Chlamydial Particles Research Articles

An optimal control model of the treatment of chronic Chlamydia trachomatis infection using a combination treatment with antibiotic and tryptophan

We develop a mathematical model of within-host Chlamydia dynamics to investigate the effects of different treatment combinations on the within-host dynamics of chronic genital chlamydial infections characterised by the presence of IFN-γ-induced chlamydial persistence. Our modelling framework is informed by recent studies which have demonstrated that tryptophan and some of its biosynthetic analogues are able to both reverse and inhibit IFN-γ-induced chlamydial persistence. Our aim is to find the optimal combination of treatments/drugs that will minimise the systemic cost of two controls/treatments, the concentration of extracellular Chlamydia, infected epithelial host cells, and importantly the development of chlamydial persistence. Pontryagin’s Maximum Principle is used to characterise the optimal controls and the resulting optimal control system is numerically solved. Optimal control solutions that eradicate the pathogen and its persistence are demonstrated. Our numerical results indicate that the optimal way to clear and truncate the progression of a chronic Chlamydia infection may be the administration of a combination therapy that is bacteriostatic to chlamydial particles and that concurrently inhibits and reverses chlamydial persistence, using a cocktail of tryptophan supplementation and levo-1-methyl tryptophan. Our approach provides a framework for the design of new therapeutic regimens and guidelines for the treatment of chronic chlamydial infections.

Virulence-related comparative transcriptomics of infectious and non-infectious chlamydial particles

BackgroundMembers of the phylum Chlamydiae are obligate intracellular pathogens of humans and animals and have a serious impact on host health. They comprise several zoonotic species with varying disease outcomes and prevalence. To investigate differences in virulence, we focused on Chlamydia psittaci, C. abortus and Waddlia chondrophila. Most threatening is C. psittaci, which frequently infects humans and causes psittacosis associated with severe pneumonia. The closest relative of C. psittaci is C. abortus, which shares the vast majority of genes but less frequently infects humans, and causes stillbirth and sepsis. W. chondrophila is more distantly related, and occasional human infections are associated with respiratory diseases or miscarriage. One possible explanation for differences in virulence originate from species-specific genes as well as differentially expressed homologous virulence factors.ResultsRNA-sequencing (RNA-Seq) was applied to purified infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs) in order to elucidate the transcriptome of the infectious and replicative chlamydial states. The results showed that approximately half of all genes were differentially expressed. For a descriptive comparison, genes were categorised according to their function in the RAST database. This list was extended by the inclusion of inclusion membrane proteins, outer membrane proteins, polymorphic membrane proteins and type III secretion system effectors. In addition, the expression of fifty-six known and a variety of predicted virulence and immunogenic factors with homologs in C. psittaci, C. abortus and W. chondrophila was analysed. To confirm the RNA-Seq results, the expression of nine factors was validated using real-time quantitative polymerase chain reaction (RT-qPCR). Comparison of RNA-Seq and RT-qPCR results showed a high mean Pearson correlation coefficient of 0.95.ConclusionsIt was shown that both the replicative and infectious chlamydial state contained distinctive transcriptomes and the cellular processes emphasised in EBs and RBs differed substantially based on the chlamydial species. In addition, the very first interspecies transcriptome comparison is presented here, and the considerable differences in expression of homologous virulence factors might contribute to the differing infection rates and disease outcomes of the pathogens. The RNA-Seq results were confirmed by RT-qPCR and demonstrate the feasibility of interspecies transcriptome comparisons in chlamydia.

Purification of infectious and non-infectious chlamydial particles using iodixanol for density gradient preparation

Chlamydiae are obligate intracellular bacteria with two distinct morphological stages, the infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs). Here we describe a rapid and straightforward protocol for the purification of EBs and RBs involving special density gradients. It has been successfully applied to three chlamydial species.

The novel chlamydial adhesin CPn0473 mediates the lipid raft-dependent uptake of Chlamydia pneumoniae.

Summary Chlamydiae are Gram‐negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down‐stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473‐coupled fluorescent latex beads adhere to human epithelial HEp‐2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp‐2 cells with rCPn0473 does not attenuate adhesion but promotes dose‐dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473‐dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae.

