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Biomedical subjects

Richard S Stephens

Publications and source records attributed to Richard S Stephens.

9 recordsLinked to original sources
PubMed2003

A developmentally regulated two-component signal transduction system in Chlamydia.

Two-component systems allow bacteria to adapt to changing environmental conditions and may induce developmental changes necessary for survival. Chlamydia trachomatis alternates between two distinct developmental forms, each optimized for survival in a separate niche. Transcriptional regulation of development is not understood. The C. trachomatis genome sequence revealed a single pair of genes (ctcB-ctcC) predicted to encode proteins with sequence conservation to bacterial two-component systems. Sequence analysis revealed that the sensor kinase, CtcB, possessed an energy-sensing PAS domain and phosphorylation site. The response regulator, CtcC, had homology to sigma(54) activators, possessing conserved receiver and ATPase domains and phosphorylation site, but lacked the C-terminal DNA-binding domain. ctcB and ctcC were expressed late in the developmental cycle, and both proteins were detected in EB lysates. Recombinant CtcB and CtcC were purified from denatured Escherichia coli inclusion bodies and refolded. CtcC was found to aggregate as dimers and tetramers in solution. In vitro phosphorylation assays showed that CtcB autophosphorylated in the presence of Mg(2+), Mn(2+), and Fe(2+) and transferred the phosphoryl group in the presence of CtcC. Collectively, these results show that CtcB and CtcC function as a two-component system and are likely responsible for transcriptional regulation by sigma(54) holoenzyme during late-stage chlamydial development.

Amino Acid Sequence↗
PubMed2003

The cellular paradigm of chlamydial pathogenesis.

Diseases caused by Chlamydia are based on intense and chronic inflammation elicited and maintained by reinfection or persistent infection. The traditional view in the field is that disease is mediated by antigen-dependent delayed-type hypersensitivity or autoimmunity. This immunological paradigm has served as the basis for years of chlamydial research but the mechanism or the antigen that causes pathology has yet to be unequivocally revealed. Recent research on responses elicited in Chlamydia-infected cells defines a new direction for our understanding of this microorganism-host interaction and provides the basis for a reassessment of disease mechanisms. Chlamydia-infected non-immune mammalian cells produce proinflammatory chemokines, cytokines, growth factors and other cellular modulators. This cellular response to infection supports an alternative hypothesis for chlamydial pathogenesis: the inflammatory processes of chlamydial pathogenesis are elicited by infected host cells and are necessary and sufficient to account for chronic and intense inflammation and the promotion of cellular proliferation, tissue remodeling and scarring, the ultimate cause of disease sequelae.

Autoimmunity↗
PubMed2003

Global stage-specific gene regulation during the developmental cycle of Chlamydia trachomatis.

Distinct morphological changes associated with the complex development cycle of the obligate intracellular bacterial pathogen Chlamydia trachomatis have been historically well characterized by microscopy. A number of temporally regulated genes have been characterized previously, suggesting that the chlamydial developmental cycle is regulated at the transcriptional level. This hypothesis was tested by microarray analysis in which the entire C. trachomatis genome was analyzed, providing a comprehensive assessment of global gene regulation throughout the chlamydial developmental cycle. Seven temporally cohesive gene clusters were identified, with 22% (189 genes) of the genome differentially expressed during the developmental cycle. The correlation of these gene clusters with hallmark morphological events of the chlamydial developmental cycle suggests three global stage-specific networks of gene regulation.

Chlamydia trachomatis↗
PubMed2002

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity.

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.

Amino Acid Sequence↗
PubMed2002

Phage-display antibody detection of Chlamydia trachomatis-associated antigens.

A phage-displayed human single-chain Fv antibody library (6.7x10(9) members) was used to select probes specific to components associated with the surface of Chlamydia trachomatis elementary bodies (EBs). Each of 15 antibodies was characterized by ELISA, dot-blot, immunoblot and immunocytochemistry, resulting in the identification of several new chlamydial components associated with the surface of EBs. In addition, six antibodies were specific for host-cell components associated with the surface of EBs. While phage display has been used effectively to produce specific antibodies for purified components, these data show that this technology is suitable for selection of specific probes from complex antigens such as the surface of a microbial pathogen.

Animals↗
PubMed2002

Selection of mutant cell lines resistant to infection by Chlamydia spp [corrected].

The lytic outcome of natural infection by Chlamydia trachomatis was exploited to select CHO (Chinese hamster ovary) cells, following chemical mutagenesis, that were deficient in their ability to sustain productive chlamydial infection. Four distinct mutant cell phenotypes with defects in either attachment or postattachment mechanisms that are required for infection by C. trachomatis and Chlamydia pneumoniae were characterized.

Animals↗
PubMed2002

Recruitment of mammalian cell fibronectin to the surface of Chlamydia trachomatis.

Pathogenic bacteria exploit the presence of various host cell molecules in order to colonize new tissues. Fibronectin is involved in a wide range of cell functions in vivo, and staphylococci, streptococci, and gonococci have evolved mechanisms to utilize this glycoprotein to mediate host cell binding. We show that elementary bodies (EB) from two biovars of Chlamydia trachomatis recruit fibronectin to their surfaces upon lysis of the host cell. We also demonstrate that a heparan sulfate lyase-sensitive molecule on chlamydial EB is responsible for binding at least a portion of this fibronectin.

Animals↗
PubMed1999

DNA structure and novel amino and carboxyl termini of the Chlamydia sigma 70 analogue modulate promoter recognition.

Genes from the eubacterium Chlamydia typically do not share promoter consensus sequences with those of Escherichia coli and are not expressed when cloned in E. coli; nevertheless, the major sigma-subunit identified from Chlamydia trachomatis has nearly identical amino acid sequence to E. coli sigma 70 in regions that contact DNA. Following expression of the chlamydial sigma-subunit gene in E. coli, expression was specifically initiated from chlamydial promoter regions. Selective recognition of chlamydial promoters by holoenzyme was dependent upon the structure of the promoter DNA coupled with novel amino- and carboxyl-terminal extensions of the chlamydial sigma-subunit.

Bacterial Outer Membrane Proteins↗