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.Members of the Chlamydiaceae are ubiquitous bacterial pathogens in humans and animals. While many primary chlamydial infections are asymptomatic or of limited severity, severe disease and the most serious sequelae are thought to be associated with chronic or persistent infection or repeat infections that may occur over years or decades. In the laboratory, cultured eukaryotic cells such as HEp-2 or McCoy cells and various animal models are used as suitable model systems for primary chlamydial infection. Under these optimized conditions, chlamydiae undergo a typical developmental cycle, which is highly conserved across the genus. Initial internalization of the infectious chlamydial elementary body (EB) particle occurs within the first 2 h, followed shortly by differentiation of the EB into the chlamydial replicative form, the reticulate body (RB). RBs are thought to multiply exponentially, replicating their DNA every 2 to 3 h for approximately 6 to 10 generations. At 16+ h postinfection (hpi), an unknown signal provokes the onset of RB to EB differentiation, whereby individual RBs engage in a cellular condensation process, progressing through a poorly defined intermediate body (IB) form and ending with the metabolically inactive but highly infectious EB, thereby closing the developmental “cycle.” Unlike the initial differentiation step which can be reasonably well synchronized, the late differentiation step is always asynchronous. Indeed, few EBs can be observed in relatively young inclusions, while significant numbers of RBs can often be seen in late inclusions (for C. trachomatis, these stages occur at approximately 20 and 48 hpi, respectively). Another perennial observation is that RBs are often observed in association with the chlamydial inclusion membrane (CIM) (21, 22, 27, 37), the membrane of the parasitophorous vacuole that contains the chlamydiae and is derived from the host cell plasma membrane (14). This is further supported by various imaging methods including cryo-electron microscopy (14), indirect immunofluorescence, confocal electron microscopy, and Nomarski differential interference contrast imaging (10, 15, 16, 31).Models for persistent or chronic infection have also been established both in vitro and in animal models. Under conditions that induce a classical stress response in many bacteria, such as exposure to gamma interferon (3) or penicillin (23, 34), infection with phage (18), or deprivation of iron (30) or amino acids (9), chlamydial RBs undergo a dramatic morphological change to nondividing, aberrantly enlarged RBs (termed maxiRBs or mRBs) that will not differentiate into EBs (3, 23, 30, 34). Coincidental to the morphological change, expression of stress response genes is upregulated (e.g., hsp60) while expression of genes thought to be involved in late differentiation (e.g., omcB) is blocked (5, 6, 13). Because mRBs may be kept in culture for several weeks (except for phage-induced stress) and removal of the stressor “unlocks” development and allows resumption of late differentiation to EBs, the stress response of the chlamydiae is thought to represent a suitable in vitro model for persistent infection (1, 2, 4, 25).We have previously proposed a model for chlamydial development that reconciles many of the observations outlined above (26, 35). Two essential features of the model are the replication of RBs in type III secretion (T3S)-mediated contact with the CIM and the disruption of T3S activity through physical detachment from the CIM, which is associated with RB to EB differentiation. This so-called “contact-dependent” model has several important theoretical implications. First, an RB that actively translocates T3S effector proteins through the CIM (i.e., an RB tethered to the CIM) should not differentiate into an EB. Second and correlated to the first, an RB whose T3S system remains active for extended periods of time, by definition, should represent a persisting chlamydial cell. Biomathematical simulations predict two situations under which detachment of RBs from the CIM is physically restricted: the case of normal size RBs in a small, tight inclusion and that of abnormally large RB(s) in normal size inclusions (17, 35). The biological relevance of these simulations lies in the frequent occurrence of multiple small or lobar inclusions, e.g., for Chlamydia pneumoniae and Chlamydia caviae, in a single infected cell and the observation of stress-induced mRBs, respectively. In either case, because of the imposed spatial constraints, disruption of contact-induced T3S activity through physical RB detachment becomes a statistical rarity as the RB/mRB size approaches that of the inclusion that contains it. Remarkably, therefore, the observed in vitro persistence is not only a prediction of the biomathematical model but an implication of it as well.We now present experimental evidence using innovative real-time light microscopy that provides some support to the contact-dependent hypothesis in its most fundamental aspects. We captured images of C. trachomatis-infected McCoy cells at key times during development using a Richardson RTM3 microscope optimized for live-cell imaging in extreme dark field (28). Live images were obtained with a high-resolution color analogue output video camera and recorded with Volocity software (Improvision, Coventry, United Kingdom). Taped imaging sequences were captured in Final Cut Pro and converted to Quicktime movies. We then analyzed these movies to obtain the spatial time-dependent trajectories of the movement of individual chlamydial particles in each infected cell, allowing kinematic calculations of the displacement and speed of individual particles.     