A model of workplace safety incorporating worker interactions and simple interventions

Although there was substantial research into the occupational health and safety sector over the past forty years, this generally focused on statistical analyses of data related to costs and/or fatalities and injuries. There is a lack of mathematical modelling of the interactions between workers and the resulting safety dynamics of the workplace. There is also little work investigating the potential impact of different safety intervention programs prior to their implementation. In this article, we present a fundamental, differential equation-based model of workplace safety that treats worker safety habits similarly to an infectious disease in an epidemic model. Analytical results for the model, derived via phase plane and stability analysis, are discussed. The model is coupled with a model of a generic safety strategy aimed at minimising unsafe work habits, to produce an optimal control problem. The optimal control model is solved using the forward-backward sweep numerical scheme implemented in Matlab. References Australian Bureau of Statistics. Forms of employment. Commonwealth of Australia, 2010. http://www.abs.gov.au/AUSSTATS/abs@.nsf/DetailsPage/6359.0November%202010?OpenDocument Bahn, S. Power and Influence: Examining the Communication Pathways that Impact on Safety in the Workplace. J. Occup. Health Safety–-Aust. N.Z., 25(3):213–222, 2009. Bird, P. Reducing Manual Handling Workers Compensation Claims in a Public Health Facility. J. Occup. Health Safety–-Aust. N.Z., 25(6):451–459, 2009. Breslin, P. Improving ohs Standards in the Building and Construction Industry through safe design. J. Occup. Health Safety–-Aust. N.Z., 23(4):89–99, 2007. Breslin, P. National Harmonisation: Designers' Duties of Care in the Australian Building and Construction Industry. J. Occup. Health Safety–-Aust. N.Z., 25(6):495–504, 2009 . Driscoll, T., Mitchell, T., Mandryk, J., Healey, S., Hendrie, L. and Hull, B. Trends in Work-Related Fatalities in Australia, 1982 to 1992. J. Occup. Health Safety–-Aust. N.Z., 18(1):21–33, 2002. Driscoll, T. Fatal Injury of young workers in Australia. J. Occup. Health Safety–-Aust. N.Z., 22(2):151–161, 2006. Foley, G., Gale, J. and Gavenlock, L. The Cost of Work-Related Injury and Disease. J. Occup. Health Safety–-Aust. N.Z., 11(2):171–194, 1995. Glendon, I. and Waring, A. Risk management as a framework for occupational health and safety. J. Occup. Health Safety–-Aust. N.Z., 13(6):525–532, 1997. Gunningham, N. and Healy, P. Agricultural ohs Policy: Towards Systemic Reform. J. Occup. Health Safety–-Aust. N.Z., 20(4):311–318, 2004. Hawkins, A., Eather, J. and Fragar, L. Improving Health and Safety in the Farm Workshop. J. Occup. Health Safety–-Aust. N.Z., 24(2):155–160, 2008. Heads of Workers' Compensation Authorities. 2008/09 Australia and New Zealand Return to Work Monitor. http://www.hwca.org.au/documents/Australia%20and%20New%20Zealand%20Return%20to%20Work%20Monitor%202008-2009.pdf Kermack, W. O. and McKendrick, A. G. A Contribution to the Mathematical Theory of Epidemics. Proc. R. Soc. Lond. A., 115(772):700–721, 1927. doi:10.1098/rspa.1927.0118 Lenhart, S. and Workman, J. T. Optimal control applied to biological models. Chapman and Hall CRC Mathematical and Computational Biology Series, 2007. Mallet, D. G., Bagher-Oskouei, M., Farr, A. C., Simpson, D. P. and Sutton, K-J. A mathematical model of Chlamydial infection incorporating movement of Chlamydial particles. B. Math. Biol., 75(11):2257–2270, 2013. doi:10.1007/s11538-013-9891-9 Murray, J. D. Mathematical Biology, I: An Introduction. Springer, 2002. Raggett, G. F. Modelling the Eyam plague. B. I. Math. Appl., 18:221–226, 1982. Safe Work Australia. The cost of work-related injury and illness for australian employers, workers and the community:2008-2009. Commonwealth of Australia, 2012. http://www.safeworkaustralia.gov.au/sites/SWA/about/Publications/Documents/660/Cost%20of%20Work-related%20injury%20and%20disease.pdf Shulgin, B., Stone, L. and Agur, Z. Pulse vaccination strategy in the sir Epidemic Model. B. Math. Biol., 60(6):1123–1148, 1998. doi:10.1006/S0092-8240(98)90005-2 Vanderkruk, R. Workplace health and safety officers: a Queensland success story. J. Occup. Health Safety–-Aust. N.Z., 15(6):557–563, 1999. Winder, C. The development of ohs legislation in Australia. J. Occup. Health Safety–-Aust. N.Z., 25(4):277–287, 2009.

