Cyclic AMP inhibits protein synthesis in Chlamydia trachomatis at a transcriptional level.

Cyclic AMP (cAMP) has an inhibitory effect on the developmental cycle of Chlamydia trachomatis. We examined its influence on the synthesis of chlamydial protein, using the major outer membrane protein (MOMP) as a marker for general chlamydial protein synthesis. During normal development MOMP synthesis accelerates from 18 h post-infection and peaks by 36 h. Cyclic AMP blocks this normal progression of the chlamydial growth cycle. At a concentration of 1 mM, nearly 75% of the total MOMP synthesis was inhibited by 36 h, as monitored by radiolabel uptake. However, no difference was observed during the first 12 h between cAMP-treated and control groups, a finding which is in keeping with correlation between developmental inhibition and protein synthesis. Hybridization studies carried out with a cloned MOMP gene demonstrate a drastic decrease in MOMP mRNA in cAMP-treated cells. Low levels of cAMP utilized in conjunction with a 100,000 x g supernatant from reticulate bodies (RBs) blocked the transcription of the recombinant MOMP gene in an in vitro transcription system. These results suggest that the inhibition of chlamydial protein synthesis, assessed by MOMP synthesis, is due to regulation at a transcriptional level.

Detection of Chlamydia pneumoniae DNA in CD3+ lymphocytes from healthy blood donors and patients with coronary artery disease.

BACKGROUND: Chlamydia pneumoniae is an intracellular bacterium responsible for respiratory tract infections. Recent studies have implicated this organism in the pathogenesis of atherosclerosis. METHODS AND RESULTS: To address how the organism is transported from lungs to cardiac vessels, we characterized the cell population within peripheral blood mononuclear cells (PBMCs) that harbor C pneumoniae DNA. Adherent and nonadherent PBMCs from 28 patients with coronary artery disease (CAD) and 19 healthy blood donors were evaluated for the presence of C pneumoniae DNA by touchdown nested polymerase chain reaction (nPCR). Of the 28 patients, 10 (36%) had detectable PCR product in their nonadherent and 3 (10%) in their adherent PBMC population. C pneumoniae-specific PCR results were positive for 5 of 19 (26%) healthy blood donors. PCR positivity was detected only in the nonadherent cell population among this group of individuals. Fractionation of nonadherent PBMCs identified C pneumoniae-specific PCR signal among the CD3+ T-cell population exclusively. Of the 18 PCR-positive subjects (13 patients and 5 healthy control subjects), 67% (8 patients and 4 healthy blood donors) tested positive for C pneumoniae-specific IgG serology. Interestingly, 2 patients became PCR negative on a repeated blood draw 5 months after initial detection of C pneumoniae DNA despite retaining C pneumoniae-specific antibodies. CONCLUSIONS: Our results demonstrate marginally significant prevalence of C pneumoniae DNA in patients with CAD compared with healthy subjects (P=0.082). In contrast, the prevalence of IgG seropositivity among the 2 groups did not reach statistical significance (P=0.306). We also provide unequivocal evidence for the presence of C pneumoniae DNA predominantly among the circulating CD3+ T-cell population.

Interspecies structural diversity among chlamydial genes encoding histone H1.

Recently, a eukaryotic histone H1-like protein has been detected in Chlamydia trachomatis serovar L2 [Hackstadt et al., Proc. Natl. Acad. Sci. USA 88 (1991) 3937-3941; Tao et al., J. Bacteriol. 173 (1991) 2818-2822]. We have cloned the corresponding gene from C. trachomatis serovar J and the Chlamydia psittaci strain mn. Sequencing demonstrated absolute gene identity between the two C. trachomatis serovars L2 and J, but divergence in the C. psittaci strain mn. These differences resulted in altered aa residues (in particular no cysteines) and a smaller molecular mass for H1 from C. psittaci strain mn. The amino acid (aa) sequence comparisons with other histone proteins show best alignment to sea urchin H1, notably in the C terminus, for both C. trachomatis and C. psittaci histones. Chlamydial interspecies aa homology, however, is most conserved at the N terminus, suggestive of a bi-functional role for these unique histone proteins.

Three tandemly repeated 5S ribosomal RNA-encoding genes identified, cloned and characterized from Cryptosporidium parvum.

