Elizabeth S. Stuart

Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells

Obligate intracellular bacterial pathogens of the genus Chlamydia are reported to enter host cells by both clathrin-dependent and clathrin-independent processes. C. trachomatis serovar K recently was shown to enter cells via caveolae-like lipid raft domains. We asked here how widespread raft-mediated entry might be among the Chlamydia. We show that C. pneumoniae, an important cause of respiratory infections in humans that additionally is associated with cardiovascular disease, and C. psittaci, an important pathogen in domestic mammals and birds that also infects humans, each enter host cells via cholesterol-rich lipid raft microdomains. Further, we show that C. trachomatis serovars E and F also use these domains to enter host cells. The involvement of these membrane domains in the entry of these organisms was indicated by the sensitivity of their entry to the raft-disrupting agents Nystatin and filipin, and by their intracellular association with caveolin-1, a 22-kDa protein associated with the formation of caveolae in rafts. In contrast, caveolin-marked lipid raft domains do not mediate entry of C. trachomatis serovars A, 36B, and C, nor of LGV serovar L2 and MoPn. Finally, we show that entry of each of these chlamydial strains is independent of cellular expression of caveolin-1. Thus, entry via the Nystatin and filipin-sensitive pathway is dependent on lipid rafts containing cholesterol, rather than invaginated caveolae per se.

Occurrence of Chlamydia trachomatis and Chlamydia pneumoniae in paediatric respiratory infections.

An emerging body of evidence suggests that half of asthma in both children and adults is associated with chronic lung infection. The aim of the present study was to determine the frequency of viable Chlamydia pneumoniae (Cp) and C. trachomatis (Ct) in the respiratory tracts of paediatric patients with chronic respiratory diseases. Bronchoalveolar lavage fluid (BALF) samples obtained from 182 children undergoing bronchoscopy for clinical reasons were assayed using PCR analysis, in vitro tissue culture and immunofluorescence staining for the presence of Cp and Ct. Chlamydia-specific DNA was detected by PCR in 124 (68%) out of 182 patients; 79 were positive for Cp, 77 positive for Ct and 32 for both organisms; 75 patients had cultivable Chlamydia. Ct DNA prevalence decreased, whereas Cp positivity generally increased with age. A total of 59 out of 128 asthma patients and 16 out of 54 nonasthmatics were Chlamydia culture positive. When the patients were divided into inflammatory versus noninflammatory airway disease, there were 69 (46%) out of 150 and six (18%) out of 32 BALF samples with cultivable Chlamydia, respectively. Viable Chlamydia pneumoniae and Chlamydia trachomatis occur frequently in children with chronic respiratory diseases and may be more prevalent in asthma patients. To the current authors’ knowledge, this is the first report of viable Chlamydia trachomatis in the lungs of children.

Protein organization on the PHA inclusion cytoplasmic boundary

Polyhydroxyalkanoate (PHA) cellular inclusions consist of polyesters, phospholipids, and proteins. Both the polymerase and the depolymerase enzymes are active components of the structure. Recently, proteins associated with these inclusions have been described in a number of bacterial species. In order to further clarify the structure and function of these proteins in relation to polymer inclusions, ultrastructural studies of isolated polymer inclusions were initiated. The surface boundary characteristics of polymer inclusions, produced by several genera of bacteria, two different Pseudomonas putida deletion mutants and by Escherichia coli recombinants, were examined. The recombinant E. coli carried either the PHB biosynthesis operon (phaCAB) from Ralstonia eutropha alone, or both this operon and a gene encoding an inclusion surface protein of R. eutropha (phaP). The results support two suggestions: (i) specific genes in the PHA gene cluster code for the proteins forming the surface boundary arrays which characterize the polymer inclusion; and (ii) transfer of such a gene would result in subcellular compartmentalization of accumulating polymer. Although the proteins appear to serve a similar function among different genera, nevertheless, the different surface proteins are encoded by a variety of non-homologous genetic sequences.