A Mathematical Model of Chlamydial Infection Incorporating Movement of Chlamydial Particles

We present a spatiotemporal mathematical model of chlamydial infection, host immune response, and movement of infectious particles. The resulting partial differential equations model both the dynamics of the infection and changes in infection profile observed spatially along the length of the host genital tract. This model advances previous Chlamydia modelling by incorporating spatial change. Numerical solutions and model analysis are carried out, and we present a hypothesis regarding the potential for treatment and prevention of infection by increasing chlamydial particle motility.

P1.038 Chlamydial Antigen and Nucleic Acid Detection in Liver Biopsies from Patients with Chronic Cholelithiasis

Background It is known that chlamydial species can propagate in hepatocyte cell lines. Moreover, some clinical cases of chlamydial infection involve liver abnormalities. This study was to clarify whether chlamydial markers (protein and nucleic acids) could be detected in liver biopsies from patients with calculous cholecystitis. Material and Methods Liver biopsies were obtained from 39 patients during cholecystectomy and analysed with immunohistochemical, nucleic acid amplification and serological protocols. Liver specimens from 8 trauma victims served as controls. Results It was shown that from 39 patients with cholecystitis 19 gave considerable signal generated by antibodies against C. trachomatis (15 patients) or C. pneumoniae (4 patients). 10.2% (4/39) of the samples contained detectable 16S rRNA genomic sequence from C. pneumoniae while amplifiable fragments of 16S rRNA and pLGV cryptic plasmid from C. trachomatis were found in 20.5% (8/39) of DNA specimens. The control group had a zero detection rate for chlamydial genetic markers in the liver. Simultaneous detection of genetic and immunohistochemical markers validated by positive serological status took place in a very limited number of the patients (4 cases for C. trachomatis and 2 cases for C. pneumoniae). Moreover, it was shown that C. trachomatis and C. pneumoniae can efficiently propagate in freshly isolated rat primary hepatocytes forming infectious progeny. Conclusions Identification of chlamydial markers in liver biopsies along with the ability of the chlamydial pathogens to propagate in native hepatocytes may suggest the possible involvement of chlamydial species in inflammatory hepatobiliary disease. We have assumed previously that abnormalities of cholesterol homeostasis associated with the increase of ApoB-containing lipoproteins may promote enhanced uptake of chlamydial particles in the liver. Thus, it is conceivable that the appearance of chlamydial markers in hepatic biopsies of patients with cholelithiasis takes place due to proatherogenic changes in plasma lipoprotein profile.

Rapamycin can Inhibit the Development of Chlamydia pneumoniae, which Might Partly Contribute to the Prevention of In-stent Restenosis

Rapamycin, an immunosuppressive and antiproliferative drug, is used to prevent neointima formation to reduce the risk of in-stent restenosis with rapamycin eluting-stents. Chronic infection of Chlamydia pneumoniae has been associated with cardiovascular diseases, and could play an important role in the mechanism of restenosis. We examined the effect of rapamycin on the growth of C. pneumoniae in cell cultures. HL cell monolayers were inoculated with C. pneumoniae CWL029 or C. trachomatis L2. Different concentrations of rapamycin were present in the culture medium continuously or for 8-hour periods. After incubation the infected cells were repassaged to fresh HL cell monolayers and incubated in the medium without rapamycin. The newborn inclusions from both passages were checked by fluorescent microscope or electron microscope. The presence of 23 microg/ml rapamycin restricted over 90% of the growth of C. pneumoniae. Continuous presence of 11 microg/ml rapamycin inhibited the growth of C. pneumoniae up to 80% and caused smaller inclusions, fewer chlamydial particles and fewer matured EBs. 11 microg/ml rapamycin presented in first passage caused the reduction of C. pneumoniae to 57% at first passage and 24% at second passage. Sufficient rapamycin can inhibit the growth of C. pneumoniae effectively, but it should be applied at the early stage of the chlamydial infections. Rapamycin eluting-stents can induce a high enough local concentration of rapamycin. This provides a possibility for us to suppose that the beneficial effect of rapamycin in preventing in-stent restenosis might partly be explained by its inhibitory effects on the growth of C. pneumoniae.

