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PubMed1995

Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells.

Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inclusion membrane protein A) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.

Amino Acid Sequence↗

PubMed2006

Multifunctional analysis of Chlamydia-specific genes in a yeast expression system.

Our understanding of how obligate intracellular pathogens co-opt eukaryotic cellular functions has been limited by their intractability to genetic manipulation and by the abundance of pathogen-specific genes with no known functional homologues. In this report we describe a gene expression system to characterize proteins of unknown function from the obligate intracellular bacterial pathogen Chlamydia trachomatis. We have devised a homologous recombination-based cloning strategy to construct an ordered array of Saccharomyces cerevisiae strains expressing all Chlamydia-specific genes. These strains were screened to identify chlamydial proteins that impaired various yeast cellular functions or that displayed tropism towards eukaryotic organelles. In addition, to identify bacterial factors that are secreted into the host cell, recombinant chlamydial proteins were screened for reactivity towards antisera raised against vacuolar membranes purified from infected mammalian cells. We report the identification of 34 C. trachomatis proteins that impact yeast cellular functions or are tropic for a range of eukaryotic organelles including mitochondria, nucleus and cytoplasmic lipid droplets, and a new family of Chlamydia-specific proteins that are exported from the parasitopherous vacuole. The versatility of molecular manipulations and protein expression in yeast allows for the rapid construction of comprehensive protein expression arrays to explore the function of pathogen-specific gene products from microorganisms that are difficult to genetically manipulate, grow in culture or too dangerous for routine analysis in the laboratory.

Bacterial Proteins↗

PubMed2006

Chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection.

Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of diseases in humans. C. trachomatis has a complex developmental cycle that depends on host cells for replication, during which gene expression is tightly regulated. Here we identify two C. trachomatis proteases that possess deubiquitinating and deneddylating activities. We have designated these proteins ChlaDub1 and ChlaDub2. The genes encoding ChlaDub1 and ChlaDub2 are present in all Chlamydia species except for Chlamydia pneumoniae, and their catalytic domains bear similarity to the catalytic domains of other eukaryotic ubiquitin-like proteases (Ulp). The C. trachomatis DUBs react with activity-based probes and hydrolyse ubiquitinated and neddylated substrates. ChlaDub1 and ChlaDub2 represent the first known bacterial DUBs that possess both deubiquitinating and deneddylating activities.

Amino Acid Sequence↗

PubMed2006

Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle.

The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.

Bacterial Proteins↗

PubMed2004

Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells.

Chlamydiae are gram-negative obligate intracellular pathogens to which access to an intracellular environment is paramount to their survival and replication. To this end, chlamydiae have evolved extremely efficient means of invading nonphagocytic cells. To elucidate the host cell machinery utilized by Chlamydia trachomatis in invasion, we examined the roles of the Rho GTPase family members in the internalization of chlamydial elementary bodies. Upon binding of elementary bodies on the cell surface, actin is rapidly recruited to the sites of internalization. Members of the Rho GTPase family are frequently involved in localized recruitment of actin. Clostridial Toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells. Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA. Rac but not Cdc42 was found to be activated by chlamydial attachment. The effect of dominant negative Rac expression on chlamydial uptake is manifested through the inhibition of actin recruitment to the sites of chlamydial entry. Studies utilizing Green Fluorescent Protein fusion constructs of Rac, Cdc42 and RhoA, showed Rac to be the sole member of the Rho GTPase family recruited to the site of chlamydial entry.

Actins↗

PubMed1994

Prevalence of Chlamydia pneumoniae in acute lower respiratory infection in the pediatric population in Japan.

Chlamydia pneumoniae has been established as an important etiologic agent of acute respiratory tract infection in humans, especially in adults. However, there is little information available on C. pneumoniae infection in the pediatric age group. The microimmunofluorescence test and Chlamydia pneumoniae-specific polymerase chain reaction (PCR) method were applied to reveal the role of C. pneumoniae as an etiologic agent of acute lower respiratory tract infection in children in Japan. Among 136 patients aged between 2 months and 15 years with acute lower respiratory tract infection, five patients with pneumonia were found to have recent C. pneumoniae infection by the microimmunofluorescence test. C. pneumoniae-specific PCR products were detected from specimens of four patients among these five. Neither C. trachomatis nor C. psittaci infection was found in this population. These results suggest that C. pneumoniae is an important causative agent of acute childhood pneumonia and may be the most prevalent pathogen among the genus Chlamydia that causes acute lower respiratory disease in this age group in Japan.

Acute Disease↗

PubMed2006

An early event in the herpes simplex virus type-2 replication cycle is sufficient to induce Chlamydia trachomatis persistence.

Epidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Transmission electron microscopic (TEM) analyses demonstrated that in both HeLa and HEC-1B cells, co-infection caused developing chlamydiae to exhibit swollen, aberrantly shaped reticulate bodies (RBs), characteristically observed in persistence. Additionally, HSV-2 co-infection suppressed production of infectious chlamydial elementary bodies (EBs) in both host cell types. Co-infection with HSV type 1 (HSV-1) produced similar morphologic alterations and abrogated infectious EB production. These data indicate that virus-induced chlamydial persistence was neither host cell- nor virus strain-specific. Purification of crude HSV-2 stocks demonstrated that viral particles were required for coinfection-induced chlamydial persistence to occur. Finally, co-infection with either UV-inactivated, replication-incompetent virus or replication-competent HSV-2 in the presence of cyclohexamide reduced chlamydial infectivity without altering chlamydial genomic DNA accumulation. These data demonstrate that productive viral replication is not required for the induction of chlamydial persistence and suggest that HSV attachment and entry can provide the necessary stimulus to alter C. trachomatis development.

Animals↗

PubMed2000

Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex.

The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH.

Amino Acid Sequence↗

PubMed2002

A Chlamydia pneumoniae infection model using established human lymphocyte cell lines.

Since current studies indicate possible infection of human lymphocytes with Chlamydia (Chlamydophila) pneumoniae, establishment of an in vitro C. pneumoniae infection model using lymphocyte cell lines was demonstrated. Human lymphoid cell lines (Molt 4 [T-cell] and P3HR1 [B-cell]) were utilized for this purpose besides human monocyte cell line (THP-1) and human epithelial cell line (HEp-2), as a reference of monocyte/macrophage cells and a positive control for support of C. pneumoniae growth, respectively. Both lymphoid cells (Molt 4 and P3HR1) supported the growth of C. pneumoniae as demonstrated by Chlamydia inclusion formation, detection of increased infective progenies and increased bacterial antigen levels. Similar data were obtained using monocyte THP-1 cells. However, the bacterial growth in these cells was less than that in HEp-2 cells. The electron microscopic study showed typical inclusions with many Chlamydia elementary bodies in lymphoid cells tested, similar to that seen in HEp-2 cells. These results indicate that C. pneumoniae can infect cells with lymphocyte properties and this infection model with lymphoid cell line cells could be valuable to study details of lymphocyte-C. pneumoniae interaction.

Cell Line↗

PubMed1981

Maternal and infantile infection with Chlamydia in a Swedish population.

Chlamydia trachomatis was isolated from 2.4% of 1 328 puerperal women. The frequency was highest in the age group below 20 years and thereafter decreased with increasing age. Chlamydial conjunctivitis was confirmed in 0.4% of the infants. Two additional cases of conjunctivitis occurred among the exposed infants but chlamydia cultures were not obtained. In a separate ophthalmological material of neonatal conjunctivitis a third of the cases developing within the first month of life was associated with chlamydia. The early and sharp incidence peak for chlamydial conjunctivitis suggested that transmission occurred at delivery. No cases of chlamydial pneumonia were noted. Peroral chemotherapy is recommended in infants for systemic eradication of C. trachomatis.

Adult↗

PubMed1991

[Detection of Chlamydia trachomatis by polymerase chain reaction].

A polymerase chain reaction (PCR) procedure was developed for detection of Chlamydia trachomatis. Two oligonucleotide primers based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used. A single DNA fragment was amplified, when C. trachomatis DNA was template for the PCR. No amplified product was detected in Chlamydia psittaci DNA, Chlamydia pneumoniae DNA or other bacterial DNAs. The amplified DNA fragment was detected, when DNA of greater than or equal to 10(2) C. trachomatis per reaction was used as template for the PCR. Thus, the PCR was shown to be specific for C. trachomatis and more sensitive than the enzyme immunoassays for detection of chlamydial antigen and the chlamydial rRNA:DNA probe hybridization method.

Chlamydia trachomatis↗

PubMed1992

[Isolation of Chlamydia pneumoniae from a patient with acute bronchitis].

Chlamydia was isolated from the throat of a 15-year-old male patient with acute bronchitis. The Chlamydia isolate, YK-41, was stained with FITC-conjugated monoclonal antibodies specific to C. pneumoniae and the genus Chlamydia, whereas staining of monoclonal antibody specific to C. trachomatis was negative. These results indicated that the strain YK-41 could be identified as C. pneumoniae. Serum IgM antibody against C. pneumoniae was detected in high titer in the patient in the acute phase using the microimmunofluorescence (MIF) test, serum IgG antibody against C. pneumoniae demonstrated a fourfold antibody titer rise between acute and convalescent serum using MIF test. Thus, our patient almost certainly contracted acute bronchitis caused by C. pneumoniae.

Acute Disease↗

PubMed1993

[Detection of Chlamydia pneumoniae by polymerase chain reaction].

