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PubMed2005

Shotgun proteomic analysis of Chlamydia trachomatis.

Chlamydiae are widespread bacterial pathogens responsible for a broad range of diseases, including sexually transmitted infections, pneumonia and trachoma. To validate the existence of hitherto hypothetical proteins predicted from recent chlamydial genome sequencing projects and to examine the patterns of expression of key components at the protein level, we have surveyed the expressed proteome of Chlamydia trachomatis strain L2. A combination of two-dimensional gel analysis, multi-dimensional protein identification (MudPIT) and nanocapillary liquid chromatography-tandem mass spectrometry allowed a total of 328 chlamydial proteins to be unambiguously assigned. Proteins identified as being expressed in the metabolically inert form, elementary body, of Chlamydia include the entire set of predicted glycolytic enzymes, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. An enzyme central to cell wall biosynthesis was also detected in the intracellular form, reticulate body, of Chlamydia, suggesting that the peptidoglycan is produced during growth within host cells. Other sets of proteins identified include 17 outer membrane-associated proteins of potential significance in vaccine studies and 67 proteins previously annotated as hypothetical or conserved hypothetical. Taken together, >/=35% of the predicted proteome for C. trachomatis has been experimentally verified, representing the most extensive survey of any chlamydial proteome to date.

Animals↗
PubMed1994

Taxonomic studies of Rickettsiella, Rickettsia, and Chlamydia using genomic DNA.

Invertebrate pathogens of the genus Rickettsiella (Order Rickettsiales) undergo a developmental cycle more characteristic of chlamydial organisms (Order Chlamydiales) than of typical rickettsiae. Moreover, among recognized species of Rickettsiella, there is considerable variation in host range and in the ultrastructure and development of the infectious stage, i.e., the elementary body. To begin an analysis of the taxonomic relationships of the invertebrate pathogens belonging to the genus Rickettsiella and of the relationship of these to chlamydiae and other rickettsiae, genomic DNA of representative species was compared with respect to restriction enzyme site polymorphism, DNA-DNA hybridization, G + C ratios, and fine melting profiles. The strains studied included isolates currently classified as Rickettsiella grylli, R. popilliae, R. chironomi, Chlamydia psittaci, C. trachomatis, Coxiella burnetii, and Rickettsia conorii. Reciprocal DNA hybridization studies carried out under high-stringency conditions (65 degrees C) showed homology between R. grylli and R. popilliae isolates but not with the others, including two isolates of R. chironomi from the midge, Chironomus dorsalis, and the scorpion, Buthus occitanus. The R. chironomi isolates shared no detectable homology with each other or with any of the other strains or species. Additionally, no homology was detected between any of the Rickettsiella isolates and the species of Coxiella, Rickettsia, or Chlamydia. Results obtained from the DNA fine melting profiles and G + C ratios corresponded with the results obtained from the DNA hybridization studies. Thus, the present study indicates that, despite similarities in developmental cycles, Rickettsiella and Chlamydia are taxonomically distinct groups appropriately placed in different orders.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Sulfated polysaccharides block chlamydia infection in vitro, but do not protect mice from vaginal inoculation.

There is considerable interest in developing a vaginal product that women could use to protect themselves from sexually transmitted pathogens, including chlamydia. It has been suggested that sulfated polysaccharides would be effective in a prophylactic product because they have been shown to block infection of cultured cells by sexually transmitted pathogens, including chlamydia. In order to compare in vitro findings with animals, we placed sulfated polysaccharides into the vaginae of mice prior to inoculation with chlamydia. The surfactant nonoxynol 9 (N9) was used as a positive control as it has been previously shown to protect mice from infection by chlamydia. In this study, N9 also protected the mice from infection. However, sulfated polysaccharides which had been shown to be efficacious in vitro did not block infection.

Animals↗
PubMed1992

Outbreak of Chlamydia pneumoniae infection in four farm families.