Identification ofChlamydia trachomatisOuter Membrane Complex Proteins by Differential Proteomics

The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.

Chlamydia trachomatisgrowth inhibition and restoration of LDL-receptor level in HepG2 cells treated with mevastatin

BackgroundPerihepatitis is rare but consistently occurring extragenital manifestation of untreated Chlamydia trachomatis infection. Despite of possible liver involvement in generalized C. trachomatis infection, the ability of the pathogen to propagate in the hepatic cells and its impact on liver functions is not thoroughly investigated. The effect of mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, on C. trachomatis growth in human hepatoma cell line HepG2 has been studied. Bacterial growth was assessed by immunostaining with FITC-labeled monoclonal antibody against chlamydial lipopolysaccharide and by RT-PCR for two chlamydial genetic markers (16S rRNA and euo).ResultsChlamydial inclusion bodies were seen in approximately 50% of hepatocytes at 48 hours in the post infection period. Lysates obtained from infected hepatocytes were positive in the infective progeny test at 48 and especially in 72 hours after infection initiation. It has been shown that chlamydial infection in hepatocytes also leads to the decline of LDL-receptor mRNA which reflects infection multiplicity rate. Additions of mevastatin (1, 20 and 40 μM) 1 hour before inoculation restored and upregulated LDL-receptor mRNA level in a dose-dependent manner. Mevastatin treatment had no effect on internalization of chlamydial particles. However it reduced drastically the number of chlamydial 16S rRNA and euo transcripts as well as overall infection rate in HepG-2 cells. Complete eradication of infection has been seen by immunofluorescent staining at 40 μM mevastatin concentration, when expression level of chlamydial 16S rRNA and euo was undetectable. Lower concentration of mevastatin (20 μM) promoted euo expression level and the appearance of atypically small chlamydial inclusions, while there was a noticeable reduction in the number of infected cells and 16S rRNA transcripts.ConclusionsC. trachomatis can efficiently propagate in hepatocytes affecting transcription rate of some liver-specific genes. Ongoing cholesterol synthesis is essential for chlamydial growth in hepatocytes. Inhibitors of cholesterol biosynthesis can supplement conventional strategy in the management of C. trachomatis infection.

ApoB-containing lipoproteins promote infectivity of chlamydial species in human hepatoma cell line

To evaluate the direct binding of two main chlamydial biovars (C. trachomatis and C. pneumoniae) to plasma lipoproteins and its effect on chlamydial infection rate in human hepatoma cell line (HepG2 cells). Murine plasma lipoproteins were fractionated and isolated using fast-performance liquid chromatography (FPLC), spotted on nitrocellulose membrane and incubated with chlamydial suspensions. Direct binding of chlamydial particles to lipoprotein fractions has been studied using lipopolysaccharide-specific antibodies in immuno-dot blot binding assay and immunoprecipitation analysis. Immunostaining protocol as well as flow cytometry analysis have been employed to study the infectivity rate of chlamydial species in HepG2 cells. Elementary bodies of both C. trachomatis and C. pneumoniae bind ApoB-containing fractions of plasma lipoproteins. That binding becomes stronger when heat-denatured FPLC fractions are used, suggesting a primary role of apolipoproteins in interaction between chlamydial particle and lipoprotein. Both chlamydial biovars efficiently propagate in human hepatoma cell line - HepG2 cells even in serum free conditions forming late-stage inclusion bodies and releasing extracellular elementary bodies. Preincubation of C. trachomatis and C. pneumoniae with native ApoB-containing lipoproteins enhances the rate of chlamydial infection in HepG2 cells. A productive infection caused by C. trachomatis and C. pneumoniae may take place in human-derived hepatocytes revealing hepatic cells as possible target in chlamydial infection. Obtained results may suggest the participation of lipoprotein receptors in the mechanism of attachment and/or entry of chlamydial particles into target cells.