The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens

Background

Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.

Results

A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.

Conclusions

This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions.     

Electron tomography and cryo-SEM characterization reveals novel ultrastructural features of host-parasite interaction during Chlamydia abortus infection

Chlamydia (C.) abortus is a widely spread pathogen among ruminants that can be transmitted to women during pregnancy leading to severe systemic infection with consecutive abortion. As a member of the Chlamydiaceae, C. abortus shares the characteristic feature of an obligate intracellular biphasic developmental cycle with two morphological forms including elementary bodies (EBs) and reticulate bodies (RBs). In contrast to other chlamydial species, C. abortus ultrastructure has not been investigated yet. To do so, samples were fixed by high-pressure freezing and processed by different electron microscopic methods. Freeze-substituted samples were analysed by transmission electron microscopy, scanning transmission electron microscopical tomography and immuno-electron microscopy, and freeze-fractured samples were analysed by cryo-scanning electron microscopy. Here, we present three ultrastructural features of C. abortus that have not been reported up to now. Firstly, the morphological evidence that C. abortus is equipped with the type three secretion system. Secondly, the accumulation and even coating of whole inclusion bodies by membrane complexes consisting of multiple closely adjacent membranes which seems to be a C. abortus specific feature. Thirdly, the formation of small vesicles in the periplasmic space of RBs in the second half of the developmental cycle. Concerning the time point of their formation and the fact that they harbour chlamydial components, these vesicles might be morphological correlates of an intermediate step during the process of redifferentiation of RBs into EBs. As this feature has also been shown for C. trachomatis and C. pneumoniae, it might be a common characteristic of the family of Chlamydiaceae.     

Genomic factors related to tissue tropism in Chlamydia pneumoniae infection

Background

Chlamydia pneumoniae (Cpn) are obligate intracellular bacteria that cause acute infections of the upper and lower respiratory tract and have been implicated in chronic inflammatory diseases. Although of significant clinical relevance, complete genome sequences of only four clinical Cpn strains have been obtained. All of them were isolated from the respiratory tract and shared more than 99% sequence identity. Here we investigate genetic differences on the whole-genome level that are related to Cpn tissue tropism and pathogenicity.

Results

We have sequenced the genomes of 18 clinical isolates from different anatomical sites (e.g. lung, blood, coronary arteries) of diseased patients, and one animal isolate. In total 1,363 SNP loci and 184 InDels have been identified in the genomes of all clinical Cpn isolates. These are distributed throughout the whole chlamydial genome and enriched in highly variable regions. The genomes show clear evidence of recombination in at least one potential region but no phage insertions. The tyrP gene was always encoded as single copy in all vascular isolates. Phylogenetic reconstruction revealed distinct evolutionary lineages containing primarily non-respiratory Cpn isolates. In one of these, clinical isolates from coronary arteries and blood monocytes were closely grouped together. They could be distinguished from all other isolates by characteristic nsSNPs in genes involved in RB to EB transition, inclusion membrane formation, bacterial stress response and metabolism.

Conclusions

This study substantially expands the genomic data of Cpn and elucidates its evolutionary history. The translation of the observed Cpn genetic differences into biological functions and the prediction of novel pathogen-oriented diagnostic strategies have to be further explored.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1377-8) contains supplementary material, which is available to authorized users.     

The Effect of Multiple Rounds of Mass Drug Administration on the Association between Ocular Chlamydia trachomatis Infection and Follicular Trachoma in Preschool-Aged Children

Purpose

To examine the relationship between ocular Chlamydia trachomatis infection and follicular trachoma (TF) in children prior to and following multiple rounds of annual mass drug administration (MDA) with azithromycin.

Methodology/principal findings

Thirty-two communities with endemic trachoma in Kongwa District, Tanzania, were offered annual MDA as part of a district-wide trachoma control program. Presence of ocular C. trachomatis infection and TF were assessed in 3,200 randomly sampled children aged five years and younger, who were examined prior to each MDA. Infection was detected using the Amplicor CT/NG assay and TF was identified by clinical examination using the World Health Organization (WHO) simplified grading system. The association between chlamydial infection and TF in children was evaluated at baseline prior to any treatment, and 12 months after each of three annual rounds of mass treatment. Factors associated with infection were examined using generalized estimating equation models.At baseline, the overall prevalence of chlamydial infection and TF was 22% and 31%, respectively. Among children with clinical signs of TF, the proportion of those with infection was 49% prior to treatment and declined to 30% after three MDAs. The odds of infection positivity among children with clinical signs of TF decreased by 26% (OR 0.74, 95% CI 0.65 to 0.84, p = <0.01) with each MDA, after adjusting for age. For children aged under one year, who did not receive treatment, the relationship was unchanged.