The epidemiology of Chlamydia pneumoniae infection was studied in an outbreak in four farm families living close together in Denmark. Eleven of 20 members of the families studied had bronchitis or pneumonia characteristic of Chlamydia pneumoniae infection. Serologic evidence of Chlamydia pneumoniae as causative agent was strengthened by a high incidence of epidemic infection. Transmission within families and a high frequency of disease versus asymptomatic infection are two findings which deviate from epidemiological patterns of Chlamydia pneumoniae infection as currently known.

Adult↗
PubMed1991

Detection of Chlamydia trachomatis by direct immunofluorescence improved by centrifugation of specimens.

During a study of women with laparoscopically investigated pelvic pain, genital tract specimens were examined for Chlamydia trachomatis using a direct fluorescent antibody (DFA) technique (MicroTrak, Syva) and culture. Some smears, particularly those from the upper genital tract, contained an inadequate number of cells when examined by the DFA technique and many cell monolayers were destroyed by the specimens. To obtain results for such samples, or to confirm the original DFA result, additional specimens which had been frozen at -70 degrees C or in liquid nitrogen were centrifuged at high speed and the resulting deposit examined by the DFA technique. By this means, 32 negative results were confirmed for specimens from 10 chlamydia-negative patients with pelvic inflammatory disease or with high chlamydial antibody titres, and 26 negative results were confirmed for 19 patients who were positive at other sites. In addition, three chlamydia-positive and six chlamydia-negative results were obtained for sites where the original smear for DFA testing had been inadequate (few epithelial cells) and six specimens that were negative originally were found to be positive. Thus, of 73 specimens that were either inadequate or negative by DFA testing originally, 9 (12%) were positive by DFA testing after centrifugation.

Centrifugation↗
PubMed2000

Atherogenetically relevant cells support continuous growth of Chlamydia pneumoniae.

The obligate intracellular bacterium Chlamydia pneumoniae has been implicated in the pathogenesis of atherosclerosis since viable pathogen has been recovered from plaques. Chlamydiae are epithelial pathogens notorious for causing persistent infection. Atherosclerosis, however, is a chronic inflammatory disease involving mesenchymal cells of the vascular wall. A bacterial contribution to atherosclerosis appears more relevant if the resident mesenchymal cells of the vascular wall that constitute the plaque can support chlamydial infection continuously. Therefore we inoculated immortalized and primary mesenchymal cells with a vascular and a respiratory Chlamydia pneumoniae isolate. Primary human coronary artery endothelial and smooth muscle cells, primary human embryonic fibroblasts as well as the immortalized cell lines were permissive for continuous growth of both strains. Thus, the resident vascular cells that produce the atheromatous plaque can acquire permanent productive. Chlamydia pneumoniae infection. Immortalized monocytic cells and peripheral blood monocytes also supported chlamydial growth, though productive infection ceased after 5 passages. Monocytes/macrophages are not resident cells of the vascular wall but have an active role in plaque formation. Systemic circulation and transendothelial migration makes them a potential vector system for chlamydial distribution. These findings add further plausibility to the hypothesis of a chronic infectious component in the multifactorial condition of atherosclerosis. Further studies must precisely define chlamydial target cells in vivo and differentiate infection in resident cells of the vascular wall from a presence limited to migrating macrophages. Endovascular infection might provide an explanation for unclear phenomena of atherogenesis like mesenchymal cell proliferation and its distinct inflammatory component.

Arteriosclerosis↗
PubMed2003

Speciation in Chlamydia: genomewide phylogenetic analyses identified a reliable set of acquired genes.

Horizontal gene transfer (HGT), a process through which genomes acquire sequences from distantly related organisms, is believed to be a major source of genetic diversity in bacteria. A central question concerning the impact of HGT on bacterial genome evolution is the proportion of horizontally transferred sequences within genomes. This issue, however, remains unresolved because the various methods developed to detect potential HGT events identify different sets of genes. The present-day consensus is that phylogenetic analysis of individual genes is still the most objective and accurate approach for determining the occurrence and directionality of HGT. Here we present a genome-scale phylogenetic analysis of protein-encoding genes from five closely related Chlamydia, identifying a reliable set of sequences that have arisen via HGT since the divergence of the Chlamydia lineage. According to our knowledge, this is the first systematic phylogenetic inference-based attempt to establish a reliable set of acquired genes in a bacterial genome. Although Chlamydia are obligate intracellular parasites of higher eukaryotes, and thus suspected to be isolated from HGT more than the free-living species, our results show that their diversification has involved the introduction of foreign sequences into their genome. Furthermore, we also identified a complete set of genes that have undergone deletion, duplication, or rearrangement during this evolutionary period leading to the radiation of Chlamydia species. Our analysis may provide a deeper insight into how these medically important pathogens emerged and evolved from a common ancestor.