Lack of cell-mediated cytotoxicity towards Chlamydia trachomatis infected target cells in humans.

Induction of cell-mediated cytotoxicity towards Chlamydia trachomatis infected target cells was studied in humans. PHA-derived lymphoblasts, infected with inactivated or virulent LGV-2 chlamydial particles, were used as target cells. Chlamydial primed T cell blasts or specific, cloned T cells were used as effector cells. A highly sensitive 18 hours lytic assay employing 111In-labelled target cells was used. Specific cytotoxicity towards C. trachomatis infected target cells in humans could not be detected. This was the case regardless of the origin of the effector cells or the various protocols tested. However, this assay was capable of yielding significant positive results with effector cells primed to alloantigens. We conclude that cell-mediated cytotoxicity towards bacterial infected target cells probably does not play a major role.

Chlamydial infection and spatial ascension of the female genital tract: a novel hybrid cellular automata and continuum mathematical model

Sexually transmitted chlamydial infection initially establishes in the endocervix in females, but if the infection ascends the genital tract, significant disease, including infertility, can result. Many of the mechanisms associated with chlamydial infection kinetics and disease ascension are unknown. We attempt to elucidate some of these processes by developing a novel mathematical model, using a cellular automata-partial differential equation model. We matched our model outputs to experimental data of chlamydial infection of the guinea-pig cervix and carried out sensitivity analyses to determine the relative influence of model parameters. We found that the rate of recruitment and action of innate immune cells to clear extracellular chlamydial particles and the rate of passive movement of chlamydial particles are the dominant factors in determining the early course of infection, magnitude of the peak chlamydial time course and the time of the peak. The rate of passive movement was found to be the most important factor in determining whether infection would ascend to the upper genital tract. This study highlights the importance of early innate immunity in the control of chlamydial infection and the significance of motility-diffusive properties and the adaptive immune response in the magnitude of infection and in its ascension.

Kinematics of Intracellular Chlamydiae Provide Evidence for Contact-Dependent Development

A crucial process of chlamydial development involves differentiation of the replicative reticulate body (RB) into the infectious elementary body (EB). We present experimental evidence to provide support for a contact-dependent hypothesis for explaining the trigger involved in differentiation. We recorded live-imaging of Chlamydia trachomatis-infected McCoy cells at key times during development and tracked the temporospatial trajectories of individual chlamydial particles. We found that movement of the particles is related to development. Early to mid-developmental stages involved slight wobbling of RBs. The average speed of particles increased sharply at 24 h postinfection (after the estimated onset of RB to EB differentiation). We also investigated a penicillin-supplemented culture containing EBs, RBs, and aberrantly enlarged, stressed chlamydiae. Near-immobile enlarged particles are consistent with their continued tethering to the chlamydial inclusion membrane (CIM). We found a significantly negative, nonlinear association between speed and size/type of particles, providing further support for the hypothesis that particles become untethered near the onset of RB to EB differentiation. This study establishes the relationship between the motion properties of the chlamydiae and developmental stages, whereby wobbling RBs gradually lose contact with the CIM, and RB detachment from the CIM is coincidental with the onset of late differentiation.

Chlamydia pneumoniaeGroEL1 Protein Is Cell Surface Associated and Required for Infection of HEp-2 Cells