Conclusions/significance

The association between ocular C. trachomatis infection and TF weakened in children with each MDA, as both infection and clinical disease prevalence declined. However, there was still a significant proportion of TF cases with infection after three rounds of MDA. New strategies are needed to assess this residual infection for optimal treatment distribution.     

Mitotic defects lead to pervasive aneuploidy and accompany loss of RB1 activity in mouse LmnaDhe dermal fibroblasts

Background

Lamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood.

Results

We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (Lmna^Dhe). We found that dermal fibroblasts from heterozygous Lmna^Dhe (Lmna^Dhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, Lmna^Dhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in Lmna^{Dhe /+} cells. Lmna^{Dhe /+} fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects.

Conclusions

These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control.     

Whole genome sequencing of penicillin-resistant Streptococcus pneumoniae reveals mutations in penicillin-binding proteins and in a putative iron permease

Background

Penicillin resistance in Streptococcus pneumoniae is mediated by a mosaic of genes encoding altered penicillin-binding proteins (PBPs). Nonetheless, S. pneumoniae has also developed non-PBP mechanisms implicated in penicillin resistance. In this study, whole genome sequencing of resistant organisms was used to discover mutations implicated in resistance to penicillin.

Results

We sequenced two S. pneumoniae isolates selected for resistance to penicillin in vitro. The analysis of the genome assemblies revealed that six genes were mutated in both mutants. These included three pbp genes, and three non-pbp genes, including a putative iron permease, spr1178. The nonsense mutation in spr1178 always occurred in the first step of the selection process. Although the mutants had increased resistance to penicillin, the introduction of altered versions of PBPs into a penicillin-susceptible strain by sequential transformation led to strains with a minimal increase in resistance, thus implicating other genes in resistance. The introduction by transformation of the non-PBP recurrent mutations did not increase penicillin resistance, but the introduction of the nonsense mutation in the putative iron permease spr1178 led to a reduced accumulation of reactive oxygen species following exposure to penicillin and to other bactericidal antibiotics as well.

Conclusions

This study indicates that the selection of resistance to penicillin in S. pneumoniae involves the acquisition of mutations conferring tolerance to the antibiotic-induced accumulation of oxidants, which translates into an increased survival that putatively enables the selection of major resistance determinants such as mutations in PBPs.     

Fluorescence lifetime imaging unravels C. trachomatis metabolism and its crosstalk with the host cell

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ₂-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ₂-NAD(P)H increase inside the chlamydial inclusion. τ₂-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ₂-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.     

Interruption of CXCL13-CXCR5 Axis Increases Upper Genital Tract Pathology and Activation of NKT Cells following Chlamydial Genital Infection

Background

Regulation of immune responses is critical for controlling inflammation and disruption of this process can lead to tissue damage. We reported that CXCL13 was induced in fallopian tube tissue following C. trachomatis infection. Here, we examined the influence of the CXCL13-CXCR5 axis in chlamydial genital infection.

Methodology and Principal Findings

Disruption of the CXCL13-CXCR5 axis by injecting anti-CXCL13 Ab to BALB/c mice or using Cxcr5−/− mice increased chronic inflammation in the upper genital tract (UGT; uterine horns and oviducts) after Chlamydia muridarum genital infection (GT). Further studies in Cxcr5−/− mice showed an elevation in bacterial burden in the GT and increased numbers of neutrophils, activated DCs and activated NKT cells early after infection. After resolution, we noted increased fibrosis and the accumulation of a variety of T cells subsets (CD4-IFNγ, CD4-IL-17, CD4-IL-10 & CD8-TNFα) in the oviducts. NKT cell depletion in vitro reduced IL-17α and various cytokines and chemokines, suggesting that activated NKT cells modulate neutrophils and DCs through cytokine/chemokine secretion. Further, chlamydial glycolipids directly activated two distinct types of NKT cell hybridomas in a cell-free CD1d presentation assay and genital infection of Cd1d−/− mice showed reduced oviduct inflammation compared to WT mice. CXCR5 involvement in pathology was also noted using single-nucleotide polymorphism analysis in C. trachomatis infected women attending a sub-fertility clinic. Women who developed tubal pathology after a C. trachomatis infection had a decrease in the frequency of CXCR5 SNP +10950 T>C (rs3922).