Base Composition↗
PubMed2000

The role of chlamydia in the pathogenesis of pulmonary emphysema. Electron microscopy and immunofluorescence reveal corresponding findings as in atherosclerosis.

Chlamydia pneumoniae has been detected in atherosclerotic plaques by various means. Chlamydiae are able to cause persistent infections. Serologically elevated antibody titers are found in severe chronic obstructive pulmonary disease. In atherosclerosis and pulmonary emphysema, inflammatory reactions can be seen by means of light microscopy. Specimens from patients with obliterative arteriosclerosis undergoing thrombendarteriectomy and with advanced emphysema undergoing lung volume reduction surgery were examined using scanning (SEM) and transmission (TEM) electron microscopy, and using immunofluorescence with monoclonal antibodies and antiserum against chlamydiae. SEM shows spherical bodies (SBs) with a diameter from 0.3 microm to 0.6 microm on the surface of the alveoli and bronchioles, as well as in atherosclerotic plaques. In atherosclerosis and emphysema, SBs reveal a double membrane, adherence to collagen fibers, tissue destruction, as well as intracellular and interstitial localization in TEM. They show in parts a densely packed central structure. SBs are seen both in alpha-1-antitrypsin deficiency emphysema and smoker's emphysema. Using immunofluorescence microscopy, spots are seen in corresponding distributions to the SBs. Morphological findings are typical for aberrant chlamydiae seen in persistent infections. Chronic infection and bacterial colonization associated with progressive disease seems to be relevant not only in atherosclerosis but also in pulmonary emphysema.

Arteriosclerosis↗
PubMed1999

Demonstration of viable Chlamydia pneumoniae in atherosclerotic plaques of carotid arteries by reverse transcriptase polymerase chain reaction.

The presence of Chlamydia pneumoniae in atheromas has been demonstrated in several studies. Culture of the organism from arterial tissue has been difficult. We report the use of a reverse transcriptase polymerase chain reaction to detect viable Chlamydia pneumoniae in carotid atheromas. We analyzed 30 patients (14 females, mean age 69.6 +/- 8.8 years) who underwent surgery for the removal of atherosclerotic plaques from carotid arteries. During surgery, samples of lingual vein and superior thyroideal artery were also taken. We applied two molecular biology techniques to the carotid plaques on lingual vein or thyroideal artery samples: 1) polymerase chain reaction (PCR) and 2) reverse transcriptase-PCR (RT-PCR) for the detection of bacterial mRNA, employing PCR primers designed to detect a fragment of the 16S rRNA gene. Blood samples were obtained from the patients for determination of Chlamydia pneumoniae IgG, IgA, and IgM antibody titers by a microimmunofluorescence technique. The results of the present study confirmed the presence of viable Chlamydia pneumoniae in atheromas and support the hypothesis that the organism may be an active factor in the pathogenesis of atherosclerosis.

Aged↗
PubMed2002

In-vitro activity of moxifloxacin and other fluoroquinolones against Chlamydia species.

The in-vitro activity of moxifloxacin, a new fluoroquinolone, against Chlamydia species was investigated. The minimal inhibitory concentration of moxifloxacin for 10 standard strains of different Chlamydia species and 15 wild-type strains of Chlamydia pneumoniae isolated in Japan, which were morphologically different from clinical isolates from the United States, ranged from 0.031 to 0.125 microg/ml. The activity of moxifloxacin was almost the same as those of sparfloxacin, and it was 16, 8, 2, and 2 times more active than ciprofloxacin, levofloxacin, grepafloxacin, and tosufloxacin, respectively. The minimal chlamydiacidal concentration of moxifloxacin ranged from 0.031 to 0.125 microg/ml. These results suggest that moxifloxacin has potential effects against Chlamydia species.