Chlamydia pneumoniae is an important obligate intracellular pathogen that replicates within an inclusion in the eukaryotic cell. The initial event of a chlamydial infection is the adherence to and subsequent uptake of the infectious elementary bodies (EBs) by the human cell. These processes require yet-unidentified bacterial and eukaryotic surface proteins. The GroEL1 protein, which exhibits a very strong antigenicity and in vitro can activate various eukaryotic cells, is a potential pathogenicity factor. We localized the protein during the infection process and found it in the inclusion but outside the chlamydial particles. GroEL1 was also localized on the surface of EBs, and the protein could be washed off the EBs. Latex beads coated with recombinantly produced GroEL1 (rGroEL1) bound in a dose-dependent manner to HEp-2 cells. Likewise, GroEL1, when expressed and displayed on the yeast cell surface, mediated adhesion to HEp-2 cells. Interestingly, the homologous GroEL2 and GroEL3 proteins showed no adhesive properties. Incubation of primary umbilical vein endothelial cells with soluble GroEL1 and GroEL1-coated latex beads activated the translocation of the general transcription factor NF-kappaB into the nucleus. Finally, preincubation of HEp-2 cells with rGroEL1 significantly reduced subsequent infection with C. pneumoniae, although adhesion of infectious bacteria to eukaryotic cells was not affected. Taken together, these data support a role for extracellular GroEL1 in the establishment of the chlamydial infection.

Effects of sustained antibiotic bactericidal treatment on Chlamydia trachomatis-infected epithelial-like cells (HeLa) and monocyte-like cells (THP-1 and U-937)

Chlamydia trachomatis is a human pathogen that causes multiple diseases worldwide. Despite appropriate therapy with existing antichlamydial antibiotics, chronic exacerbated diseases often occur and lead to serious sequelae. Since C. trachomatis has been found to enter a persistent state after exposure to deleterious conditions, the role of persistence in the failure of chlamydial antibiotherapy is questioned. HeLa, THP-1 and U-937 cells were infected with 10 4 C. trachomatis serovar L2 infectious particles. Three days later the infected cells were treated with minimal bactericidal concentrations of doxycycline (DOX), erythromycin (ERY) or tetracycline (TET) for 24 days or 30 days. Antibiotic efficacy was assessed by measuring chlamydial inclusions and infectious particles, by investigating the resumption of chlamydial growth after antibiotic removal and by testing Chlamydia viability using reverse transcriptase polymerase chain reaction targeting unprocessed 16S rRNA, processed 16S rRNA and Omp-1 mRNA. Treatment of infected HeLa cells with the usual antichlamydial antibiotics suppressed chlamydial active growth. The infection remained unapparent. However, 24 days post treatment the bacterium was found to be viable, as proved by continued expression of unprocessed and processed 16S rRNA and Omp-1 mRNA. This inactive unapparent chlamydial state is not infectious, suggesting Chlamydia persistence. Chlamydia trachomatis also developed persistence both in permissive THP-1 and non-permissive U-937 cells. Unlike in HeLa cells, persistent chlamydial infection in THP-1 and U-937 cells was resolved after 30 days of DOX treatment. Of interest, we noticed that only THP-1 and U-937 cells that were persistently infected following their interaction with infected HeLa cells remained capable of transmitting active infection to HeLa cells. These findings suggest that DOX, TET and ERY, usually administered to combat chlamydial diseases, fail to resolve persistent infection occurring during treatment in non-immune HeLa cells. However, in immune THP-1 and U-937 cells, the persistent infection is resolved by therapy with DOX. Epithelial cells could be the reservoir of persistent chlamydial particles.

A mathematical model for the investigation of the Th1 immune response to Chlamydia trachomatis

Chlamydia are bacterial pathogens of humans and animals causing the important human diseases trachoma, sexually transmitted chlamydial disease and pneumonia. Of the human chlamydial diseases, sexually transmitted disease caused by Chlamydia trachomatis is a major public health concern. Chlamydia trachomatis replicates intracellularly and is characterised by a complex developmental cycle. Chlamydia is susceptible to humoral and cell-mediated immunity. Here we investigate the Th1 cell-mediated immune response against Chlamydia-infected cells as the response changes over the chlamydial developmental cycle. We suggest a form for the immune response over one developmental cycle by modelling the change in the number of intracellular chlamydial particles and assume peptides are presented in proportion to the number of replicating forms of chlamydial particles. We predict, perhaps non-intuitively, that persistent Chlamydia should be induced and forced not to return to the lytic cycle. We also suggest that extending the length of the time of the lytic cycle will effectively decrease the required efficacy of the Th1 response to eliminate the pathogen. We produce plots of active disease progression, control and clearance for varying levels of Th1 effectiveness.