Conclusions/Significance

These experiments indicate that disruption of the CXCL13-CXCR5 axis permits increased activation of NKT cells by type I and type II glycolipids of Chlamydia muridarum and results in UGT pathology potentially through increased numbers of neutrophils and T cell subsets associated with UGT pathology. In addition, CXCR5 appears to contribute to inter-individual differences in human tubal pathology following C. trachomatis infection.     

Exploration of Chlamydial Type III Secretion System Reconstitution in Escherichia coli

Background

Type III secretion system is a virulent factor for many pathogens, and is thought to play multiple roles in the development cycle and pathogenesis of chlamydia, an important human pathogen. However, due to the obligate intracellular parasitical nature of chlamydiae and a lack of convenient genetic methodology for the organisms, very limited approaches are available to study the chlamydial type III secretion system. In this study, we explored the reconstitution of a chlamydial type III secretion in Escherichia coli.

Results

We successfully cloned all 6 genomic DNA clusters of the chlamydial type III secretion system into three bacterial plasmids. 5 of the 6 clusters were found to direct mRNA synthesis from their own promoters in Escherichia coli transformed with the three plasmids. Cluster 5 failed to express mRNA using its own promoters. However, fusion of cluster 5 to cluster 6 resulted in the expression of cluster 5 mRNA. Although only two of the type III secretion system proteins were detected transformed E. coli due to limited antibody availability, type III secretion system-like structures were detected in ultrathin sections in a small proportion of transformed E. coli.

Conclusions

We have successfully generated E. coli expressing all genes of the chlamydial type III secretion system. This serves as a foundation for optimal expression and assembly of the recombinant chlamydial type III secretion system, which may be extremely useful for the characterization of the chlamydial type III secretion system and for studying its role in chlamydial pathogenicity.     

Distinct Transmission Networks of Chlamydia trachomatis in Men Who Have Sex with Men and Heterosexual Adults in Amsterdam,The Netherlands

Background

Genovar distributions of Chlamydia trachomatis based on ompA typing differ between men who have sex with men (MSM) and heterosexuals. We investigated clonal relationships using a high resolution typing method to characterize C. trachomatis types in these two risk groups.

Methods

C. trachomatis positive samples were collected at the STI outpatient clinic in Amsterdam between 2008 and 2010 and genotyped by multilocus sequence typing. Clusters were assigned using minimum spanning trees and these were combined with epidemiological data of the hosts.

Results

We typed 526 C. trachomatis positive samples: 270 from MSM and 256 from heterosexuals. Eight clusters, containing 10–128 samples were identified of which 4 consisted of samples from MSM (90%–100%), with genovars D, G, J, and L2b. The other 4 clusters consisted mainly of samples from heterosexuals (87%–100%) with genovars D, E, F, I, and J. Genetic diversity was much lower in the MSM clusters than in heterosexual clusters. Significant differences in number of sexual partners and HIV-serostatus were observed for MSM–associated clusters.

Conclusions

C. trachomatis transmission patterns among MSM and heterosexuals were largely distinct. We hypothesize that these differences are due to sexual host behavior, but bacterial factors may play a role as well.     

Successive microsporogenesis affects pollen aperture pattern in the tam mutant of Arabidopsis thaliana

Background and Aims

The tam (tardy asynchronous meiosis) mutant of Arabidopsis thaliana, which exhibits a modified cytokinesis with a switch from simultaneous to successive cytokinesis, was used to perform a direct test of the implication of cytokinesis in aperture-pattern ontogeny of angiosperm pollen grains. The aperture pattern corresponds to the number and arrangement of apertures (areas of the pollen wall permitting pollen tube germination) on the surface of the pollen grain.

Methods

A comparative analysis of meiosis and aperture distribution was performed in two mutant strains of arabidopsis: quartet and quartet-tam.

Key Results

While the number of apertures is not affected in the quartet-tam mutant, the arrangement of the three apertures is modified compared with the quartet, resulting in a different aperture pattern.

Conclusions

These results directly demonstrate the relationship between the type of sporocytic cytokinesis and pollen aperture-pattern ontogeny.     

