Biphasic Developmental Cycle Research Articles

Chlamydia-infected cells shed Gp96 to prevent chlamydial re-infection.

Chlamydia trachomatis is an obligate intracellular human pathogen with a biphasic developmental life cycle. The infectious elementary bodies (EBs) enter a host cell where they transform into reticulate bodies (RBs) that use cellular metabolites to multiply. Re-infection of an infected cell during the replicative phase of chlamydial development may prevent formation of infectious EBs, interrupting the infectious cycle. Here, we report that Glucose Regulated Protein 96 (Gp96), a chaperone for cell surface receptors, binds to and facilitates adherence and entry of C. trachomatis. Gp96 expression was increased early in infection in a MAP kinase-dependent way, thereby increasing chlamydial adherence and invasion. Gp96 co-precipitated with Protein Disulphide Isomerase (PDI), known to be involved in chlamydial host cell entry. During the replicative phase, Gp96 was depleted from infected cells and shed into the supernatant by activation of metalloproteinase TACE (ADAM17). Loss of Gp96 also reduced the activity of PDI on the cell surface. Reduced surface display of Gp96 prevented chlamydial re-infection in a TACE-dependent manner in cell lines but also in primary cells derived from human fimbriae, the natural site of chlamydial infection. Our data suggest a role of infection-induced Gp96 shedding in the protection of the chlamydial replicative niche.

The Rsb Phosphoregulatory Network Controls Availability of the Primary Sigma Factor in Chlamydia trachomatis and Influences the Kinetics of Growth and Development.

Chlamydia trachomatis is an obligate intracellular human pathogen that exhibits stage-specific gene transcription throughout a biphasic developmental cycle. The mechanisms that control modulation in transcription and associated phenotypic changes are poorly understood. This study provides evidence that a switch-protein kinase regulatory network controls availability of σ66 , the main sigma subunit for transcription in Chlamydia. In vitro analysis revealed that a putative switch-protein kinase regulator, RsbW, is capable of interacting directly with σ66, as well as phosphorylating its own antagonist, RsbV1, rendering it inactive. Conversely, the putative PP2C-like phosphatase domain of chlamydial RsbU was capable of reverting RsbV1 into its active state. Recent advances in genetic manipulation of Chlamydia were employed to inactivate rsbV1, as well as to increase the expression levels of rsbW or rsbV1, in vivo. Representative σ66-dependent gene transcription was repressed in the absence of rsbV1 or upon increased expression of RsbW, and increased upon elevated expression of RsbV1. These effects on housekeeping transcription were also correlated to several measures of growth and development. A model is proposed where the relative levels of active antagonist (RsbV1) and switch-protein anti-sigma factor (RsbW) control the availability of σ66 and subsequently act as a molecular 'throttle' for Chlamydia growth and development.

Damage/Danger Associated Molecular Patterns (DAMPs) Modulate Chlamydia pecorum and C. trachomatis Serovar E Inclusion Development In Vitro.

Persistence, more recently termed the chlamydial stress response, is a viable but non-infectious state constituting a divergence from the characteristic chlamydial biphasic developmental cycle. Damage/danger associated molecular patterns (DAMPs) are normal intracellular components or metabolites that, when released from cells, signal cellular damage/lysis. Purine metabolite DAMPs, including extracellular ATP and adenosine, inhibit chlamydial development in a species-specific manner. Viral co-infection has been shown to reversibly abrogate Chlamydia inclusion development, suggesting persistence/chlamydial stress. Because viral infection can cause host cell DAMP release, we hypothesized DAMPs may influence chlamydial development. Therefore, we examined the effect of extracellular ATP, adenosine, and cyclic AMP exposure, at 0 and 14 hours post infection, on C. pecorum and C. trachomatis serovar E development. In the absence of de novo host protein synthesis, exposure to DAMPs immediately post or at 14 hours post infection reduced inclusion size; however, the effect was less robust upon 14 hours post infection exposure. Additionally, upon exposure to DAMPs immediately post infection, bacteria per inclusion and subsequent infectivity were reduced in both Chlamydia species. These effects were reversible, and C. pecorum exhibited more pronounced recovery from DAMP exposure. Aberrant bodies, typical in virus-induced chlamydial persistence, were absent upon DAMP exposure. In the presence of de novo host protein synthesis, exposure to DAMPs immediately post infection reduced inclusion size, but only variably modulated chlamydial infectivity. Because chlamydial infection and other infections may increase local DAMP concentrations, DAMPs may influence Chlamydia infection in vivo, particularly in the context of poly-microbial infections.

Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms.

Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.

Effects of Mentha suaveolens essential oil on Chlamydia trachomatis.

