, most demonstrable after removal of Lyt-2.2+ cells and cytotoxic to uninfected targets, also is present.