Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.     

Influence of cysteine deprivation on chlamydial differentiation from reproductive to infective life-cycle forms

The effects of omission of individual amino acids from growth medium on the differentiation of Chlamydia trachomatis DK-20 (serotype E) during infection of cycloheximide-treated McCoy cells are described. As judged by inclusion body staining with acridine orange, omission of cysteine from the medium severely retarded differentiation of reproductive reticulate body (RB) to infective elementary body (EB) forms. The effect appeared specific to cysteine in that omission of other amino acids had little or no effect on differentiation once RBs appeared. On restoration of cysteine, culture infectivity increased and inclusions contained organisms which, by cytochemical and morphological criteria, were differentiating to infective forms, indicating that cysteine deprivation did not irreversibly inhibit differentiation. Impairment of RB to EB differentiation in cysteine-less medium was also observed for three strains of Chlamydia psittaci and 10 other strains of C. trachomatis. It is suggested that the effect arises via the biosynthetic requirement for cysteine for provision of three cysteine-rich proteins, whose synthesis and insertion into the outer membrane have previously been shown to accompany RB to EB differentiation of C. psittaci 6BC and C. trachomatis 434 (serotype L2). Synthesis of cysteine-rich outer membrane proteins during differentiation may thus be common to all chlamydiae.     

Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms

Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography - tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyse the membranes of the pathogen-containing vacuole ('inclusion'). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of > 54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient type III secretion (T3S) in the RB stage at 18 h post infection, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a 'per organism' basis. Our results show that EB and RB proteomes are streamlined to fulfil their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs.     

Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae?

The effects of exogenous reducing agents on a number of biological properties of purified Chlamydia trachomatis LGV-434 and Chlamydia psittaci meningopneumonitis elementary bodies (EBs) have been examined in an attempt to identify in vitro correlates of early events in the differentiation of the infectious EB to the replicative cell type, the reticulate body (RB). Treatment of EBs with dithiothreitol elicited a number of changes normally associated with differentiation to the RB. EBs in the presence of 10 mM dithiothreitol displayed enhanced rates of [14C]glutamate oxidation, reduced infectivity, and decreased osmotic stability, and their Machiavello staining properties changed to those characteristic of the RB. A true differentiation of EB to RB did not take place under these conditions, since EBs treated in this manner and examined by transmission electron microscopy did not demonstrate increased size or decreased electron density as do isolated RBs. Additional studies were initiated to identify the macromolecules involved in this process. With polyacrylamide gel electrophoresis and immunoblotting procedures with monoclonal and polyclonal monospecific antibodies, the chlamydial major outer membrane protein was found to be the predominant component that varied under reducing versus nonreducing conditions. Furthermore, the extent of disulfide-mediated cross-linking of the major outer membrane protein varied between the infective and replicative forms of the C. trachomatis LGV-434 life cycle. Implications of disulfide interactions in the life cycle of chlamydiae are discussed.     

Immunoelectron microscopic localization of chlamydial lipopolysaccharide (LPS) in McCoy cells inoculated with Chlamydia trachomatis.

Interactions between Chlamydia trachomatis, host cells, and the immune system are believed to involve lipopolysaccharide (LPS). We used immunogold techniques to study the distribution of chlamydial LPS in cultured cells infected with C. trachomatis LGV-L1. McCoy cells inoculated with C. trachomatis were cultured and then fixed and embedded in situ with acrylic resins. Sections were immunolabeled with a protein A-gold method using antisera to the genus-specific, periodate-sensitive epitope on chlamydial LPS. Pre-embedding immunogold labeling on permeabilized cells was also done. By post-embedding methods, labeling for LPS was equally abundant over the outer membranes of elementary (EB) and reticulate bodies (RB). By post-embedding labeling, the sub-surface side of the EB outer membrane was more heavily labeled than the surface side. By pre-embedding labeling, LPS was found to be less abundant on the surface of EBs than RBs. Labeling for LPS was found over apparent lysosomes in McCoy cells and over electron-dense blebs on or near the surface of the plasma membranes of McCoy cells. These results indicate that the concentration of LPS in chlamydial membranes is constant during development but that with development its location changes from being mostly cell-surface to sub-surface. These results show that the post-embedding immunogold technique can be a useful approach for the cell culture-based study of chlamydial LPS.     