Chlamydia trachomatis, the most common cause of sexually transmitted bacterial infection worldwide, has a unique biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body. C. trachomatis is responsible for severe reproductive complications including pelvic inflammatory disease, ectopic pregnancy, and obstructive infertility. The aim of our study was to evaluate whether Mentha suaveolens essential oil (EOMS) can be considered as a promising candidate for preventing C. trachomatis infection. Specifically, we investigated the in vitro effects of EOMS towards C. trachomatis analysing the different phases of chlamydial developmental cycle. Our results demonstrated that EOMS was effective towards C. trachomatis, whereby it not only inactivated infectious elementary bodies but also inhibited chlamydial replication. Our study also revealed the effectiveness of EOMS, in combination with erythromycin, towards C. trachomatis with a substantial reduction in the minimum effect dose of antibiotic. In conclusion, EOMS treatment may represent a preventative strategy since it may reduce C. trachomatis transmission in the population and, thereby, reduce the number of new chlamydial infections and risk of developing of severe sequelae.

The many forms of a pleomorphic bacterial pathogen-the developmental network of Legionella pneumophila.

Legionella pneumophila is a natural intracellular bacterial parasite of free-living freshwater protozoa and an accidental human pathogen that causes Legionnaires' disease. L. pneumophila differentiates, and does it in style. Recent experimental data on L. pneumophila's differentiation point at the existence of a complex network that involves many developmental forms. We intend readers to: (i) understand the biological relevance of L. pneumophila's forms found in freshwater and their potential to transmit Legionnaires' disease, and (ii) learn that the common depiction of L. pneumophila's differentiation as a biphasic developmental cycle that alternates between a replicative and a transmissive form is but an oversimplification of the actual process. Our specific objectives are to provide updates on the molecular factors that regulate L. pneumophila's differentiation (Section The Differentiation Process and Its Regulation), and describe the developmental network of L. pneumophila (Section Dissecting Lp's Developmental Network), which for clarity's sake we have dissected into five separate developmental cycles. Finally, since each developmental form seems to contribute differently to the human pathogenic process and the transmission of Legionnaires' disease, readers are presented with a challenge to develop novel methods to detect the various L. pneumophila forms present in water (Section Practical Implications), as a means to improve our assessment of risk and more effectively prevent legionellosis outbreaks.

Twenty years of research into Chlamydia-like organisms: a revolution in our understanding of the biology and pathogenicity of members of the phylum Chlamydiae.

Chlamydiae are obligate intracellular bacteria that share a unique but remarkably conserved biphasic developmental cycle that relies on a eukaryotic host cell for survival. Although the phylum was originally thought to only contain one family, the Chlamydiaceae, a total of nine families are now recognized. These so-called Chlamydia-like organisms (CLOs) are also referred to as 'environmental chlamydiae', as many were initially isolated from environmental sources. However, these organisms are also emerging pathogens, as many, such as Parachlamydia sp., Simkania sp. and Waddlia sp., have been associated with human disease, and others, such as Piscichlamydia sp. and Parilichlamydia sp., have been documented in association with diseases in animals. Their strict intracellular nature and the requirement for cell culture have been a confounding factor in characterizing the biology and pathogenicity of CLOs. Nevertheless, the genomes of seven CLO species have now been sequenced, providing new information on their potential ability to adapt to a wide range of hosts. As new isolation and diagnostic methods advance, we are able to further explore the richness of this phylum with further research likely to help define the true pathogenic potential of the CLOs while also providing insight into the origins of the 'traditional' chlamydiae.

Conserved features and major differences in the outer membrane protein composition of chlamydiae.

Chlamydiae are a highly successful group of obligate intracellular bacteria infecting a variety of eukaryotic hosts. Outer membrane proteins involved in attachment to and uptake into host cells, and cross-linking of these proteins via disulfide bonds are key features of the biphasic chlamydial developmental cycle. In this study, we used a consensus approach to predict outer membrane proteins in the genomes of members of three chlamydial families. By analysing outer membrane protein fractions of purified chlamydiae with highly sensitive mass spectrometry, we show that the protein composition differs strongly between these organisms. Large numbers of major outer membrane protein-like proteins are present at high abundance in the outer membrane of Simkania negevensis and Waddlia chondrophila, whereas yet uncharacterized putative porins dominate in Parachlamydia acanthamoebae. Simkania represents the first case of a chlamydia completely lacking stabilizing cysteine-rich proteins in its outer membrane. In agreement with this, and in contrast to Parachlamydia and Waddlia, the cellular integrity of Simkania is not impaired by conditions that reduce disulfide bonds of these proteins. The observed differences in the protein composition of the outer membrane among members of divergent chlamydial families suggest different stabilities of these organisms in the environment, probably due to adaption to different niches or transmission routes.

