Biomedical subjects

W M Wenman

Publications and source records attributed to W M Wenman.

At least 19 recordsLinked to original sources

PubMed1998

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.

Animals↗

PubMed1997

The chlamydial EUO gene encodes a histone H1-specific protease.

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.

Amino Acid Sequence↗

PubMed1996

Sequence specific binding of chlamydial histone H1-like protein.

Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear homology to eukaryotic histone H1. Changes in macromolecular conformation of DNA mediated by the histone H1-like protein (Hc1) appear to regulate stage specific differentiation. We have developed a cross-linking immunoprecipitation protocol to examine in vivo protein-DNA interaction by immune precipitating chlamydial Hc1 cross linked to DNA. Our results strongly support the presence of sequence specific binding sites on the chlamydial plasmid and hc1 gene upstream of its open reading frame. The preferential binding sites were mapped to 520 bp BamHI-XhoI and 547 bp BamHI-DraI DNA fragments on the plasmid and hc1 respectively. Comparison of these two DNA sequences using Bestfit program has identified a 24 bp region with >75% identity that is unique to the chlamydial genome. Double-stranded DNA prepared by annealing complementary oligonucleotides corresponding to the conserved 24 bp region bind Hc1, in contrast to control sequences with similar A+T ratios. Further, Hc1 binds to DNA in a strand specific fashion, with preferential binding for only one strand. The site specific affinity to plasmid DNA was also demonstrated by atomic force microscopy data images. Binding was always followed by coiling, shrinking and aggregation of the affected DNA. Very low protein-DNA ratio was required if incubations were carried out in solution. However, if DNA was partially immobilized on mica substrate individual strands with dark foci were still visible even after the addition of excess Hc1.

Bacterial Proteins↗

PubMed1996

Functional domains of chlamydial histone H1-like protein.

Chlamydial trachomatis is one of the few prokaryotic organisms known to contain proteins that bear amino acid similarity to eukaryotic histone H1. It is also appreciated that chlamydial histone-like proteins, designated Hc1 and Hc2, can bind DNA and are presumably involved in the condensation of infectious elementary bodies. However, there is no information on either the orientation of Hc1 and Hc2 or the mechanism of their DNA-protein and protein-protein interactions. Whereas the C-terminal domain of Hc1 between amino acids 63 and 125 shows best alignment with sea-urchin histone H1, and N-terminus between amino acids 1 and 62 is highly conserved among various chlamydial species, suggesting a bifunctional role for this unique protein. In order to delineate the regions responsible for the Hc1 characteristics, we have expressed these two fragments independently in Escherichia coli and studied the binding of double-stranded DNA to either whole Hc1 protein or its two termini. Our results support the role of the carboxyl portion in DNA-protein interaction, a function similar to its eukaryotic counterpart. Although this interaction initiates DNA condensation in the absence of the N-terminal domain, it is not sufficient to produce complete compaction. Intra- or inter-molecular protein-protein interactions may be necessary to achieve such an effect.

Amino Acid Sequence↗

PubMed1995

Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis.

We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial alpha subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.

Amino Acid Sequence↗

PubMed1994

Cloning and characterization of a secY homolog from Chlamydia trachomatis.

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc-alpha ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50,195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SecY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.

Amino Acid Sequence↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Alberta↗

PubMed1994

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.

Anti-Bacterial Agents↗

PubMed1994

Identification of low-molecular-mass coproantigens of Cryptosporidium parvum.

Cryptosporidium parvum coproantigens (CCAg) of 18 and 20 kDa were identified in the stool eluates of calves and humans infected with Cryptosporidium species. Monospecific antibodies raised against the 20-kDa antigen recognized both 18- and 20-kDa CCAg in all positive but no negative control samples. These antibodies reacted with C. parvum sporozoites in an immunofluorescence assay. Human immune sera recognized the 20-kDa antigen in infected calf stool eluates. Both 18- and 20-kDa CCAg remained intact in commonly used preservatives and at various temperatures. These CCAg may be useful in designing sensitive, reliable methods for diagnosing cryptosporidiosis.

Animals↗

PubMed1993

Characterization of an immunodominant Giardia lamblia protein antigen related to alpha giardin.