We have characterized the 5S rRNA of Cryptosporidium parvum. The gene (rDNA) encoding this 5S rRNA was identified, mapped, the primary and secondary structures determined, and the copy number estimated. Using a PCR-amplified 5S rDNA as a probe, it was shown that this gene can specifically recognize C. parvum genomic DNA, but not other intestinal and environmental organisms tested. Three repeat units of the 5S rDNA found in genomic C. parvum oocyst DNA are within the 2012-bp EcoRI-HindIII fragment and are identical in coding sequence, but differ in flanking regions. Flanking regions are A+T rich (78-89%). The termination signal for polymerase III consists of five thymidine residues at the 3' end of each of three units.

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability.

The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.

Eukaryotic-like histones in Chlamydia.

A fundamental process in all organisms is their ability to regulate gene expression in response to developmental and environmental signals. In Chlamydia, changes in gene expression are closely linked to the presence or to undetectability of eukaryotic-like histones observed late in the parasites life cycle. It is becoming increasingly clear that these histone-like proteins are involved in macromolecular confirmation of DNA. However, their functional role(s) in chlamydial development and the underlying mechanism(s) involved in their degradation and dissociation are largely unknown. It is not surprising therefore that eukaryotic-like histones are a focus of intense research in several laboratories around the world. Recent studies on the interaction of eukaryotic- like histones with DNA, the role of phosphorylation and identification of a histone specific protease are beginning to unravel the mechanism of stage specific differentiation and gene expression in Chlamydia. In this article we review recent advances on the eukaryotic-like histones that have set the stage for elucidation of the chlamydial developmental cycle.

Geographical variation in 18S rRNA gene sequence of Cryptosporidium parvum.

Using 18S rRNA as a probe, an EcoR1 fragment containing 1507 nt of 18S rRNA from C. parvum was identified, cloned and sequenced. Comparison of this sequence with the partial sequence of the small subunit rRNA of Cryptosporidium published by Johnson, Fielke, Lumb & Baverstock (International Journal for Parasitology 20: 141-147, 1990) and 1516 bp of the 18S rRNA nucleotide sequence of C. parvum published by Cai, Collins, McDonald & Thompson (Biochimica et Biophysica Acta, 1131: 317-320, 1992) revealed 97% and 91.6% sequence homology, respectively. These data suggest that differences exist among the same species of Cryptosporidium from different geographical areas.

Adenovirus bronchiolitis in Manitoba: epidemiologic, clinical, and radiologic features.

We reviewed our experience with 41 children hospitalized from 1974 to 1978 for adenovirus (ADV) bronchiolitis. Thirty-two patients (78 percent) were native Indians between four and 12 months old. In 18 of the 41 patients (43.9 percent) acute complications developed. The five fatal cases (12.2 percent) were confined to native children. The initial chest roentgenograms showed lobar consolidation in 35 patients (85.4 percent). Atelectasis developed in five (12.2 percent) during hospitalization. Sixteen of 25 patients (64 percent) with adequate radiologic follow-up examination had subsequent pneumonias or showed residual chronic changes. The reasons for the predilection of ADV bronchiolitis in native Indian children and the precise effect on subsequent airway function in survivors are unknown and require further study. We emphasize the importance of ADV as a cause of bronchiolitis in native Indian children. Furthermore, this report focuses attention on the contribution of this disease to the spectrum of chronic pulmonary disorders in the pediatric group.

In vitro activity of rifaximin, a topical rifamycin derivative, against Chlamydia trachomatis.

Rifaximin is a rifamycin derivative that possesses in vitro activity against a wide range of bacteria. Its antimicrobial spectrum plus poor intestinal absorption have led to consideration of this compound as a topical agent. We evaluated its in vitro activity against clinical and laboratory strains of Chlamydia trachomatis and found that rifaximin exhibits minimum inhibitory concentrations (MICs) at concentrations that would be greatly exceeded in a topical preparation.

In vitro activity of dirithromycin, a new macrolide antibiotic, against Mycoplasma species.