Identification of novel small RNAs and characterization of the 6S RNA of Coxiella burnetii.

Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated (≥2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 5′ UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription.

Imaging of Chlamydia and host cell metabolism.

Chlamydial infections cause a wide range of acute and chronic diseases. Chlamydia trachomatis is the most common sexually transmitted bacterium while Chlamydia pneumoniae causes infections of the upper and lower respiratory tract. Chlamydia are obligate, intracellular bacteria with a biphasic developmental cycle that involves unique metabolic changes. Aside from entering an actively replicating state, Chlamydia may also implement persistent infections depending on different microenvironmental factors. In addition, changes in local oxygen availability and the composition of surrounding host microbiota are suggested to affect chlamydial growth and metabolism. Both bacteria and host cells endure characteristic metabolic changes during infection. Technical developments in recent years enable us to separately characterize chlamydial and host cell metabolism in living cells. This article focuses on novel approaches to analyze chlamydial metabolism such as NAD(P)H fluorescence lifetime imaging by two-photon microscopy. In addition, we provide an overview regarding promising future possibilities to further elucidate host-pathogen metabolic 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.

Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov.

The family Chlamydiaceae with the recombined single genus Chlamydia currently comprises nine species, all of which are obligate intracellular organisms distinguished by a unique biphasic developmental cycle. Anecdotal evidence from epidemiological surveys in flocks of poultry, pigeons and psittacine birds have indicated the presence of non-classified chlamydial strains, some of which may act as pathogens. In the present study, phylogenetic analysis of ribosomal RNA and ompA genes, as well as multi-locus sequence analysis of 11 field isolates were conducted. All independent analyses assigned the strains into two different clades of monophyletic origin corresponding to pigeon and psittacine strains or poultry isolates, respectively. Comparative genome analysis involving the type strains of currently accepted Chlamydiaceae species and the designated type strains representing the two new clades confirmed that the latter could be classified into two different species as their average nucleotide identity (ANI) values were always below 94%, both with the closest relative species and between themselves.In view of the evidence obtained from the analyses, we propose the addition of two new species to the current classification: Chlamydia avium sp. nov. comprising strains from pigeons and psittacine birds (type strain 10DC88T; DSMZ: DSM27005T, CSUR: P3508T) and Chlamydia gallinacea sp. nov. comprising strains from poultry (type strain 08-1274/3T; DSMZ: DSM27451T, CSUR: P3509T).

A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis

Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is consistent with the presence of chlamydial PG modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence to date that chlamydial species possess functional PG.

Developmental transitions of Coxiella burnetii grown in axenic media

Coxiella burnetii undergoes a biphasic developmental cycle within its host cell that generates morphologically and physiologically distinct large cell variants (LCV) and small cell variants (SCV). During the lag phase of the C. burnetii growth cycle, non-replicating SCV differentiate into replicating LCV that in turn differentiate back into SCV during stationary phase. Nearly homogeneous SCV are observed in infected Vero cells after extended incubation (21 to 28days). In the current study, we sought to establish whether C. burnetii developmental transitions in host cells are recapitulated during host cell-free (axenic) growth in first and second generation acidified citrate cysteine media (ACCM-1 and ACCM-2, respectively). We show that ACCM-2 supported developmental transitions and viability. Although ACCM-1 also supported SCV to LCV transition, LCV to SCV transition did not occur after extended incubation (21days). Instead, C. burnetii exhibited a ghost-like appearance with bacteria containing condensed chromatin but otherwise devoid of cytoplasmic content. This phenotype correlated with a near total loss in viability between 14 and 21days of cultivation. Transcriptional profiling of C. burnetii following 14days of incubation revealed elevated expression of oxidative stress genes in ACCM-1 cultivated bacteria. ACCM-2 differs from ACCM-1 by the substitution of methyl-β-cyclodextrin (Mβ-CD) for fetal bovine serum. Addition of Mβ-CD to ACCM-1 at 7days post-inoculation rescued C. burnetii viability and lowered expression of oxidative stress genes. Thus, Mβ-CD appears to alleviate oxidative stress in ACCM-2 to result in C. burnetii developmental transitions and viability that mimic host cell-cultivated organisms. Axenic cultivation of C. burnetii in ACCM-2 and new methods of genetic manipulation now allow investigation of the molecular basis of C. burnetii biphasic development.