We aimed at developing a method for early detection of Chlamydia pneumoniae (C. pneumoniae) from clinical specimens. For this purpose, we amplified C. pneumoniae-specific DNA fragments by polymerase chain reaction. A pair of 24 mer of oligonucleotides which were complementary to the sequences within the region encoding the major outer membrane were synthesized and used as primers. As the results, all three standard strains of C. pneumoniae (AR-39, TW-183 and AR-388) were identified by the detection of the amplified products of 174 base pairs, while each strain of Chlamydia trachomatis and Chlamydia psittaci and a total of 11 strains of bacteria and a strain of Mycoplasma pneumoniae were not. Thus, the present method was found to provide a very high specificity and also a high sensitivity of a detectable level of 10 x 10(-9) inclusion-forming units.

Base Sequence↗

PubMed1998

Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis.

Analysis of the 1,042,519-base pair Chlamydia trachomatis genome revealed unexpected features related to the complex biology of chlamydiae. Although chlamydiae lack many biosynthetic capabilities, they retain functions for performing key steps and interconversions of metabolites obtained from their mammalian host cells. Numerous potential virulence-associated proteins also were characterized. Several eukaryotic chromatin-associated domain proteins were identified, suggesting a eukaryotic-like mechanism for chlamydial nucleoid condensation and decondensation. The phylogenetic mosaic of chlamydial genes, including a large number of genes with phylogenetic origins from eukaryotes, implies a complex evolution for adaptation to obligate intracellular parasitism.

Aerobiosis↗

PubMed1984

Comparison of the in vitro activities of ofloxacin and tetracycline against Chlamydia trachomatis as assessed by indirect immunofluorescence.

The in vitro activity of ofloxacin (DL8280), a new pyridone carboxylic acid, against Chlamydia trachomatis serotype E was assessed by growth of chlamydia in cycloheximide-treated McCoy cells. Chlamydia-infected monolayers were exposed to the drug for 48 and 96 h and for 48 h followed by 48 h of further incubation after withdrawal of ofloxacin. Inclusions were visualized by indirect immunofluorescence, and the effect of the drug was assessed both by inclusion development and by the number of inclusion-forming units generated in the monolayers. The action of ofloxacin was completely lethal when monolayers were exposed to a concentration of 1 microgram/ml throughout one chlamydial developmental cycle. In contrast, tetracycline, when examined by similar techniques, inhibited inclusion development at a concentration of 0.3 micrograms/ml, but was only lethal at 2.4 micrograms/ml. Ofloxacin may prove to be a useful new antichlamydial agent.

Anti-Bacterial Agents↗

PubMed1985

Inhibition of Chlamydia trachomatis growth in McCoy, HeLa, and human prostate cells by zinc.

Zinc salts (10(-4) and 10(-5) M) inhibited the number of Chlamydia trachomatis inclusions in McCoy, HeLa, and primary human prostate epithelial cell cultures. Addition of zinc salts 1 h before or 24 h after inoculation with C. trachomatis was found to inhibit growth. Both C. trachomatis serotype D and a lymphogranuloma venereum strain were inhibited by the zinc salts. Although the mechanism of inhibition is not known, the continued presence of the zinc appeared necessary for maximal effect. At the concentrations tested, zinc was not directly toxic to the McCoy cells. These results suggest that the levels of zinc in prostatic secretions may be sufficient to preclude the recovery of chlamydia in the diagnostic laboratory or to inhibit chlamydia from infecting the prostate in vivo.

Cell Line↗

PubMed1973

Use of gentamicin in the isolation of subgroup A Chlamydia.

The use of gentamicin to control contamination in a tissue culture system for the isolation of Chlamydia was investigated. Gentamicin, at concentrations up to 100 mug/ml, did not appear to inhibit the growth of stock chlamydial strains, as judged by assays for iodine-staining inclusions. When 343 cervical and urethral specimens were examined in the presence and in the absence of gentamicin, significantly more isolations of chlamydiae were obtained in the presence of 10 mug of gentamicin/ml, and significantly fewer unsatisfactory results due to contaminating organisms were seen. Therefore, this aminoglycoside antibiotic appears to be a useful addition to a cell system for isolation of Chlamydia.

Chlamydia↗

PubMed1992

In vitro activity of nonoxynol 9 on HeLa 229 cells and primary monkey cervical epithelial cells infected with Chlamydia trachomatis.

Nonoxynol 9 (non-9) is the active ingredient in a wide variety of vaginal contraceptive preparations. The manufacturer recommendation for optimal contraceptive practice is repeated application every 6 h. We studied the in vitro activity of non-9 against Chlamydia trachomatis (E/UW-5/Cx) and its toxicity against HeLa 229 cells and monkey cervical epithelial cells. With a contact time of 6 h, non-9 was toxic to HeLa cells at concentrations of 50 micrograms/ml or greater and to monkey cervical cells at 100 micrograms/ml or greater. Inhibitory effects of non-9 on extracellular chlamydiae were observed at concentrations of 50 micrograms/ml or greater. Inhibition of intracellular growth of chlamydiae in monkey cervical cells was observed at a nontoxic concentration of 50 micrograms/ml. Our studies show that non-9 has antichlamydial activity. However, owing to its toxicity to cervical cells in vitro, the effects of prolonged use of non-9 in vivo should be further studied.

Animals↗