Biomedical subjects

Robert J Belland

Publications and source records attributed to Robert J Belland.

14 recordsLinked to original sources

PubMed2006

Global transcriptional upregulation in the absence of increased translation in Chlamydia during IFNgamma-mediated host cell tryptophan starvation.

The developmentally regulated intracellular pathogen Chlamydia pneumoniae is a natural tryptophan auxotroph. These organisms survive tryptophan starvation induced by host cell activation with IFNgamma by blocking maturation to the infectious form. In most bacteria, the stringent response is induced during amino acid starvation to promote survival. However, the response of obligate intracellular pathogens, which are predicted to lack stringent responses to amino acid starvation, is poorly characterized. Chlamydial transcription and translation were analysed during IFNgamma-mediated tryptophan starvation using genomic normalization methods, and the data revealed the novel findings that: (i) global chlamydial transcription was upregulated; and (ii) protein synthesis was dramatically reduced. These results indicate a dysregulation of developmental gene expression and an uncoupling of transcription from translation. These observations represent an alternative survival strategy for host-adapted obligate intracellular bacterial pathogens that have lost the genes for stringent control during reductive evolution.

Cell Line↗

PubMed2006

Different growth rates of Chlamydia trachomatis biovars reflect pathotype.

BACKGROUND: Despite small genomic differences, Chlamydia trachomatis biovars exhibit diverse disease manifestations and different growth rates in vivo and in cell culture models. METHODS: Chlamydial inclusion-forming units were enumerated over time in HeLa cells, to evaluate the length of the developmental cycle for C. trachomatis strains A, B, C, and E/Bour (ocular strains) as well as D, E/UW5/Cx, F, and L2 (genital strains). Prototype strains A, D, and L2 were selected for detailed analysis of reticulate body growth, division, and genomic replication. The impact that changing host cells and that coinfection with different strains has on growth was also assessed. RESULTS: The genital strains completed the developmental cycle in 36-44 h, whereas the ocular strains lagged behind considerably. Differences were the result of a longer lag phase (entry plus differentiation) and generation time for the ocular strains. A prototype ocular strain grew faster in conjunctival cells than in cervical cells. Coinfection with genital (D or L2) and ocular strains expedited recovery of the ocular strain. CONCLUSIONS: Precise temporal evaluation of the chlamydial developmental cycle for selected genital and ocular C. trachomatis biovars provides a means for investigating genomic differences that define chlamydial pathotype.

Chlamydia trachomatis↗

PubMed2005

The chlamydial developmental cycle.

Intracellular parasitism by bacterial pathogens is a complex, multi-factorial process that has been exploited successfully by a wide variety of organisms. Members of the Order Chlamydiales are obligate intracellular bacteria that are transmitted as metabolically inactive particles and must differentiate, replicate, and re-differentiate within the host cell to carry out their life cycle. Understanding the developmental cycle has been greatly advanced by the availability of complete genome sequences, DNA microarrays, and advanced cell biology techniques. Measuring transcriptional changes throughout the cycle has allowed investigators to determine the nature of the temporal gene expression changes required for bacterial growth and development.

Animals↗

PubMed2005

The Chlamydia pneumoniae type III secretion-related lcrH gene clusters are developmentally expressed operons.

Two chlamydial homologues of the Yersinia lcrH chaperone for type III secretion system structural components are present within separate gene clusters. Quantitative transcriptional analyses demonstrated that each cluster is differentially regulated and expressed as an operon using major sigma factor elements, suggesting the presence of more elaborate developmental regulation mechanisms in chlamydiae.

Bacterial Proteins↗

PubMed2004

Chlamydia pneumoniae and atherosclerosis.

Exposure to Chlamydia pneumoniae is extremely common, and respiratory infections occur repeatedly among most people. Strong associations exist between C. pneumoniae infection and atherosclerosis as demonstrated by: (i) sero-epidemiological studies showing that patients with cardiovascular disease have higher titres of anti-C. pneumoniae antibodies compared with control patients; (ii) detection of the organism within atherosclerotic lesions, but not in adjacent normal tissue by immunohistochemistry, polymerase chain reaction and electron microscopy and by culturing the organism from lesions; and (iii) showing that C. pneumoniae can either initiate lesion development or cause exacerbation of lesions in rabbit and mouse animal models respectively. The association of this organism with atherosclerosis has also provided sufficient impetus to conduct a variety of human secondary prevention antibiotic treatment trials. The results of these studies have been mixed and, thus far, no clear long-lasting benefit has emerged from these types of investigations. Studies of C. pneumoniae pathogenesis have shown that the organism can infect many cell types associated with both respiratory and cardiovascular sites, including lung epithelium and resident alveolar macrophages, circulating monocytes, arterial smooth muscle cells and vascular endothelium. Infected cells have been shown to exhibit characteristics associated with the development of cardiovascular disease (e.g. secretion of proinflammatory cytokines and procoagulants by infected endothelial cells and foam cell formation by infected macrophages). More detailed analysis of C. pneumoniae pathogenesis has been aided by the availability of genomic sequence information. Genomic and proteomic analyses of C. pneumoniae infections in relevant cell types will help to define the pathogenic potential of the organism in both respiratory and cardiovascular disease.