Dirithromycin is a new macrolide antibiotic that achieves high tissue concentration. We compared its in vitro activity against Mycoplasma species with that of erythromycin and tetracycline. Clinical isolates of M. pneumoniae (40), M. hominis (40), and Ureaplasma urealyticum (40) were tested against serial dilutions of three antibiotics using a microtiter plate method. Minimum inhibitory concentrations (MIC) were read as the lowest concentration of antibiotic yielding no color change in the broth. Neither macrolide antibiotic exhibited antimicrobial activity against M. hominis; MIC50 and MIC90 for tetracycline were 0.6 and 32 micrograms/ml, respectively. MIC50 for U. urealyticum was 4.0 micrograms/ml for dirithromycin, 2.0 micrograms/ml for erythromycin, and 1.0 micrograms/ml for tetracycline. MIC90 for U. urealyticum was > 128 micrograms/ml for all three agents. Against M. pneumoniae dirithromycin exhibited MIC50 of 0.1 micrograms/ml and MIC90 of 0.1 micrograms/ml. Both values for erythromycin were 0.2 micrograms/ml; for tetracycline they were 0.1 and 1.0 micrograms/ml, respectively. These results demonstrate the high in vitro activity of dirithromycin against M. pneumoniae and suggest that this agent may have a role in the treatment of respiratory Mycoplasma infections.

Initial characterization of a chlamydial receptor on mammalian cells.

We have examined characteristics of the binding of eukaryotic cells to chlamydial elementary body (EB)-specific proteins. A wide variety of eukaryotic cell lines bound to representatives of both Chlamydia trachomatis lymphogranuloma venereum (LGV) and trachoma biovars and a C. psittaci strain meningopneumonitis (Mn) suggesting the presence of a common host cell receptor. Neither tunicamycin nor neuraminidase treatment of HeLa cells impaired binding to C. trachomatis EB, implying that host cell N-linked carbohydrate domains and sialic acid moieties, respectively, are not involved in attachment. However, trypsinized HeLa cells do not bind to EB, suggestive of a proteinaceous host cell receptor. The trypsin sensitivity of two EB-specific binding proteins Mr = 18,000 and 31,000) was also examined, and the finding that 125I-labeled HeLa cells bind both the 18,000 and 31,000-dalton proteins after chlamydial trypsinization corroborates our earlier observation that these EB binding proteins mediate attachment.

Molecular epidemiology of Candida albicans colonization and fungemia in very low birthweight infants.

OBJECTIVE: This study investigated the relationship between colonization and fungemia. DESIGN: This was a prospective study involving surveillance cultures of the nares, base of umbilicus, point of entry of umbilical catheter and parenteral fluids. Blood cultures were done when sepsis was suspected. All Candida albicans isolates were typed using restriction enzyme analysis of DNA. SETTING: Patients were from the neonatal intensive care unit of a tertiary care hospital. POPULATION STUDIED: Twenty-nine very low birthweight infants. MAIN RESULTS: Eleven babies were colonized with C albicans and five of these babies developed fungemia, including five of seven who were colonized at the point of entry of the umbilical catheter. Three different strains of C albicans caused fungemia. In four of the five patients, initial catheter entry site isolates were identical to the subsequent blood isolates. Occasionally, infants were colonized with more than one strain of C albicans. CONCLUSIONS: Preceding colonization with C albicans and, in particular, colonization at the site of entry of umbilical vascular catheters are risk factors for subsequent development of C albicans fungemia. Fungemic and colonizing isolates are usually identical to one another by DNA typing.

The effect of ions on the adhesion and internalization of Chlamydia trachomatis by HeLa cells.

The adhesion and internalization of Chlamydia trachomatis by HeLa cells was unaffected by removal of K+, Mg2+, or glucose from the incubation medium, slightly reduced by removal of Na+, and significantly reduced by omission of Ca2+, Sr2+, Mg2+, and Mn2+ could replace Ca2+ in the adhesion but only Sr2+ supported internalization, and La3+, Co2+, Fe3+, Ba2+, and Zn2+ all reduced internalization more than adhesion. During initial infection there was no measurable difference in the uptake or release of 45Ca2+ or 86Rb+ between infected and noninfected HeLa monolayers. Infection was not prevented by pretreatment of the monolayers with the calcium channel blockers, verapamil, D600, and nitrendipine, or the calmodulin inhibitors, TMB-8 or trifluperazine. The results suggest that divalent cations are not essential for chlamydial infection but that the process of internalization is facilitated by the presence of cations, particularly Na+ and Ca2+.