Intramuscular Immunisation with Chlamydial Proteins Induces Chlamydia trachomatis Specific Ocular Antibodies

Background

Ocular infection with Chlamydia trachomatis can cause trachoma, which is the leading cause of blindness due to infection worldwide. Despite the large-scale implementation of trachoma control programmes in the majority of countries where trachoma is endemic, there remains a need for a vaccine. Since C. trachomatis infects the conjunctival epithelium and stimulates an immune response in the associated lymphoid tissue, vaccine regimens that enhance local antibody responses could be advantageous. In experimental infections of non-human primates (NHPs), antibody specificity to C. trachomatis antigens was found to change over the course of ocular infection. The appearance of major outer membrane protein (MOMP) specific antibodies correlated with a reduction in ocular chlamydial burden, while subsequent generation of antibodies specific for PmpD and Pgp3 correlated with C. trachomatis eradication.

Methods

We used a range of heterologous prime-boost vaccinations with DNA, Adenovirus, modified vaccinia Ankara (MVA) and protein vaccines based on the major outer membrane protein (MOMP) as an antigen, and investigated the effect of vaccine route, antigen and regimen on the induction of anti-chlamydial antibodies detectable in the ocular lavage fluid of mice.

Results

Three intramuscular vaccinations with recombinant protein adjuvanted with MF59 induced significantly greater levels of anti-MOMP ocular antibodies than the other regimens tested. Intranasal delivery of vaccines induced less IgG antibody in the eye than intramuscular delivery. The inclusion of the antigens PmpD and Pgp3, singly or in combination, induced ocular antigen-specific IgG antibodies, although the anti-PmpD antibody response was consistently lower and attenuated by combination with other antigens.

Conclusions

If translatable to NHPs and/or humans, this investigation of the murine C. trachomatis specific ocular antibody response following vaccination provides a potential mouse model for the rapid and high throughput evaluation of future trachoma vaccines.     

Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells

Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inc lusion membrane protein A ) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.     

CT358蛋白在沙眼衣原体感染细胞中的定位及特性分析

确定沙眼衣原体CT358蛋白在衣原体感染细胞中的位置并初步鉴定其生物学功能．采用PCR方法从D型沙眼衣原体的基因组中扩增CT358基因,并克隆入pGEX和pDSRedC1表达载体中．将重组质粒pGEX-CT358转化到XL1-blue宿主菌,并诱导表达融合蛋白GST-CT358．纯化后的CT358融合蛋白免疫小鼠制备抗体,应用间接免疫荧光技术对CT358蛋白在衣原体感染细胞内的定位及表达模式进行分析．同时,pDSRedC1-CT358重组质粒瞬时转染HeLa细胞,观察CT358蛋白对衣原体感染的影响．实验结果证明CT358蛋白为沙眼衣原体包涵体膜蛋白．该蛋白质在衣原体感染12 h后就表达定位于包涵体膜上,直至持续到整个感染周期,转基因在胞浆表达的CT358融合蛋白不影响其后的衣原体感染．该研究为深入研究衣原体与宿主细胞间相互作用提供了新的线索,并可为衣原体性的治疗、预防提供新方向．     

Immunobiological Outcomes of Repeated Chlamydial Infection from Two Models of Within-Host Population Dynamics

Background

Chlamydia trachomatis is a common human pathogen that mediates disease processes capable of inflicting serious complications on reproduction. Aggressive inflammatory immune responses are thought to not only direct a person''s level of immunity but also the potential for immunopathology. With human immunobiology being debated as a cause of prevailing epidemiological trends, we examined some fundamental issues regarding susceptibility to multiple chlamydial infections that could have implications for infection spread. We argue that, compared to less-frequent exposure, frequent exposure to chlamydia may well produce unique immunobiological characteristics that likely to have important clinical and epidemiological implications.

Methods and Results

As a novel tool for studying chlamydia, we applied principles of modeling within-host pathogen dynamics to enable an understanding of some fundamental characteristics of an individual''s immunobiology during multiple chlamydial infections. While the models were able to reproduce shorter-term infection kinetics of primary and secondary infections previously observed in animal models, it was also observed that longer periods between initial and second infection may increase an individual''s chlamydial load and lengthen their duration of infectiousness. The cessation of short-term repeated exposure did not allow for the formation of long-lasting immunity. However, frequent re-exposure non-intuitively linked the formation of protective immunity, persistent infection, and the potential for immunopathology.

Conclusions

Overall, these results provide interesting insights that should be verified with continued study. Nevertheless, these results appear to raise challenges for current evidence of the development of long-lasting immunity against chlamydia, and suggest the existence of a previously unidentified mechanism for the formation of persistent infection. The obvious next goal is to investigate the qualitative impact of these results on the spread of chlamydia.