Anti-Infective Agents↗
PubMed1987

Prevalence of antibody to Chlamydia trachomatis in spontaneous abortion and infertility.

A higher prevalence of Chlamydia trachomatis antibody occurred in 57.6% of women with recurrent abortion, but not in their male partners, compared to 33.7% of normal pregnant women (p less than 0.01) and 44.2% of infertile women (NS). The mean titer for the recurrent abortion group was not significantly elevated, compared to controls. Women with blocked fallopian tubes had the highest prevalence of elevated titers (p less than 0.01) and the highest mean titer (p less than 0.001). Despite multiple testing, no women or men were chlamydia culture-positive. The lack of isolation among patients with antibody could be due to cryptic infection at a site not amenable to culture or to inhibition by secretory IgA. There could also be nonspecific stimulation of chlamydial antibody caused by other infections such as mycoplasma. The presence, though at a low level, of antibody in culture-negative patients suggests chlamydia may not be directly associated with recurrent abortion but reflect previous exposure to chlamydia or an altered immune system.

Abortion, Habitual↗
PubMed1991

Cowdria ruminantium is recognized by a monoclonal antibody directed against the major outer membrane protein of Chlamydia trachomatis.

The relationship between Cowdria ruminantium and Chlamydia trachomatis was studied by immunofluorescence. A monoclonal antibody directed against the major outer membrane protein of C. trachomatis recognized rickettsial colonies of C. ruminantium in infected goat brain. No specific fluorescence was observed in non-infected brain. Two commercial Chlamydia-specific monoclonal antibodies as well as polyvalent anti-Chlamydia rabbit serum recognized C. trachomatis, but did not recognize Cowdria. Moreover, polyvalent Cowdria antiserum failed to recognize C. trachomatis cultivated in HeLa cells. It is concluded that Cowdria and Chlamydia are to a certain extent related, confirming similarities in ultrastructure and developmental cycle.

Animals↗
PubMed1995

Chlamydia psittaci infections: a review with emphasis on avian chlamydiosis.

In the first part of this article the general characteristics of Chlamydia psittaci namely the history, taxonomy, morphology, reproductive cycle, metabolism and genetics are reviewed. For the taxonomy in particular, a considerable amount of new information has become available in recent years, following the application of monoclonal antibodies and restriction enzymes. Using these techniques isolates of Chlamydia psittaci from birds have been subdivided in different serovars, a number of isolates have been classified in a new species (Chlamydia pecorum) and isolates from animals have been classified as Chlamydia trachomatis. In the second part of the article, the current knowledge on avian chlamydiosis is summarized. Emphasis is put on clinical signs, lesions, pathogenesis, epizootiology, immunity, diagnosis, prevention and treatment. Also the public health considerations are reviewed. It is concluded that the diagnosis of avian chlamydiosis is laborious and that there is still a need for more accurate, simple and rapid diagnostic tools, both for antigen and antibody detection in various species of birds.

Animals↗
PubMed2006

Effects of sustained antibiotic bactericidal treatment on Chlamydia trachomatis-infected epithelial-like cells (HeLa) and monocyte-like cells (THP-1 and U-937).