Cyclic AMP inhibits developmental regulation of Chlamydia trachomatis.

The effect of cyclic AMP (cAMP) on the chlamydial growth cycle was studied with Chlamydia trachomatis-infected HeLa cells. At concentrations of 1 mM, cAMP had a profound effect on the chlamydial developmental cycle, resulting in small, immature inclusions. Immunoblot analysis revealed the absence of elementary body (EB)-specific antigens in the cAMP-treated cells. This effect was observed only if cAMP was added within the first 12 h of incubation and continued thereafter. Its withdrawal at any time from the medium led to the reappearance of fully mature, infectious organisms. Analogs or breakdown products of cAMP exerted no inhibitory effect on chlamydial development. Intracellular inclusions from the cAMP-treated cells were unable to infect fresh HeLa monolayers, in contrast to the completely infectious nontreated inclusions. Protein profiles of the cAMP-treated organisms (at any time point) resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis very closely resembled reticulate bodies (RB) and did not possess characteristic EB-binding proteins. Collectively, these observations suggest an inhibitory role for cAMP at the RB stage of intracellular development. We also identified a cAMP receptor protein which is associated with RB and not with EB, further supporting a role for this system in the developmental regulation of chlamydiae.

Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes.

Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L2 were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. Our data suggest that these putative chlamydial adhesins play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.

Chlamydia pneumoniae facilitates monocyte adhesion to endothelial and smooth muscle cells.

Chlamydia pneumoniae has been linked to atherosclerotic heart disease. However, there is a limited knowledge by which C. pneumoniae gain access to atheromatous lesions. The adhesion of C. pneumoniae -infected circulatory component(s) to endothelium and smooth muscle cells represents the first step in an inflammatory response. We examined the ability of viable as well as heat inactivated C. pneumoniae to infect human monocytes and subsequently the ability of infected monocytes to adhere to human coronary artery endothelial cells (HCAEC) and human coronary smooth muscle cells (HCSMC). Our results demonstrate susceptibility of monocytes to in vitro chlamydial infection. Inclusions of varying sizes and intensities were observed 3-5 days after inoculation with viable C. pneumoniae. Monocytes infected with heat inactivated organisms revealed no inclusions, in keeping with the observations of uninfected monocytes. Moreover, monocytes infected with viable C. pneumoniae adhered preferentially to HCAEC and HCSMC, as compared to uninfected monocytes or monocytes harbouring heat inactivated Chlamydia.

Surgical and postoperative management of two neonates with necrotizing fasciitis.

Two neonates who presented with necrotizing fasciitis (NF) secondary to omphalitis were treated by radical excision of the anterior abdominal wall. Postoperatively, renal failure in both children was managed by continuous arteriovenous hemofiltration (CAVH). One child survived; the other died of sepsis. Serum lactate levels, which were 10 to 15 times the normal levels in both infants postoperatively, decreased rapidly in the survivor, but never approached normal levels in the infant who died. Although the best prospect for survival in neonatal NF remains prompt, radical, surgical excision and is associated with a low threshold for repeat débridement, modern supportive measures (including CAVH) may enhance survival.

Cloning, expression, and primary structure of a Chlamydia trachomatis binding protein.

The gene encoding an 18,000-dalton eucaryotic cell-binding protein of Chlamydia trachomatis serovar L2 was cloned into Escherichia coli, and the nucleotide sequence of a 1,658-base-pair PstI restriction endonuclease fragment encoding this protein was determined. The recombinant chlamydial gene consists of a 486-base-pair open reading frame encoding a polypeptide of molecular weight 18,314. The resultant polypeptide, comprising 162 amino acids, possesses a highly charged carboxy-terminal end. The expression of this recombinant protein is under the control of a vector promoter. The recombinant 18,000-dalton protein possessed the same eucaryotic cell-binding characteristics as did the native chlamydial 18,000-dalton protein when electrophoresed and transferred to nitrocellulose. Polyclonal antibodies to the recombinant protein exhibited neutralizing activity.