Animals↗

PubMed2004

Polymorphisms in the Chlamydia trachomatis cytotoxin locus associated with ocular and genital isolates.

Chlamydia trachomatis is a strict human pathogen producing infections that cause medically important chronic inflammatory diseases, such as blinding trachoma and tubal factor infertility. Isolates exist as serotypes that fall into distinct biologic and pathological groups corresponding to differences in infection tissue tropism and invasion properties. Paradoxically, genome sequencing of several diverse strains has revealed a remarkable level of genomic synteny, suggesting that minor genetic differences determine the pathogen host- and tissue-specific infection characteristics. To better understand the genetic basis of chlamydial pathobiologic diversity, we performed comparative DNA-DNA microarray genomic hybridizations with all 15 C. trachomatis serovariants. We found there are few major genetic differences among the 15 serovars. An exception was the cytotoxin locus located in the plasticity zone, a region that exhibited significant polymorphisms among serovars. We therefore sequenced this region from all 15 serovars. The cytotoxin gene was interrupted by extensive mutations and deletions among the different serovars; however, three basic open reading frame motifs were discovered that correlated with noninvasive oculotropic, urogenitotropic, and invasive serovars. Of interest, only noninvasive genitotropic serovars possessed an intact N-terminal portion of the putative toxin gene. This region contains the UDP-glucose binding domain and the glycosyltransferase domain required for enzymatic activity of the clostridial toxin homologs, suggesting a role in urogenital infection or pathogenesis.

Bacterial Toxins↗

PubMed2003

Transcriptome analysis of chlamydial growth during IFN-gamma-mediated persistence and reactivation.

Chlamydia trachomatis is an obligatory intracellular prokaryotic parasite that causes a spectrum of clinically important chronic inflammatory diseases of humans. Persistent infection may play a role in the pathophysiology of chlamydial disease. Here we describe the chlamydial transcriptome in an in vitro model of IFN-gamma-mediated persistence and reactivation from persistence. Tryptophan utilization, DNA repair and recombination, phospholipid utilization, protein translation, and general stress genes were up-regulated during persistence. Down-regulated genes included chlamydial late genes and genes involved in proteolysis, peptide transport, and cell division. Persistence was characterized by altered but active biosynthetic processes and continued replication of the chromosome. On removal of IFN-gamma, chlamydiae rapidly reentered the normal developmental cycle and reversed transcriptional changes associated with cytokine treatment. The coordinated transcriptional response to IFN-gamma implies that a chlamydial response stimulon has evolved to control the transition between acute and persistent growth of the pathogen. In contrast to the paradigm of persistence as a general stress response, our findings suggest that persistence is an alternative life cycle used by chlamydiae to avoid the host immune response.

Cell Division↗

PubMed2003

Genotypic differences in the Chlamydia pneumoniae tyrP locus related to vascular tropism and pathogenicity.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes respiratory infections and has been associated with cardiovascular disease. We compared respiratory and cardiovascular isolates to find genetic differences associated with pathogenicity. A polymorphic region encoding a tyrosine/tryptophan permease was found to differ between disease isolates. Respiratory strains contained multiple copies of the tyrP gene, and vascular strains contained a single copy. Single-nucleotide polymorphism analysis revealed the duplication to be a phylogenetically old event. Gene amplification was associated with higher mRNA levels and higher uptake of the substrate tyrosine, indicating an amino-acid transport-related phenotype associated with the tyrP genotype. Vascular strains, despite their reduced ability to transport tyrosine, do not appear to have a reduced growth rate in vitro. We hypothesize that the important difference between strains of vascular and respiratory origin may lie in the increased tendency of vascular strains to elicit persistent infection that is triggered by amino-acid starvation.

Amino Acid Transport Systems↗

PubMed2003

Micromanipulation of the Chlamydia pneumoniae inclusion: implications for cloning and host-pathogen interactions.

The Chlamydia trachomatis inclusion is fragile, rendering it incompatible to micromanipulation. We show that the Chlamydia pneumoniae inclusion differs, being resistant to micromanipulation as shown by direct microinjection of the infected host cytosol or the inclusion itself. We have used micromanipulation to clone C. pneumoniae and to free it from mycoplasma contamination.

Cell Membrane↗

PubMed2003

Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis.

Chlamydia trachomatis is one of the most common bacterial pathogens and is the etiological agent of debilitating sexually transmitted and ocular diseases in humans. The organism is an obligate intracellular prokaryote characterized by a highly specialized biphasic developmental cycle. We have performed genomic transcriptional analysis of the chlamydial developmental cycle. This approach has led to the identification of a small subset of genes that control the primary (immediate-early genes) and secondary (late genes) differentiation stages of the cycle. Immediate-early gene products initiate bacterial metabolism and potentially modify the bacterial phagosome to escape fusion with lysosomes. One immediate early gene (CT147) is a homolog of the human early endosomal antigen-1 that is localized to the chlamydial phagosome; suggesting a functional role for CT147 in establishing the parasitophorous vacuole in a nonfusogenic pathway. Late gene products terminate bacterial cell division and constitute structural components and remodeling activities involved in the formation of the highly disulfide cross-linked outer-membrane complex that functions in attachment and invasion of new host cells. Many of the genes expressed during the immediate-early and late differentiation stages are Chlamydia-specific and have evolutionary origins in eukaryotic lineages.

Bacterial Outer Membrane Proteins↗

PubMed2003

Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates.

We previously reported that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogen's tryptophan synthase (trpBA) genes. Here, we have applied functional genomics to extend this work and find that the paradigm established for reference serovars also applies to clinical isolates - specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme. Moreover, functional enzyme activity was directly correlated to IFN-gamma resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We speculate that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-gamma-mediated eradication and thus establish persistent infection.

Antigens, Bacterial↗

PubMed2002

Molecular basis defining human Chlamydia trachomatis tissue tropism. A possible role for tryptophan synthase.

Here we report the cloning and sequencing of a region of the chlamydiae chromosome termed the "plasticity zone" from all the human serovars of C. trachomatis containing the tryptophan biosynthesis genes. Our results show that this region contains orthologues of the tryptophan repressor as well as the alpha and beta subunits of tryptophan synthase. Results from reverse transcription-PCR and Western blot analyses indicate that the trpBA genes are transcribed, and protein products are expressed. The TrpB sequences from all serovars are highly conserved. In comparison with other tryptophan synthase beta subunits, the chlamydial TrpB subunit retains all conserved amino acid residues required for beta reaction activity. In contrast, the chlamydial TrpA sequences display numerous mutations, which distinguish them from TrpA sequences of all other prokaryotes. All ocular serovars contain a deletion mutation resulting in a truncated TrpA protein, which lacks alpha reaction activity. The TrpA protein from the genital serovars retains conserved amino acids required for catalysis but has mutated several active site residues involved in substrate binding. Complementation analysis in Escherichia coli strains, with defined mutations in tryptophan biosynthesis, and in vitro enzyme activity data, with cloned TrpB and TrpA proteins, indicate these mutations result in a TrpA protein that is unable to utilize indole glycerol 3-phosphate as substrate. In contrast, the chlamydial TrpB protein can carry out the beta reaction, which catalyzes the formation of tryptophan from indole and serine. The activity of the chlamydial Trp B protein differs from that of the well characterized E. coli and Salmonella TrpBs in displaying an absolute requirement for full-length TrpA. Taken together our data indicate that genital, but not ocular, serovars are capable of utilizing exogenous indole for the biosynthesis of tryptophan.

Amino Acid Sequence↗

PubMed2002

Isolation of Chlamydia pneumoniae clonal variants by a focus-forming assay.

Chlamydia pneumoniae is an obligate intracellular prokaryotic human pathogen that causes community-acquired respiratory infection and has been associated with atherosclerosis and cardiovascular disease. Unexpected results from genomic sequencing indicate that significant intrastrain polymorphism exists for some C. pneumoniae isolates. These polymorphisms could reflect genotypes with differing disease-causing characteristics. A definitive means to test this hypothesis is to obtain genetically homogeneous clonal populations of the pathogen and test them in models of infection and disease. To date, methods for cloning C. pneumoniae have not been reported. In this study, we describe the isolation of clonal variants with genetic differences in the tyrP locus from a polymorphic respiratory isolate, using a novel focus-forming assay. These results now allow investigations on the biology and pathogenesis of C. pneumoniae clonal genovars that could lead to new insights into the pathogenesis of this important human pathogen.

Animals↗

PubMed2002

Carcinoembryonic antigen family receptor recognition by gonococcal Opa proteins requires distinct combinations of hypervariable Opa protein domains.

Neisserial Opa proteins function as a family of adhesins that bind heparan sulfate proteoglycan (HSPG) or carcinoembryonic antigen family (CEACAM) receptors on human host cells. In order to define the CEACAM binding domain on Opa proteins, we tested the binding properties of a series of gonococcal (strain MS11) recombinants producing mutant and chimeric Opa proteins with alterations in one or more of the four surface-exposed loops. Mutagenesis demonstrated that the semivariable domain, present in the first loop, was completely dispensable for CEACAM binding. In contrast, the two hypervariable (HV) regions present in the second and third loops were essential for binding; deletion of either domain resulted in loss of receptor recognition. Deletion of the fourth loop resulted in a severe decrease in Opa expression at the cell surface and could therefore not be tested for CEACAM binding. Chimeric Opa variants, containing combinations of HV regions derived from different CEACAM binding Opa proteins, lost most of their receptor binding activity. Some chimeric variants gained HSPG binding activity. Together, our results indicate that full recognition of CEACAM receptors by Opa proteins requires a highly coordinate interplay between both HV regions. Furthermore, shuffling of HV regions may result in novel HSPG receptor binding activity.

Amino Acid Sequence↗