Dynamics of the development of Chlamydophila psittaci inclusions in epithelial and fibroblast host cells.

The development of Chlamydophila psittaci (formerly Chlamydia psittaci, avian strains) inclusions in fibroblast L-929 and epithelial BGM cell lines was studied along the bacterial growth cycle using a BGM cell-adapted strain in the presence or absence of cycloheximide and cycloheximide + polyethylene glycol. Evolution of the inclusions was determined in terms of their number and size at 24, 30, 36, 48 and 54 h after infection. Significant differences in the chlamydial growth were found between both host cells, throughout the study. Higher numbers of inclusions (P < 0.05) were observed in L cells while larger inclusions (P < 0.01) were found in BGM cells. In both fibroblast and epithelial cells, inclusions showed a significant (P < 0.001) increase in size at the later times studied. Free extracellular chlamydial particles were noticed at 48 and 54 h post-infection (p.i.) in infected L cells, and at 54 h p.i. in BGM cells. Addition of cycloheximide or cycloheximide + polyethylene glycol had no significant effect on the number of inclusions or their size. The results suggest that host cell characteristics and innate compatibility between Chlamydophila strain and host cell are more important than host cell adaptation for the development of the microorganism.

Chlamydia pneumoniae infection in human monocytes.

Chlamydia pneumoniae infection has been associated with cardiovascular diseases in seroepidemiological studies and by demonstration of the pathogen in atherosclerotic lesions. It has the capacity to infect several cell types, including monocyte-derived macrophages, which play an essential role in the development of atherosclerosis. However, the persistence of C. pneumoniae in mononuclear cells is poorly understood. To study the morphology and biological characteristics of the infection, human peripheral blood monocytes were infected with C. pneumoniae. Freshly isolated monocytes resisted the development of infectious progeny, and confocal and transmission electron microscopy showed that the morphology of the inclusions and chlamydial particles was abnormal. Addition of tryptophan or antibodies against gamma interferon did not diminish the inhibition of C. pneumoniae, suggesting that other factors are involved in the chlamydiostatic activity of the monocytes. Chlamydial mRNA was expressed at least 3 days after infection, however, and a capability for infected monocytes to induce a positive lymphocyte proliferative response was detected for up to 7 days, indicating that C. pneumoniae remains metabolically active in the monocytes in vitro. These results are in accordance with the hypothesis that C. pneumoniae may participate in the maintenance of local immunological response and inflammation via infected monocytes and thus enhance atherosclerosis.

Studies of persistent infection by Chlamydia trachomatis serovar K in TPA-differentiated U937 cells and the role of IFN-gamma.

Inoculation of phorbol ester-differentiated U937 cells as a model for human macrophages with Chlamydia trachomatis of the urogenital serovar K resulted in a persistent infection, with maximal growth at day 7, until day 10 post-infection. At these times inclusion bodies were present in 0.5-2% of the cells. Typical inclusion bodies containing elementary bodies and reticulate bodies were observed by electron microscopy. Furthermore, single chlamydial particles resembling atypical elementary or intermediate bodies were identified in the cytoplasm in > 80% of the host cells. IFN-gamma exerts antichlamydial activity in epithelial and fibroblastoid cells, but the infection of U937 cells by C. trachomatis was not affected by IFN-gamma. The activity of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) was not detected in untreated or in IFN-gamma-treated or chlamydiae-infected or mock-infected U937 cells. The presence of atypical persisting chlamydiae and the lack of IDO expression in U937 cells indicates that the development of these atypical bacteria is independent from IFN-gamma-mediated tryptophan deprivation and other IFN-gamma-mediated effects. Evaluation of persistently infected cells revealed that the expression of the chlamydial major outer-membrane protein, heat-shock protein (hsp60) and lipopolysaccharide (LPS) antigens was not significantly altered in the course of the culture. An intense staining of the LPS on the surface of the host cells was demonstrated by immunofluorescence. The data show that phorbol ester-differentiated U937 cells restrict chlamydial growth strongly but not completely through a mechanism distinct from IDO-mediated tryptophan deprivation. The mechanisms of persistence of chlamydiae in monocytes, which differ considerably from those described for other cells, require further investigation.