Chlamydia trachomatis is a human pathogen that causes multiple diseases worldwide. Despite appropriate therapy with existing antichlamydial antibiotics, chronic exacerbated diseases often occur and lead to serious sequelae. Since C. trachomatis has been found to enter a persistent state after exposure to deleterious conditions, the role of persistence in the failure of chlamydial antibiotherapy is questioned. HeLa, THP-1 and U-937 cells were infected with 10(4)C. trachomatis serovar L2 infectious particles. Three days later the infected cells were treated with minimal bactericidal concentrations of doxycycline (DOX), erythromycin (ERY) or tetracycline (TET) for 24 days or 30 days. Antibiotic efficacy was assessed by measuring chlamydial inclusions and infectious particles, by investigating the resumption of chlamydial growth after antibiotic removal and by testing Chlamydia viability using reverse transcriptase polymerase chain reaction targeting unprocessed 16S rRNA, processed 16S rRNA and Omp-1 mRNA. Treatment of infected HeLa cells with the usual antichlamydial antibiotics suppressed chlamydial active growth. The infection remained unapparent. However, 24 days post treatment the bacterium was found to be viable, as proved by continued expression of unprocessed and processed 16S rRNA and Omp-1 mRNA. This inactive unapparent chlamydial state is not infectious, suggesting Chlamydia persistence. Chlamydia trachomatis also developed persistence both in permissive THP-1 and non-permissive U-937 cells. Unlike in HeLa cells, persistent chlamydial infection in THP-1 and U-937 cells was resolved after 30 days of DOX treatment. Of interest, we noticed that only THP-1 and U-937 cells that were persistently infected following their interaction with infected HeLa cells remained capable of transmitting active infection to HeLa cells. These findings suggest that DOX, TET and ERY, usually administered to combat chlamydial diseases, fail to resolve persistent infection occurring during treatment in non-immune HeLa cells. However, in immune THP-1 and U-937 cells, the persistent infection is resolved by therapy with DOX. Epithelial cells could be the reservoir of persistent chlamydial particles.

Anti-Bacterial Agents↗
PubMed1992

Antibodies to Chlamydia trachomatis in sera of women with recurrent spontaneous abortions.

OBJECTIVE: The relationship between high-titer immunoglobulin G antibodies to Chlamydia trachomatis and recurrent spontaneous abortions was evaluated. STUDY DESIGN: Sera from the female partners of 258 couples with unexplained infertility, no history of chlamydial infection, and negative cervical cultures were diluted 1:128 and tested for immunoglobulin G antibodies to Chlamydia trachomatis. A subset of patients was also tested for antibodies to cytomegalovirus, cardiolipin, nuclear antigens, lactoferrin, and spermatozoa. RESULTS: Seven (41%) of 17 women with three abortions and 6 (60%) of 10 women with four abortions had chlamydial antibodies as opposed to 20 (13.5%) of 148 with no abortions, 6 (12.8%) of 47 with one abortion, and 4 (12.1%) of 33 with two abortions (p < 0.01). The incidence of > or = 3 spontaneous abortions was 31.8% among women with high-titer chlamydial antibodies and 7.5% among women who had seronegative results (p < 0.001). There was no relation between any of the other antibodies and > or = 3 abortions or antibodies to Chlamydia trachomatis. CONCLUSION: High-titer immunoglobulin G antibody to Chlamydia trachomatis was associated with recurrent spontaneous abortions. The mechanism may involve reactivation of a latent chlamydial infection, endometrial damage from a past chlamydial infection, or an immune response to an epitope shared by a chlamydial and a fetal antigen.

Abortion, Habitual↗
PubMed2004

Chlamydia pneumoniae and future risk in patients with coronary heart disease.

AIM: To evaluate the association between previous exposure to Chlamydia pneumoniae and future coronary risk in patients with coronary heart disease. METHODS: A prospective, nested, case-control design was used. The patient sample was derived from a trial study of bezafibrate for the treatment of coronary heart disease. Anti-Chlamydia pneumoniae antibodies (IgG and IgA) in the baseline sera of 136 patients who had coronary events during follow-up (mean 6.2 years) were compared with those in 136 age- and gender-matched patients from the same trial without subsequent coronary events. RESULTS: Mean titers of IgG and IgA antibodies were similar in cases and controls. The relative odds of future coronary events in patients who were seropositive at baseline were 1.0 (95% CI, 0.54-1.84) for IgG and 0.74 (95% CI, 0.41-1.31) for IgA. The relative odds did not change after adjustment for multiple confounding variables. The risk of future coronary events did not increase with increasing anti-Chlamydia pneumoniae antibody titers. CONCLUSIONS: Prior exposure to Chlamydia pneumoniae in patients with chronic coronary heart disease is not associated with increased risk of recurrent coronary events.