The progeny ofLegionella pneumophilain human macrophages shows unique developmental traits

The Gram-negative bacterium Legionella pneumophila is an intracellular parasite of amoebae and an accidental human pathogen that causes a noncommunicable atypical pneumonia known as Legionnaires' disease (LD). In some mammalian cells (e.g. HeLa), L.pneumophila follows a biphasic developmental cycle, differentiating between a replicative form that actively multiplies intracellularly, and a mature infectious form (MIF) that emerges as progeny. To date, it is not known whether the L.pneumophila progenies that emerge from amoebae and human macrophages reach similar developmental stages. Here, we demonstrate that in relation to the fully differentiated and highly infectious MIFs that emerge from amoebae, the L.pneumophila progeny that emerges from macrophages is morphologically undifferentiated, less resistant to antibiotics and less able to initiate infections. However, the L.pneumophila progeny from macrophages did not show any defects in intracellular growth. We thus concluded that macrophage infection with L.pneumophila yields a low number of bona fide MIFs. Because MIFs are the transmissive forms of L.pneumophila produced in vivo, our results showing that they are not efficiently produced in cultured macrophages provide an initial insight into why LD is not communicable.

Conditional Gene Expression in Chlamydia trachomatis Using the Tet System

Chlamydia trachomatis is maintained through a complex bi-phasic developmental cycle that incorporates numerous processes that are poorly understood. This is reflective of the previous paucity of genetic tools available. The recent advent of a method for transforming Chlamydia has enabled the development of essential molecular tools to better study these medically important bacteria. Critical for the study of Chlamydia biology and pathogenesis, is a system for tightly controlled inducible gene expression. To accomplish this, a new shuttle vector was generated with gene expression controlled by the Tetracycline repressor and anhydryotetracycline. Evaluation of GFP expression by this system demonstrated tightly controlled gene regulation with rapid protein expression upon induction and restoration of transcription repression following inducer removal. Additionally, induction of expression could be detected relatively early during the developmental cycle and concomitant with conversion into the metabolically active form of Chlamydia. Uniform and strong GFP induction was observed during middle stages of the developmental cycle. Interestingly, variable induced GFP expression by individual organisms within shared inclusions during later stages of development suggesting metabolic diversity is affecting induction and/or expression. These observations support the strong potential of this molecular tool to enable numerous experimental analyses for a better understanding of the biology and pathogenesis of Chlamydia.

Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium

Chlamydia trachomatis is among the most clinically significant human pathogens, yet their obligate intracellular nature places severe restrictions upon research. Chlamydiae undergo a biphasic developmental cycle characterized by an infectious cell type known as an elementary body (EB) and an intracellular replicative form called a reticulate body (RB). EBs have historically been described as metabolically dormant. A cell-free (axenic) culture system was developed, which showed high levels of metabolic and biosynthetic activity from both EBs and RBs, although the requirements differed for each. EBs preferentially used glucose-6-phosphate as an energy source, whereas RBs required ATP. Both developmental forms showed increased activity when incubated under microaerobic conditions. Incorporation of isotopically labeled amino acids into proteins from both developmental forms indicated unique expression profiles, which were confirmed by genome-wide transcriptional analysis. The described axenic culture system will greatly enhance biochemical and physiological analyses of chlamydiae.

Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen) and surE (stationary-phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis SurE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in co-ordinating development of the stress resistance for passage from the present to the next host cells through its regulon.

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 Bonding within Components of the Chlamydia Type III Secretion Apparatus Correlates with Development

Chlamydia spp. exhibit a unique biphasic developmental cycle whereby infectious elementary bodies (EBs) invade host epithelial cells and differentiate into noninfectious, metabolically active reticulate bodies (RBs). EBs posses a unique outer envelope where rigidity is achieved by disulfide bonding among cysteine-rich envelope-associated proteins. Conversely, these disulfide bonds become reduced in RBs to accommodate vegetative growth, thereby linking the redox status of cysteine-rich envelope proteins with progression of the developmental cycle. We investigated the potential role of disulfide bonding within the chlamydial type III secretion system (T3SS), since activity of this system is also closely linked to development. We focused on structural components of the T3S apparatus that contain an unusually high number of cysteine residues compared to orthologs in other secretion systems. Nonreducing SDS-PAGE revealed that EB-localized apparatus proteins such as CdsF, CdsD, and CdsC form higher-order complexes mediated by disulfide bonding. The most dramatic alterations were detected for the needle protein CdsF. Significantly, disulfide bonding patterns shifted during differentiation of developmental forms and were completely reduced in RBs. Furthermore, at later time points during infection following RB to EB conversion, we found that CdsF is reoxidized into higher-order complexes. Overall, we conclude that the redox status of specific T3SS apparatus proteins is intimately linked to the developmental cycle and constitutes a newly appreciated aspect of functionally significant alterations within proteins of the chlamydial envelope.