Initial characterization of a chlamydial receptor on mammalian cells

We have examined characteristics of the binding of eukaryotic cells to chlamydial elementary body (EB)-specific proteins. A wide variety of eukaryotic cell lines bound to representatives of both Chlamydia trachomatis lymphogranuloma venereum (LGV) and trachoma biovars and a C. psittaci strain meningopneumonitis (Mn) suggesting the presence of a common host cell receptor. Neither tunicamycin nor neuraminidase treatment of HeLa cells impaired binding to C. trachomatis EB, implying that host cell N-linked carbohydrate domains and sialic acid moieties, respectively, are not involved in attachment. However, trypsinized HeLa cells do not bind to EB, suggestive of a proteinaceous host cell receptor. The trypsin sensitivity of two EB-specific binding proteins Mr = 18,000 and 31,000) was also examined, and the finding that 125I-labeled HeLa cells bind both the 18,000 and 31,000-dalton proteins after chlamydial trypsinization corroborates our earlier observation that these EB binding proteins mediate attachment.     

Detection of surface-exposed epitopes on Chlamydia trachomatis by immune electron microscopy

The cell surfaces of two Chlamydia trachomatis serovars were explored by immune electron microscopy with monoclonal antibodies that recognize a number of chlamydial outer-membrane components. Species, subspecies and serovar-reactive epitopes on the major outer-membrane protein (MOMP) of a lymphogranuloma venereum biovar strain, L2/434/Bu, and a trachoma biovar strain, F/UW-6/Cx, were exposed on the surfaces of both elementary bodies (EBs) and reticulate bodies (RBs). Three epitopes on MOMP were inaccessible on EBs and RBs of both strains. These included a genus-reactive, species-reactive, and a subspecies-reactive epitope. In contrast, genus-specific epitopes on lipopolysaccharide (LPS) were not detected on the EB surface, but were clearly expressed on RBs of both L2/434/Bu and F/UW-6/Cx chlamydiae. Antibodies specific for the 60 kDa and 12 kDa 'cysteine-rich' outer-membrane proteins did not react with surface epitopes on either EBs or RBs. These data provide evidence that MOMP is a major surface antigen of both morphological forms, whereas some portions of the LPS molecule are exposed on the RB surface but become inaccessible to antibody after conversion to the infectious EB form.     

Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle

The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.     

Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.     

An RpoH-like heat shock sigma factor is involved in stress response and virulence in Brucella melitensis 16M

B. melitensis 16M genome analysis revealed the presence of six putative sigma factor-encoding genes: rpoD, rpoH1, rpoH2, rpoE1, rpoE2, and rpoN. We mutated all these genes except rpoD. Phenotypic analysis of the mutants reveals that a strain carrying an rpoH2 null mutation (DeltarpoH2) is impaired for growth at 21 and 42 degrees C and shows increased sensitivity to hydrogen peroxide. Compared to the wild-type strain, the DeltarpoH2 mutant is attenuated in all virulence models tested. Three other null mutants (DeltarpoH1, DeltarpoE1, and DeltarpoE2 mutants) are also defective for survival in mice at 4 weeks postinfection. We also demonstrated that rpoH2 deletion strongly reduces the expression of two major virulence factors in B. melitensis, the type IV secretion system and the flagellum.     