Antibodies, Bacterial↗
PubMed1996

[Clinical manifestations of Chlamydia pneumoniae infections].

Chlamydia pneumoniae is a newly described and ubiquitous bacterium. Most infections are asymptomatic as shown by a high worldwide seroprevalence (> 50% of cases). It is a common cause of acute respiratory infections, mainly pneumonia (> 50% of cases) and other acute respiratory tract infections (25% of acute bronchitis, < 5% of sinusitis, otitis and pharyngitis). About 10% of the community acquired pneumonia cases have been associated with Chlamydia pneumoniae infection. This incidence depends on a cyclic epidemiology with a high incidence for 2 to 3 years followed by a low prevalence for 3 to 4 years. Most chlamydial infections are mild but occasionally severe with death especially in old people. Mostly acute infections are recurrent infections. The seroprevalence is higher in asthmatic patients, its role in acute exacerbation of chronic bronchitis is not definitely established. Extra-respiratory acute infections are less frequent, either fever alone, or cardiovascular diseases (acute myocarditis, pericarditis and endocarditis) or neurological (encephalitis, meningitis or Guillain-Barré syndrome). In addition, seroepidemiology studies have shown an association with coronary artery disease, Chlamydia pneumoniae was detected in coronary atheroma by immunochemistry, polymerase chain reaction and by electron microscopy. Chlamydia pneumoniae may be involved in the atherosclerotic process. To define the clinical spectrum of infection requires precise laboratory diagnosis, the most efficient tests (PCR, direct immunofluorescence and culture) are done in specialized laboratories, serological tests are less reliable. Macrolides, cyclines and fluoroquinolones are the most potent antibiotics but with differences in vitro within and between these families of antibiotics. Bacteriological failures are described despite the in vitro activity. A lot of questions on clinical aspects, epidemiology and treatment are unanswered, we need more studies.

Anti-Bacterial Agents↗
PubMed1997

Detection of Chlamydia pneumoniae in human nonrheumatic stenotic aortic valves.

OBJECTIVES: We sought to study the possible presence of Chlamydia pneumoniae in aortic valve stenosis (AVS). BACKGROUND: Inflammation and immune mechanisms are considered important for the pathogenesis of nonrheumatic AVS. All chlamydial species are able to cause heart infections, and seroepidemiologic studies have indicated an association between chronic C. pneumoniae infection and coronary artery disease. Furthermore, the organism has been demonstrated in atherosclerotic lesions. METHODS: Aortic valve specimens with varying degrees of macroscopic disease were obtained from 35 subjects--17 consecutive patients undergoing aortic valve replacement for treatment of nonrheumatic AVS and 18 age-matched subjects at autopsy. The possible presence of C. pneumoniae in aortic valves was studied by immunohistochemical analysis, polymerase chain reaction or transmission electron microscopy, or a combination of these. RESULTS: Positive immunohistochemical staining with C. pneumoniae specific antibody was found in 9 (53%) of 17 patients with advanced aortic valve disease requiring surgical treatment (group A), 8 (80%) of 10 cadavers with clearly macroscopic aortic valve pathology (group B) and 1 (12%) of 8 grossly normal cadaver control subjects (group C). Statistical significance with regard to the presence of C. pneumoniae was found when combined diseased subjects (groups A and B: total 17 of 27 subjects) were compared with group C (p = 0.018). However, when group A was compared with group C, there was only marginal statistical significance (p = 0.088). Finally, there was a strong statistical significance (p = 0.015) when groups B and C were compared. Chlamydia pneumoniae DNA was also found in three stenotic valves, and in two of the three tested valve specimens chlamydia-like particles were seen by electron microscopy. CONCLUSIONS: Chlamydia pneumoniae is frequently present in nonrheumatic AVS. Similarly, the high number of C. pneumoniae infections detected in the early lesions of "degenerative" AVS suggest that this pathogen may play an etiologic role in the development of this disease. The validity of this relation requires additional study.

Aged↗