The trophozoites of Giardia lamblia possess several protein antigens, predominant among them a protein of approximately 32,000 Da. In the present study, we used monospecific antibodies that recognize this protein to demonstrate its presence on a variety of G. lamblia isolates from human and animal sources. Immune electron microscopy was used to localize 32-kDa antigen on the trophozoite membrane and disk. Immunofluorescent assays employing monospecific antibodies confirmed the presence of 32-kDa antigen on the membrane and disk and its absence on flagella or nuclei. The N-terminal 17 amino acids of the 32-kDa antigen are identical to alpha-1-giardin, a protein component of microribbons on the ventral disk. These results suggest that the 32-kDa immunodominant trophozoite antigen is alpha-1-giardin.

Amino Acid Sequence↗

PubMed1993

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.

Administration, Topical↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1991

Isolation and molecular characterization of the ribosomal protein L6 homolog from Chlamydia trachomatis.

The cloning of a Chlamydia trachomatis eukaryotic cell-binding protein reported earlier from our laboratory (R. Kaul, K. L. Roy, and W. M. Wenman, J. Bacteriol. 169:5152-5156, 1987) represents an artifact generated by nonspecific recombination of chromosomal DNA fragments. However, the amino terminus of this plasmid-encoded fusion product demonstrated significant homology to Escherichia coli ribosomal protein L6. By using a 458-bp PstI-HindIII fragment of recombinant pCT161/18 (representing the 5' end of the cloned gene), we isolated and characterized a C. trachomatis homolog of the ribosomal protein L6 gene of E. coli. Sequence analysis of an 1,194-bp EcoRI-SacI fragment that encodes chlamydial L6 (designated CtaL6e) revealed a 552-bp open reading frame comprising 183 amino acids and encodes a protein with a molecular weight of 19,839. Interestingly, complete gene homology between C. trachomatis serovars L2 and J, each of which exists as a single copy per genome, was observed. Expression of a plasmid-encoded gene product is dependent on the lac promoter, since no product was obtained if the open reading frame was oriented in opposition to the lac promoter. Immunoblotting of purified ribosomes revealed functional, as well as antigenic, homology between the E. coli and C. trachomatis ribosomal L6 proteins.

Amino Acid Sequence↗

PubMed1991

Identification and nucleotide sequence of a developmentally regulated gene encoding a eukaryotic histone H1-like protein from Chlamydia trachomatis.

A lambda gt11 recombinant library of Chlamydia trachomatis serovar L2 chromosomal DNA was screened with a 29-mer synthetic oligonucleotide specific to the N-terminal amino acids of a predominant 18-kDa chlamydial protein. One recombinant clone, designated lambda gt11/L2/RKA10, was selected on the basis of its strong hybridization signal. Restriction endonuclease analysis and complete nucleotide sequencing of the recombinant revealed a 2,633-bp insert containing one complete open reading frame (ORF2) and two partial ORFs (ORF1 and ORF3). The deduced amino acid sequence of ORF2 matched perfectly at its N-terminal end with the derived amino acid sequence. The 375-bp ORF is capable of encoding a protein comprising 125 amino acids with a molecular mass of 13,689. A sequence compatible with a Shine-Dalgarno ribosome-binding site was located 9 bp upstream from the initiation codon, while the sequence distal to ORF2 revealed a rho-independent terminator. The protein, designated CTH1, possesses an estimated pI of 10.71 due to its high lysine content. This highly basic protein contains no tryptophan or phenylalanine. A protein data base search identified significant homology between CTH1 and painted sea urchin histone H1. Northern (RNA) blot analysis of Chlamydia-infected host cells demonstrated transcripts at 12 h postinfection. The recombinant plasmid encoding ORF2 expressed a gene product of approximately 18 kDa, similar to the native chlamydial protein as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein appears to represent one of the few eukaryotic histonelike proteins described to date in prokaryotes.

Amino Acid Sequence↗

PubMed1990

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.

Bacterial Outer Membrane Proteins↗

PubMed1990

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.

Amino Acid Sequence↗

PubMed1990

Detection of the surface-exposed 18-kilodalton binding protein in Chlamydia trachomatis by immunogold staining.

Dot-blot analysis of Chlamydia trachomatis elementary bodies (EBs) with monospecific polyclonal antibodies demonstrated that the 18-kilodalton binding protein is surface exposed. Immunoelectron microscopy with whole serovar L2 EBs and ultrathin sections confirmed this finding. In addition, only the extracellular EBs and not the intracellular reticulate bodies were labeled with immunogold.

Antibodies, Monoclonal↗