The persistence of Chlamydia trachomatis elementary body cell walls in human polymorphonuclear leucocytes and induction of a chemiluminescent response

Human polymorphonuclear leucocytes (HPMN) were incubated with [35S]methionine-labelled Chlamydia trachomatis (serovar L2/434/Bu) elementary bodies (EB) and EB cell walls. No net loss in the TCA-precipitable radioactivity was observed over 24 h in the HPMN that had taken up EB cell walls. SDS-polyacrylamide gel electrophoresis of the labelled C. trachomatis EB and EB cell wall proteins extracted from the HPMN at 2 and 24 h after infection demonstrated the persistence of most of the chlamydial cell wall polypeptides. Analysis of extracts of the HPMN that had taken up either EB or EB cell walls on Urografin density gradients at 2 and 24 h after infection, and electron microscopic observations on fractions representing the peaks, demonstrated the persistence of the EB cell walls in the HPMN. Electron microscopic observations of HPMN that had taken up EB or EB cell walls demonstrated EB cell walls in the HPMN phagosomes at 24 h after infection. The HPMN exposed to EB and EB cell walls of C. trachomatis gave chemiluminescent (CL) responses with peaks respectively 12 and 7 times greater than the peak value of the control. The significance of the persistence of the EB cell wall polypeptides and cell walls in the HPMN and activation of the HPMN to produce oxygen radicals (i.e. a CL response), and its possible relation to rheumatic diseases, is discussed.     

Drosophila melanogaster S2 cells: a model system to study Chlamydia interaction with host cells

Chlamydia spp. are major causes of important human diseases, but dissecting the host-pathogen interactions has been hampered by the lack of bacterial genetics and the difficulty in carrying out forward genetic screens in mammalian hosts. RNA interference (RNAi)-based methodologies for gene inactivation can now be easily carried out in genetically tractable model hosts, such as Drosophila melanogaster, and offer a new approach to identifying host genes required for pathogenesis. We tested whether Chlamydia trachomatis infection of D. melanogaster S2 cells recapitulated critical aspects of mammalian cell infections. As in mammalian cells, C. trachomatis entry was greatly reduced by heparin and cytochalasin D. Inclusions were formed in S2 cells, acquired Golgi-derived sphingolipids, and avoided phagolysosomal fusion. Elementary body (EB) to reticulate body (RB) differentiation was observed, however, no RB to EB development or host cell killing was observed. RNAi-mediated inactivation of Rac, a Rho GTPase recently shown to be required for C. trachomatis entry in mammalian cells, inhibits C. trachomatis infection in S2 cells. We conclude that Drosophila S2 cells faithfully mimic early events in Chlamydia host cell interactions and provides a bona fide system to systematically dissect host functions important in the pathogenesis of obligate intracellular pathogens.     

Interactions between CdsD, CdsQ, and CdsL, three putative Chlamydophila pneumoniae type III secretion proteins

Chlamydophila pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes pneumonia and bronchitis and may contribute to atherosclerosis. The developmental cycle of C. pneumoniae includes a morphological transition from an infectious extracellular elementary body (EB) to a noninfectious intracellular reticulate body (RB) that divides by binary fission. The C. pneumoniae genome encodes a type III secretion (T3S) apparatus that may be used to infect eukaryotic cells and to evade the host immune response. In the present study, Cpn0712 (CdsD), Cpn0704 (CdsQ), and Cpn0826 (CdsL), three C. pneumoniae genes encoding yersiniae T3S YscD, YscQ, and YscL homologs, respectively, were cloned and expressed as histidine- and glutathione S-transferase (GST)-tagged proteins in Escherichia coli. Purified recombinant proteins were used to raise hyper-immune polyclonal antiserum and were used in GST pull-down and copurification assays to identify protein-protein interactions. CdsD was detected in both EB and RB lysates by Western blot analyses, and immunofluorescent staining demonstrated the presence of CdsD within inclusions. Triton X-114 solubilization and phase separation of chlamydial EB proteins indicated that CdsD partitions with cytoplasmic proteins, suggesting it is not an integral membrane protein. GST pull-down assays indicated that recombinant CdsD interacts with CdsQ and CdsL, and copurification assays with chlamydial lysates confirmed that native CdsD interacts with CdsQ and CdsL. To the best of our knowledge, this is the first report demonstrating interactions between YscD, YscQ, and YscL homologs of bacterial T3S systems. These novel protein interactions may play important roles in the assembly or function of the chlamydial T3S apparatus.