Detection of Chlamydia in the Peripheral Blood Cells of Normal Donors Using in Vitro Culture, Immunofluorescence Microscopy and Flow Cytometry Techniques

BackgroundChlamydia trachomatis (Ct) and Chlamydia pneumoniae (Cp) are medically significant infectious agents associated with various chronic human pathologies. Nevertheless, specific roles in disease progression or initiation are incompletely defined. Both pathogens infect established cell lines in vitro and polymerase chain reaction (PCR) has detected Chlamydia DNA in various clinical specimens as well as in normal donor peripheral blood monocytes (PBMC). However, Chlamydia infection of other blood cell types, quantification of Chlamydia infected cells in peripheral blood and transmission of this infection in vitro have not been examined.MethodsCp specific titers were assessed for sera from 459 normal human donor blood (NBD) samples. Isolated white blood cells (WBC) were assayed by in vitro culture to evaluate infection transmission of blood cell borne chlamydiae. Smears of fresh blood samples (FB) were dual immunostained for microscopic identification of Chlamydia-infected cell types and aliquots also assessed using Flow Cytometry (FC).ResultsELISA demonstrated that 219 (47.7%) of the NBD samples exhibit elevated anti-Cp antibody titers. Imunofluorescence microscopy of smears demonstrated 113 (24.6%) of samples contained intracellular Chlamydia and monoclonals to specific CD markers showed that in vivo infection of neutrophil and eosinophil/basophil cells as well as monocytes occurs. In vitro culture established WBCs of 114 (24.8%) of the NBD samples harbored infectious chlamydiae, clinically a potentially source of transmission, FC demonstrated both Chlamydia infected and uninfected cells can be readily identified and quantified.ConclusionNBD can harbor infected neutrophils, eosinophil/basophils and monocytes. The chlamydiae are infectious in vitro, and both total, and cell type specific Chlamydia carriage is quantifiable by FC.

Antigen presentation using novel particulate organelles from halophilic archaea

A presentation vehicle was developed based on particulate gas vesicles produced by halophilic archaea. Gas vesicle epitope displays were prepared using standard coupling methods or recombinant DNA technology. When presented in the context of gas vesicle preparations, either the hapten, TNP, or a model six amino acid recombinant insert in the outer gas vesicle protein, GvpC was rendered immunogenic. Assays to quantify humoral responses indicated that each preparation elicited strong antibody responses in the absence of exogenous adjuvant. Thus, each preparation elicited a humoral response when injected into mice and this response was long lived and exhibited immunologic memory. Recombinant gas vesicle preparations therefore constitute a new, self-adjuvanting carrier/display vehicle for presentation of an array of peptidyl epitopes.

The anti-idiotypic antibody to chlamydial glycolipid exoantigen (GLXA) protects mice against genital infection with a human biovar of Chlamydia trachomatis.

Despite more than three decades of anti-chlamydial vaccine research and improved vaccine strategies with new technologies, no vaccine candidate has protected against heterologous challenge, nor at more than one site of infection. The majority of experimental anti-chlamydial vaccines to date have targeted the chlamydial major outer membrane protein (MOMP). Many MOMP-directed vaccine candidates have been highly immunogenic, but have failed to protect against infectious challenge. We have extended our previous studies of a different anti-chlamydial vaccine, a monoclonal anti-idiotypic antibody (anti-Id; mAb2) which is a molecular mimic of the chlamydial glycolipid exoantigen (GLXA). The present studies demonstrate that the mAb2 vaccine is protective in a murine genital infection model utilizing a human urogenital strain. After either mucosal (oral or intranasal) or systemic (subcutaneous) immunization with the poly (lactide) encapsulated-mAb2 to GLXA, C3H/HeJ mice were significantly protected against topical vaginal challenge with Chlamydia trachomatis (K serovar; UW-31). Reduced vaginal shedding of organism and genital tract inflammation were associated with GLXA-specific and/or anti-EB neutralizing serum antibody. Our results demonstrate that the anti-Id (mAb2) vaccine is protective against an additional human biovar of C. trachomatis in C3H/HeJ mice, which are allogeneic to the source of mAb2 (BALB/c).

Investigation of the function of proteins associated to polyhydroxyalkanoate inclusions in Pseudomonas putida BMO1

Polyhydroxyalkanoate (PHA) granule associated proteins from Pseudomonas oleovorans were purified and the N-terminal sequences of two major proteins migrating in sodium dodecyl sulfate polyacrylamide gels with a relative molecular mass of 18 and 43 kDa (GA1 and GA2, respectively) were analyzed. Radiolabeled degenerate probes deduced from these amino acid sequences were used to identify genomic DNA fragments from P. oleovorans and Pseudomonas putida encoding GA1 and GA2. DNA sequence analysis of the fragments obtained from P. putida revealed that the genes encoding these proteins were adjacent to phaC2 and ORF3, the PHA synthase II gene and an open reading frame of unknown function, respectively, found at the P. oleovorans and P. aeruginosa PHA synthase gene locus. The open reading frames encoding GA1, GA2 and ORF3 or smaller fragments beginning at GA1 were inactivated by chromosomal insertion of the Tn5 kanamycin resistance gene block (neo). When these mutants were grown on mineral salts agar media under nitrogen limitation, containing gluconate or decanoate as carbon sources, they appeared more translucent than the wild-type grown under similar conditions. Gas-chromatographic analysis of the cellular dry mass revealed that the mutant strains accumulated 30-50% less PHA than the P. putida wild type.

Recombinant gas vesicles from Halobacterium sp . displaying SIV peptides demonstrate biotechnology potential as a pathogen peptide delivery vehicle

BackgroundPrevious studies indicated that recombinant gas vesicles (r-GV) from a mutant strain of Halobacterium sp. NRC-1 could express a cassette containing test sequences of SIVmac gag derived DNA, and function as an antigen display/delivery system. Tests using mice indicated that the humoral immune response to the gag encoded sequences evoked immunologic memory in the absence of an exogenous adjuvant.ResultsThe goal of this research was to extend this demonstration to diverse gene sequences by testing recombinant gas vesicles displaying peptides encoded by different SIV genes (SIVtat, rev or nef). Verification that different peptides can be successfully incorporated into the GvpC surface protein of gas vesicle would support a more general biotechnology application of this potential display/delivery system. Selected SIVsm-GvpC fusion peptides were generated by creating and expressing fusion genes, then assessing the resulting recombinant gas vesicles for SIV peptide specific antigenic and immunogenic capabilities. Results from these analyses support three conclusions: (i) Different recombinant gvpC-SIV genes will support the biosynthesis of chimeric, GvpC fusion proteins which are incorporated into the gas vesicles and generate functional organelles. (ii) Monkey antibody elicited by in vivo infection with SHIV recognizes these expressed SIV sequences in the fusion proteins encoded by the gvpC-SIV fusion genes as SIV peptides. (iii) Test of antiserum elicited by immunizing mice with recombinant gas vesicles demonstrated notable and long term antibody titers. The observed level of humoral responses, and the maintenance of elevated responses to, Tat, Rev and Nef1 encoded peptides carried by the respective r-GV, are consistent with the suggestion that in vivo there may be a natural and slow release of epitope over time.ConclusionThe findings therefore suggest that in addition to providing information about these specific inserts, r-GV displaying peptide inserts from other relevant pathogens could have significant biotechnological potential for display and delivery, or serve as a cost effective initial screen of pathogen derived peptides naturally expressed during infections in vivo.

Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

BackgroundLipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function.MethodsWe utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions.ResultsWe show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp), C. caviae (GPIC), and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi.ConclusionCaveolin-2 associates with the chlamydial inclusion independently of caveolin-1. The function of caveolin-2, either in the uninfected cell or in the chlamydial developmental cycle, remains to be elucidated. Nevertheless, this second caveolin protein can now be added to the small number of host proteins that are associated with the inclusions of this obligate intracellular pathogen.

Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development

BackgroundChlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host’s signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive.ResultsIn this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatis infection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-β (PDGFRβ) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRβ that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatis development.ConclusionCumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases.